Patent Application
20220396762
The disclosure provides compositions and methods for growing programmable enzyme-functionalized and sense-and-response bacterial cellulose living materials with engineered microbial co-cultures.
The field of engineered living materials (ELMs) aims to recreate desirable properties from natural biological materials such as, for example, self-assembly from simple raw materials, autonomous morphogenesis, diverse physical and chemical properties and the ability to sense-and-respond to environmental stimuli.
Because of its material properties, high natural yield, and genetic tractability, bacterial cellulose (BC) is an ideal natural biological material for ELM development. However, there remains a paucity of genetic tools and circuits with which to engineer BC-producing bacteria.
Accordingly, there exists a need for compositions and methods to produce bacterial cellulose living materials using engineered bacteria.
Provided herein are compositions and methods for growing programmable enzyme-functionalized and sense-and-response bacterial cellulose living materials with engineered microbial co-cultures.
Natural biological materials exhibit remarkable properties: self-assembly from simple raw materials, autonomous morphogenesis, diverse physical and chemical properties and the ability to sense-and-respond to environmental stimuli. The field of engineered living materials (ELMs) aims to recreate these properties to generate new and useful materials. Owing to its material properties, high natural yield and genetic tractability, bacterial cellulose (BC) is an ideal natural biological material for ELM development. However, there remains a paucity of genetic tools and circuits with which to engineer BC-producing bacteria. Inspired by the natural microbial community of fermented kombucha tea, the studies presented herein set out to co-culture the engineerable BC-producing bacterium Komagataeibacter rhaeticus with the model organism and synthetic biology host Saccharomyces cerevisiae. The studies provided herein first established and characterized a method for stable co-culture of K. rhaeticus and S. cerevisiae, in which the two species exhibited a symbiotic interaction. Using this system, the studies further demonstrated that S. cerevisiae can be engineered to secrete enzymes into BC, generating grown, functionalized materials. The studies presented herein further developed a method for incorporating yeast cells within the pellicle, generating living materials with tunable mechanical properties. This modular system allows for shuffling engineered S. cerevisiae strains that can sense and respond to chemical and optical inputs to be incorporated into BC, enabling a versatile biosensor platform production. As demonstrated herein, living test paper that can detect contaminants and living films that can generate images based on projected patterns has been produced. This novel and robust co-culture approach therefore empowers the sustainable growth of BC-based ELMs with programmable properties.
According to one aspect, isolated bacterial cellulose (BC)-based living compositions are provided. The BC-based living compositions include a stable co-culture of at least one bacterial cellulose (BC)-producing bacteria strain and at least one synthetic biology host organism, wherein the synthetic biology host organism has been engineered to secrete one or more enzymes into bacterial cellulose (BC), and wherein the interaction between the bacteria strain and the synthetic biology host organism in the co-culture produces a self-assembled BC-based living composition. In some embodiments, the bacteria comprises Komagataeibacter rhaeticus (K. rhaeticus). In some embodiments, the synthetic biology host organism comprises an engineered yeast strain. In some embodiments, the engineered yeast strain comprises an engineered Saccharomyces cerevisiae (S. cerevisiae) strain. In some embodiments, the BC-based living composition comprises a pellicle. In some embodiments, the pellicle comprises the BC-producing bacteria strain, the synthetic biology host organism, or both the BC-producing bacteria strain and the synthetic biology host organism.
In some embodiments, each of the one or more enzymes is linked to a cellulose binding protein or cellulose binding domain. In some embodiments, the cellulose binding protein or cellulose binding domain is CBDcex. In some embodiments, each of the one or more enzymes is linked to a secretion signal peptide. In some embodiments, expression of the one or more enzymes is controlled by a genetic circuit that responds to biological, chemical, or physical stimuli.
According to another aspect, isolated bacterial cellulose (BC)-based living compositions are provided. The compositions include a stable co-culture of Komagataeibacter rhaeticus (K. rhaeticus) and an engineered Saccharomyces cerevisiae (S. cerevisiae) strain, wherein the engineered strain of S. cerevisiae has been engineered to secrete one or more enzymes into bacterial cellulose (BC), and wherein the interaction between the K. rhaeticus and the engineered S. cerevisiae in the co-culture produces a self-assembled BC-based living composition. In some embodiments, the BC-based living composition comprises a pellicle. In some embodiments, the pellicle comprises K. rhaeticus, the engineered S. cerevisiae strain, or both K. rhaeticus and the engineered S. cerevisiae strain.
In some embodiments, each of the one or more enzymes is linked to a cellulose binding protein or cellulose binding domain. In some embodiments, the cellulose binding protein or cellulose binding domain is CBDcex. In some embodiments, each of the one or more enzymes is linked to a secretion signal peptide. In some embodiments, expression of the one or more enzymes is controlled by a genetic circuit that responds to biological, chemical, or physical stimuli.
According to another aspect, isolated engineered strains of Saccharomyces cerevisiae (S. cerevisiae) are provided. The engineered strains of S. cerevisiae secrete one or more enzymes into bacterial cellulose (BC). In some embodiments, each of the one or more enzymes is linked to a cellulose binding protein or cellulose binding domain. In some embodiments, the cellulose binding protein or cellulose binding domain is CBDcex.
In some embodiments, each of the one or more enzymes is linked to a secretion signal peptide. In some embodiments, expression of the one or more enzymes is controlled by a genetic circuit that responds to biological, chemical, or physical stimuli.
According to another aspect, isolated co-cultures of Komagataeibacter rhaeticus (K. rhaeticus) and an engineered strain of Saccharomyces cerevisiae (S. cerevisiae) are provided, wherein the engineered strain of S. cerevisiae secretes one or more enzymes into bacterial cellulose (BC). In some embodiments, each of the one or more enzymes is linked to a cellulose binding protein or cellulose binding domain. In some embodiments, the cellulose binding protein or cellulose binding domain is CBDcex.
In some embodiments, each of the one or more enzymes is linked to a secretion signal peptide. In some embodiments, expression of the one or more enzymes is controlled by a genetic circuit that responds to biological, chemical, or physical stimuli.
According to another aspect, methods of producing a bacterial cellulose (BC)-based living composition are provided. The methods include creating a stable co-culture of at least one bacterial cellulose (BC)-producing bacteria strain and at least one synthetic biology host organism, wherein the synthetic biology host organism strain has been engineered to secrete one or more enzymes into bacterial cellulose (BC), and wherein the interaction between the bacteria strain and the synthetic biology host organism in the co-culture produces a self-assembled BC-based living composition. In some embodiments, the synthetic biology host organism comprises an engineered yeast strain. In some embodiments, the engineered yeast strain comprises an engineered S. cerevisiae strain. In some embodiments, the bacterial strain comprises K. rhaeticus. In some embodiments, each of the one or more enzymes is linked to a cellulose binding protein or cellulose binding domain. In some embodiments, the cellulose binding protein or cellulose binding domain is CBDcex.
In some embodiments, each of the one or more enzymes is linked to a secretion signal peptide. In some embodiments, expression of the one or more enzymes is controlled by a genetic circuit that responds to biological, chemical, or physical stimuli.
In some embodiments, the bacteria strain and the synthetic biology host organism are co-cultured for at least about 3 days. In some embodiments, the bacteria strain and the synthetic biology host organism are co-cultured in a culture medium that has a higher density than the bacteria strain and the synthetic biology host organism. In some embodiments, the bacteria strain and the synthetic biology host organism are co-cultured in sucrose-containing media.
According to another aspect, methods of producing a bacterial cellulose (BC)-based living composition are provided. The methods include creating a stable co-culture of Komagataeibacter rhaeticus (K. rhaeticus) and an engineered Saccharomyces cerevisiae (S. cerevisiae) strain, wherein the engineered strain of S. cerevisiae has been engineered to secrete one or more enzymes into bacterial cellulose (BC), and wherein the interaction between the K. rhaeticus and the engineered S. cerevisiae in the co-culture produces a self-assembled BC-based living composition. In some embodiments, each of the one or more enzymes is linked to a cellulose binding protein or cellulose binding domain. In some embodiments, the cellulose binding protein or cellulose binding domain is CBDcex.
In some embodiments, each of the one or more enzymes is linked to a secretion signal peptide. In some embodiments, expression of the one or more enzymes is controlled by a genetic circuit that responds to biological, chemical, or physical stimuli.
In some embodiments, the K. rhaeticus and the engineered S. cerevisiae are co-cultured for at least about 3 days. In some embodiments, the K. rhaeticus and the engineered S. cerevisiae are co-cultured in a culture medium that has a higher density than the K. rhaeticus and the engineered S. cerevisiae. In some embodiments, the bacteria strain and the synthetic biology host organism are co-cultured in sucrose-containing media.
In some embodiments, the BC-based living composition comprises an engineered living material (ELM). In some embodiments, the BC-based living composition comprises a pellicle.
According to another aspect, uses of the isolated bacterial cellulose (BC)-based living compositions as a biosensor are provided.
According to another aspect, uses of the isolated BC-based living compositions for detecting microbe-microbe interactions are provided.
According to another aspect, uses of the isolated BC-based living composition in a method for the detection and/or degradation of an environmental pollutant are provided.
In some embodiments, the environmental pollutant is one or more β-lactam antibiotics, one or more estrogen hormones, or a combination thereof.
According to another aspect, uses of the isolated BC-based living composition for detecting one or more pathogens in a sample are provided.
According to another aspect, uses of the isolated BC-based living composition for detecting one or more biomarkers in a sample are provided.
According to another aspect, uses of the isolated BC-based living composition in a living test paper or living film are provided. In some embodiments, the living test paper or living film generates an image in response to one or more stimuli. In some embodiments, the one or more stimuli is light.
These and other aspects of the disclosure, as well as various embodiments thereof, will become more apparent in reference to the drawings and detailed description of the disclosure.
Provided herein are compositions and methods for growing programmable enzyme-functionalized and sense-and-response bacterial cellulose living materials with engineered microbial co-cultures.
Living organisms produce materials with remarkable properties. As cells grow and divide, they can synthesize a range of complex biopolymers while simultaneously controlling the patterning and morphogenesis of the resultant materials with incredible precision and over multiple length scales. This basic process of biological material assembly, typically fed by simple raw materials and occurring under mild conditions, enables the production of materials with a vast range of functional properties. Further, living cells within biological materials are poised to sense and respond to changes in their environment, allowing dynamic remodeling of material properties in response to defined chemical and physical stimuli.
The field of engineered living materials (ELMs) aims to take advantage of these properties by genetically-programming cells to assemble novel, useful materials1-4. However, rationally engineering cells to produce materials with the complexity of natural biological materials remains a major challenge. Numerous recent reports have described engineering biological material assembly in simple, genetically-tractable microbial systems. In particular, Escherichia coli biofilm nanomaterials have attracted great interest as model ELM systems. Curli fibers, amyloid polymers of the secreted CsgA protein, are a major component of the extracellular structural matrix of E. coli biofilms5. By engineering the CsgA monomer and controlling its expression, biofilm ELMs with electrical conductivity6-8, autonomous patterning9, metal adhesion10, catalytic activity11,12 and environmental sense-and-response functions13 have been created. However, production scalability remains a major limitation to potential applications of engineered curli nanomaterials; even under optimized conditions, the yields of E. coli biofilm ELMs remain restricted to tens of milligrams per liter of culture14.
More recently, bacterial cellulose (BC) has emerged as a promising alternative model ELM system. Various species of Gram-negative acetic acid bacteria—particularly members of the Komagataeibacter and Gluconacetobacter genera—are able to produce large quantities of extracellular cellulose. When grown in static liquid culture, these BC-producing bacteria secrete cellulose, in the form of numerous individual glucan chains bundled into ribbon-like fibrils. Over the course of several days, a thick floating mat forms—referred to as a pellicle—which is composed of a network BC fibrils, within which the BC-producing bacteria become embedded (
By contrast to curli nanomaterials, BC is naturally produced at relatively high yields—reaching in excess of 10 grams per liter. Further, BC exhibits useful natural material properties, including high crystallinity, high tensile strength, biodegradability and biocompatibility. Consequently, BC has garnered interest as a feedstock material for industrial applications, including wound dressings, acoustic diaphragms for headphones and speakers, stabilizers for foams and emulsions, and scaffolds for tissue engineering and battery separators.
However, by comparison to standard synthetic biology host organisms, there is a relative paucity of genetic tools for modification of BC-producing bacteria, limiting the engineerability of BC materials. Recent years have seen an increasing number of efforts to genetically engineer BC-producing bacteria to modify BC material properties. Several early studies focused on increasing BC yields15,16. More recently, BC-producing bacteria have been engineered to produce additional non-native polysaccharides, creating chitin-cellulose17 and curdlan-cellulose18 co-polymer materials. The studies herein have developed and utilized a modular genetic toolkit to engineer a newly-isolated BC-producing strain, Komagataeibacter rhaeticus19. In addition, by adding chemically modified glucose to the growth media the BC-producing bacteria is able to incorporate the subunits into cellulose and a change in BC could be achieved.
Although engineering BC-producing bacteria will likely continue to be a fruitful approach to developing BC-based ELMs, the studies presented herein were designed to test whether an alternative, co-culture approach could accelerate these efforts. Specifically, these studies set out to co-culture BC-producing bacteria with a standard synthetic biology host organism, for which a wealth of genetic tools and circuits are available. It was speculated that division of labor between BC-producing bacteria and a co-cultured host conferring novel functional properties, might expand the possibilities for BC-based ELMs. Previous work has shown that engineered E. coli can be incorporated into BC materials by manually adding cells partway through pellicle growth20. This approach was used to create BC-based ELMs in which engineered E. coli could sense-and-respond to chemical inducers21. However, this previous approach uses manual intervention to generate co-cultures and fails to take advantage of one of the advantages of biological material production, self-assembly. By contrast, the studies presented herein provide a stable co-culture system, which enables spontaneous self-assembly and growth of BC ELMs with programmable properties.
To achieve this, the studies presented herein took inspiration from the natural source of many of the highest-yielding BC-producing bacteria, kombucha tea. Kombucha tea is a fermented beverage produced by the action of a microbial community of bacteria and yeast (
Therefore, the studies presented herein set out to recreate kombucha-like co-cultures of an engineerable BC-producing bacterium, K. rhaeticus, and the synthetic biology host organism S. cerevisiae (
Establishing S. cerevisiae-K. rhaeticus Co-Culture Conditions
The first aim of the studies presented herein was to recreate kombucha-like co-cultures between S. cerevisiae and K. rhaeticus. These studies selected two engineered strains, an S. cerevisiae strain constitutively expressing green fluorescent protein (GFP) from a strong promoter (yWS195) and a K. rhaeticus strain constitutively expressing monomeric red fluorescent protein (mRFP) from a mid-strength promoter (Kr RFP). These strains were chosen to enable detection of and discrimination between strains within co-cultures through fluorescence measurements.
To identify conditions under which K. rhaeticus and S. cerevisiae might be efficiently co-cultured, these studies initially screened a set of co-culture conditions for growth of both strains. K. rhaeticus and S. cerevisiae were grown separately in liquid culture and then inoculated together into different media at a range of inoculation ratios. Four different culture media were selected: standard rich yeast medium with glucose (YPD) or sucrose (YPS) as the carbon source and standard medium for cultivation of BC-producing bacteria with glucose (HS-glucose) or sucrose (HS-sucrose) as the carbon source. This screen led to a number of observations regarding the growth of S. cerevisiae and K. rhaeticus (
These studies next attempted to develop a standard co-culture protocol to confirm these observations which could be used for all subsequent investigations. In this approach, liquid cultures of K. rhaeticus and S. cerevisiae were grown and diluted to fixed cell densities, chosen based on this initial screen (
Given the aim of establishing a robust method for co-culturing S. cerevisiae alongside K. rhaeticus, the observed unexpected beneficial interaction between K. rhaeticus and S. cerevisiae in sucrose media can be considered a useful trait. Specifically, since K. rhaeticus growth is dependent on the growth of S. cerevisiae, these co-culture conditions effectively ensure that K. rhaeticus cannot outcompete S. cerevisiae and thus ensure formation of a stable co-culture. Co-culture in YPS following this protocol was therefore defined as the standard co-culture condition.
This interaction likely represents either a commensal symbiotic relationship, where one partner benefits from the interaction while the other is unaffected or a parasitic symbiotic relationship, in which one partner benefits from the interaction while the other is detrimentally affected. A more desirable co-culture system might incorporate an obligate mutualistic symbiosis, where both species are unable to survive without the other. In this case, neither species can outcompete the other, resulting in a stable co-culture system. In some embodiments, such a co-dependence between a bacterial cell, such as K. rhaeticus, and a synthetic biology host cell, such as S. cerevisiae, can be engineered. For example, the synthetic biology host cell, such as S. cerevisiae, can be engineered to express one or more enzymes that metabolize a carbon source (e.g., in culture media) that otherwise is not usable by the bacterial cell, such as K. rhaeticus, to produce a carbon source that can be metabolized by the bacterial cell, such as K. rhaeticus. As another example, the synthetic biology host cell, such as S. cerevisiae, can be engineered to express one or more enzymes that degrade a molecule (e.g., in culture media) that otherwise is detrimental to the bacterial cell, such as K. rhaeticus, and thereby allow the bacterial cell to survive and grow.
Co-Culture Characterization
Having defined standard conditions for co-culture growth, these studies next attempted to characterize various properties of the co-culture system to guide BC material modification efforts. First, to determine the optimal incubation time for pellicle formation, the pellicle yield from co-cultures of S. cerevisiae and K. rhaeticus over time was measured. Pellicle formation was first detectable at low yields after 2 days, yields then plateaued at approximately 9 mg after 3 days (
One challenge of the co-culture system affecting the downstream development of BC ELMs is the distribution of S. cerevisiae and K. rhaeticus between the liquid below the pellicle and the pellicle layer itself. Therefore, co-cultures were prepared and counts of viable cells obtained from the liquid and pellicle layers for both K. rhaeticus and S. cerevisiae. For comparison, viable cell counts were also obtained from mono-cultures of S. cerevisiae grown in YPS and K. rhaeticus grown in YPD and YPS. As described in greater detail in the methods section, since the degraded pellicle volume was not measured, cell counts in pellicles were estimated by assuming a fixed pellicle volume. In all conditions, the majority of K. rhaeticus cells were found in the pellicle layer, while the majority of S. cerevisiae cells were found in the liquid layer (
Since yeast and bacterial communities are stable over many cycles of passage during kombucha tea brewing, these studies next set out to determine to what extent the co-culture system constitutes a stable co-culture. In order to assay long-term co-culture dynamics, these studies used a serial passage approach, in which the liquid below mature pellicles was inoculated into fresh media and allowed to grow for 3 days (
Without intending to be bound by theory, one possible explanation for the observed stimulatory effect of S. cerevisiae on the growth of K. rhaeticus in sucrose medium is that S. cerevisiae converts sucrose to a carbon source which K. rhaeticus is able to consume more efficiently. Several studies report that symbiotic interactions occur between the BC-producing bacteria and yeast in the kombucha microbial community. However, the exact nature of the interactions between kombucha microbes remains unclear. It is believed that yeasts in kombucha fermentations hydrolyze the majority of carbon source, sucrose, to form extracellular glucose and fructose through the action of the secreted enzyme invertase (
Without intending to be bound by theory, some studies have shown that, consistent with an interaction in which K. rhaeticus and S. cerevisiae share culture medium nutrients, pellicle yields from YPS co-cultures were reduced compared to pellicle yields from K. rhaeticus YPD mono-cultures: 47.0±4.3 mg compared to 61.7±2.7 mg, respectively (
To further explore this possibility, these studies tested whether purified S. cerevisiae invertase could enhance K. rhaeticus growth in YPS medium (
Finally, to give an idea of the robustness of the co-culture methods provided herein, these studies set out to determine the reproducibility of co-culture properties. To achieve this, identical co-cultures were prepared following the standard protocol on three separate occasions and two parameters were measured: pellicle yields and cell counts. These two parameters were chosen as they are likely to significantly impact any downstream applications. For instance, if S. cerevisiae is engineered to secrete proteins into the pellicle, both the cell density of S. cerevisiae in the co-culture and the pellicle yield will strongly influence the final titers of secreted protein. It was found that pellicle yields tended to be consistent within triplicate samples, but variable between co-cultures set up on different occasions (
The propensity for S. cerevisiae to grow in the liquid layer rather than the pellicle layer might preclude the utility of co-cultured S. cerevisiae for certain applications—for example, controlling pattern formation or creating biosensor materials. In addition, fluorescence microscopy showed that S. cerevisiae cells that were present in the pellicle layer exhibited a highly-variable distribution across the pellicle (
Engineering BC Material Functionalization
Having developed conditions for growth of co-cultures, these studies next attempted to take advantage of the wealth of biotechnological tools developed for S. cerevisiae to confer new biological functions to BC materials. Since S. cerevisiae is well-known as an effective recombinant protein secretion host, these studies first asked if S. cerevisiae strains could be engineered to secrete proteins and thereby functionalize BC materials (
To confirm that the engineered strains were able to secrete functional BLA enzyme, wild type, yCGO4 and yCGO5 S. cerevisiae strains were grown in mono-culture in YPS medium. Supernatants were harvested from these cultures and then screened for BLA activity using the colorimetric nitrocefin assay (
Having demonstrated that the engineered S. cerevisiae strains can secrete active BLA, these studies next attempted to test whether they could be co-cultured with K. rhaeticus to produce a grown, enzyme-functionalized material. Co-cultures were prepared with wild type, BLA-secreting (yCG04) or BLA-CBD-secreting (yCG05) strains and the resultant unprocessed, wet pellicles were screened for β-lactamase activity. While pellicles from co-cultures with WT S. cerevisiae showed no BLA activity, a clear signal was observed with pellicles from co-cultures with BLA-secreting and BLA-CBD-secreting strains (FIG. 2D), demonstrating that engineered S. cerevisiae can indeed direct BC functionalization. Fusion of the CBD to the BLA C-terminus resulted in an increase in the observed β-lactamase signal. Therefore, although fusion of the CBD resulted in a decreased yield of secreted BLA in mono-culture, under co-culture it results in an increase in the proportion of secreted enzyme that becomes incorporated into the pellicle layer and so a greater degree of BC functionalization.
Since native BC pellicles have a water content of ˜99%, these studies attempted to determine if enzyme-functionalized materials could be dried and re-hydrated without eliminating BLA activity. To test this, pellicles produced by co-culturing K. rhaeticus with WT, BLA-secreting (yCG04) and BLA-CBD-secreting (yCG05) S. cerevisiae strains were dried by sandwiching them between sheets of absorbent paper towel to create thin, paper-like materials (
To enable comparison of the absolute levels of β-lactamase activity between wet pellicles and dried pellicles, the nitrocefin assay was repeated alongside standard curves with a commercial BLA enzyme (
As the BLA enzyme is passively incorporated within the BC matrix by diffusion and the BLA-CBD fusion is specifically bound through the CBD-cellulose interaction, it might, be anticipated that BLA enzyme could leach out of the BC material over time, while BLA-CBD would remain bound stably. To test this, dried pellicles functionalized with BLA and BLA-CBD were subjected to multiple rounds of washes in PBS buffer and then assayed for β-lactamase activity (
Having demonstrated that the co-culture approach enables BC functionalization with β-lactamase, these studies set out to determine whether BC can be functionalized with other enzymes. A native S. cerevisiae secreted protein, the alpha-galactosidase enzyme Mel1 for which a simple colorimetric activity assay is available, was selected for these studies. In addition, two laccase enzymes, previously engineered to be expressed and secreted from S. cerevisiae29, were selected. Laccases catalyze one-electron oxidations, typically possessing a broad substrate range and have attracted interest for numerous industrial applications, such as in textile and food processing, in the pharmaceutical and chemical industries, in biofuel cells, and in the degradation of environmental pollutants30. The YTK cloning system was used to clone S. cerevisiae Mel1 and fungal laccases from Myceliophthora thermophila (MtLcc1) and from Coriolopsis troggi (CtLcc1). Variants of each protein were generated with either the native secretion signal peptide or the S. cerevisiae MFα signal peptide, all of which also possessed a C-terminal CBD (
Finally, these studies explored whether BC could be functionalized with a more challenging target protein which is not secreted from its native host, green fluorescent protein (GFP). Previous attempts to secrete GFP from S. cerevisiae have yielded mixed results, with some studies reporting inefficient secretion and others reporting low milligram per liter secretion yields31,32. Here, these studies again employed the YTK system to generate strains secreting GFP and GFP fused to CBDcex. Both GFP and GFP-CBD were fused to the S. cerevisiae MFα secretion signal peptide and expressed from a strong constitutive promoter. It was found that both GFP and GFP-CBD could be detected in the culture supernatant after 48 hours growth in shake-flask, indicating successful secretion (
In some embodiments, the co-culture approach for BC functionalization using engineered S. cerevisiae strains to functionalize BC may be limited to cases where proteins of interest can be efficiently secreted or are highly-active at low yields. In addition, in some embodiments, the applicability of enzyme-functionalized BC materials produced through this method may be limited to conditions under which functionalizing proteins and CBDs remain active. Finally, since sterilizing BC materials without inactivating functionalizing proteins poses a challenge, in some embodiments, potential applications may use BC materials containing live engineered cells.
The primary motivation of these efforts was to demonstrate that a co-culture approach enables self-assembly of BC materials with engineered functional properties. However, methods to attach enzymes to materials—also known as ‘enzyme immobilization’—have potential applications in a number of industries. Broadly, enzyme immobilization is used to improve the cost-effectiveness of industrial biocatalysts, facilitating purification of the product from the enzyme and often improving the stability and reusability of the enzyme. Immobilized enzymes are used in a variety of industrial processes: lactase in lactose-free milk production33, glucose isomerase in high-fructose corn syrup production34 and lipases in the interesterification of food fats and oils35. Moreover, BC has been proposed as a suitable material substrate for enzyme immobilization36-38. The approach provided herein enables sustainable, self-assembled enzyme immobilization under mild conditions of BC-materials which may have potential utility in decontamination of soil and wastewater from β-lactam antibiotics39,40. Therefore, 0-lactamase-functionalized BC materials could be applied to the bioremediation of these environments. Similarly, given their numerous potential uses, BC materials functionalized with laccase enzymes could be applied in areas such as bioremediation, pharmaceutical and chemical industries or textile and food processing. These and other applications might be facilitated by the fact that enzyme-functionalized BC materials can retain activity following drying and re-hydration after long-term storage.
Incorporating S. cerevisiae Cells within BC Materials
The majority of S. cerevisiae cells in the co-cultures are found in the liquid layer below the pellicle, which has to be taken into account when generating biological ELM engineering efforts. Consequently, the studies provided herein attempted to develop a co-culture method that would enable efficient incorporation of S. cerevisiae cells within BC. S. cerevisiae settles to the bottom of culture vessels because the density of the cells is greater than that of water: ˜1.11 g/mL compared to 1 g/mL26. Therefore, it was hypothesized that, if the density of the culture medium were increased to >1.11 g/mL, S. cerevisiae cells would float to the surface rather than sinking and so become incorporated into the newly-forming pellicle at the air-water interface (
Modifying BC Material's Physical Properties with Secreted Enzymes
In nature, living cells can modify or remodel their surrounding non-cellular matrix using various secreted enzymes. Such process plays an important part in the development and differentiation of multicellular organisms as well as disease progression. Inspired by the role of proteases in orchestrating the dynamics of protein-based extracellular matrix, these studies explored whether one could modify the cellulosic matrix in the pellicles with cellulases secreted from the yeast cells, thus change the physical properties of the BC living materials.
Metabolic engineering of S. cerevisiae for cellulose degradation through expressing fungal cellulases has become a well-established field in past decades. In this study, a yeast strain, yCelMix, in which cellobiohydrolases (CBH1 and CBH2), endoglucanase (EGL2), β-glucosidase (BGL1), and lytic polysaccharide monooxygenases (LPMO) are optimized to be secreted simultaneously for synergistic cellulose degradation, was constructed. (
Despite the cellulose yield is not deeply impacted by cellulase secretion, the mechanical properties of the pellicles are significantly altered. The stress-strain curves from tensile test demonstrates a clear difference between WT pellicle and yCelMix pellicle (
The weakening of microstructure in yCelMix pellicle is also reflected by its rheological properties. In the strain sweep experiment (
Engineering Sense-and-Response BC Materials
Another of the advantageous properties of natural biological materials is their ability to sense-and-respond to changes in their external environment. A variety of genetic circuits have been engineered in S. cerevisiae enabling biological, chemical, and physical stimuli to drive changes in gene expression. Therefore, these studies attempted to determine whether such engineered biosensor S. cerevisiae strains could be incorporated into BC to create materials able to sense-and-respond to environmental stimuli (
In order to generate BC materials in which the GPY093 biosensor strain was incorporated within the BC matrix, co-cultures of K. rhaeticus and wild-type or GPY093 S. cerevisiae were prepared in YPS-OptiPrep medium. The resultant pellicles were rinsed and then incubated in fresh medium in the presence or absence of BED for 24 hours. By contrast to pellicles grown with wild-type S. cerevisiae, addition of BED to pellicles containing GPY093 S. cerevisiae yielded a strong GFP signal (
A variety of other S. cerevisiae biosensor strains have been engineered to screen and detect a range of environmental pollutants and pathogens41-43. This approach could, therefore, be used to create grown biosensor materials for on-site screening of medical or environmental samples. There is increasing concern over environmental pollution with endocrine disruptors/estrogen hormones, including, by way of non-limiting example, BED. In some embodiment, biosensor BC materials are useful in the detection of BED in the environment.
However, for this approach to be feasible, biosensor materials would have to be stored without losing their functionality. Therefore, these studies attempted to determine if S. cerevisiae cells incorporated into BC materials could remain viable after drying and long-term storage (
As mentioned above, S. cerevisiae strains have been engineered to sense-and-respond to numerous other biological, chemical, and physical stimuli. One class of S. cerevisiae biosensors employs the G protein coupled receptor (GPCR) family of receptors. GPCRs are membrane protein receptors that share a common basic structure but are able to detect a remarkable range of different chemical and physical stimuli. S. cerevisiae possesses a native GPCR signaling cascade, which it uses to sense-and-respond to mating pheromones. By transplanting heterologous GPCRs into this pathway, biosensors with novel targets can be generated. To demonstrate that this approach is compatible with GPCR-based signaling, co-cultures were prepared with a S. cerevisiae biosensor strain. This biosensor strain detects the S. cerevisiae mating factor alpha (MFα) peptide through the native Ste2 GPCR, activating GFP expression in response. Pellicles into which yWS890 and wild type S. cerevisiae had been incorporated were grown, dried and re-hydrated in the presence of absence of MFα. Biosensor pellicles exhibited a clear increase in GFP signal in the presence of MFα, indicating that the GPCR-based biosensor strain does indeed function well using this grown biosensor approach (
Biosensor strains serve to sense external stimuli and provide a convenient readout, in this case in the form of fluorescent protein expression. However, living materials are also able to dynamically remodel and adapt their functional properties in response to changes in their environment. These studies took advantage of the modularity of the YTK to engineer an S. cerevisiae strain secreting the laccase CtLcc1 under control of the BED-inducible promoter, yCG23 (
Spatial Patterning of Catalytic Living Materials
Building upon the fact that living yeast cells can sense and respond to chemical inputs in the co-culture system, these studies further investigated the possibility of using other modalities of inputs to program the living materials. Optogenetic tools in bacteria such as E. coli have enabled high-resolution spatial patterning of microbial biofilm using light-inducible control of protein-protein interactions. Inspired by the recent advances in optical dimerizers in yeast, these studies started the implementation by using a blue light sensing system that is based on the CRY2/CIB transcription system. To achieve high activation and low background expression, these studies first investigated the importance of promoter strengths in driving the expression of the DNA binding component (LexA-CRY2) and the activation component (VP16-CIB1) (
These studies further explored the responsiveness of yNC and yNS pellicles to light patterning created by masking and projecting. These “living films” were grown in dark for 3 days before exposure. For mask patterning, the pellicles were covered by a black foil piece with a square carved out in the center, where bioluminescence soon appeared after 4 hours of development under a LED lamp (
These studies presented herein have demonstrated that stable co-cultures of two engineerable microbes, K. rhaeticus and S. cerevisiae can be recreated in the lab. By screening various co-culture conditions, these studies uncovered an interaction between K. rhaeticus and S. cerevisiae, resembling a commensal symbiotic interaction, in which the presence of S. cerevisiae promotes the growth of K. rhaeticus on sucrose medium. Exploiting this interaction, these studies developed and characterized a standard protocol that enabled reproducible co-culture of K. rhaeticus with S. cerevisiae and self-assembly of a BC-based biological material. There is growing interest in the field of synthetic ecology, which aims to construct and understand artificial communities of microbes. This co-culture system therefore is not only of interest for the development of biological ELMs but may also represent an interesting model system for synthetic ecology, in which further microbe-microbe interactions could be investigated and engineered.
Using this co-culture method, these studies demonstrated how the wealth of existing S. cerevisiae synthetic biology tools can be leveraged to program functional biological properties into grown BC materials. Firstly, these studies showed that a number of enzymes can be secreted from S. cerevisiae, become incorporated into the BC material and, therefore, functionalize the material. In addition, these studies found that functionalized BC materials can be dried and later rehydrated, retaining catalytic activity. The functionalized BC materials that were generated could be applied to the degradation of β-lactam antibiotics or estrogen hormones present in wastewater streams, both of which are environmental pollutants. Further, this approach is highly adaptable—numerous other protein targets could be secreted from S. cerevisiae to add various biological properties to the material. However, the feasibility of the approach strongly depends on the survivability of the enzymes in the growth media (pH 5-6, salt composition) or after sterilization procedures. Also, yeast might not be able to secrete every enzyme as efficiently. Given this modular approach it is possible to design, engineer and grow various yeast strains in co-cultures and screen the obtained ELM with high throughput methods.
Secondly, these studies showed how S. cerevisiae biosensor strains can be incorporated within the BC material to modify the bulk mechanical properties of BC as well as create living materials able to sense and respond to changes in their environment. The ability of cells to sense-and-respond to environmental stimuli underlies a number of interesting properties of natural biological materials, including autonomous patterning and dynamic, responsive physical properties. Once again, since this approach is highly-adaptable, numerous other S. cerevisiae biosensor strains able to detect pathogens41, environmental pollutants42, biomarkers44 and so on, could be used in conjunction with the co-culture method.
So far, BC serves as a storage and protection shell for yeast and enzymes as this approach, focused mainly on engineering and exchanging S. cerevisiae. However, morphology and properties of the BC scaffold are also a target through genetic engineering of K. rhaeticus, as it is genetically tractable and more tools are being developed.
In summary, the co-culture approach to grow biofunctional ELMs combines the advantage of high yield BC production up to gram per liter quantities and its versatile modification and functionalization possibilities. Past and future advances in synthetic biology related yeast strain modifications can be easily incorporated and used to sense, degrade or assemble surrounding materials. This modular approach might even allow to tackle more complex tasks by relying on more than one engineered yeast strain.
As described herein, co-cultures of the bacterial cellulose-expressing bacteria cell, and a synthetic biology host organism are provided. In some embodiments, any type of bacterial cell that expresses bacterial cellulose can be used as described herein. In some embodiments, the bacterial cell is a Komagataeibacter cell or a Gluconacetobacter cell. In one exemplary embodiment, the bacterial call is K. rhaeticus.
In some embodiments, the synthetic biology host organism is a prokaryotic cell or a eukaryotic cell. One exemplary synthetic biology host organism is S. cerevisiae. In some embodiments the synthetic biology host organism is a bacterial cell, such as Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. The bacterial cell can be a Gram-negative cell such as an Escherichia coli (E. coli) cell, or a Gram-positive cell such as a species of Bacillus. In other embodiments, the cell is a fungal cell such as a yeast cell, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains. Preferably the yeast strain is a S. cerevisiae strain. Other examples of fungi include Aspergillus spp., Pennicilium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.
As described herein, a co-culture approach can be used to grow biofunctional ELMs that provide high yield BC production and functionalized materials. High yield BC production can be gram per liter quantities, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 grams per liter, or in excess of 10 grams per liter.
Co-cultures are considered stable when neither of the two cell types outgrows the other, which can be assessed by the stability of cell numbers or cell densities in the co-culture over time, i.e., by comparing these values in at least two cycles of passage. The stability of the co-culture also can be assessed by stability of pellicle yield over time, i.e., by comparing the pellicle yield (e.g., by weight) in at least two cycles of passage. Such parameters are considered to remain stable (relatively constant) if they vary by no more than 25%, preferably no more than 20%, more preferably no more than 15%, more preferably no more than 10%, still more preferably no more than 5%. To determine stability of cell counts, they are determined in the part of the co-culture in which they are stable for each of the cell types. For example, for K. rhaeticus, cell counts were found to be consistent in the pellicle layer, where the majority of cells were detected, but more variable in the liquid layer. For S. cerevisiae, cell counts were found to be consistent in the liquid layer of the co-culture.
The co-cultures disclosed herein are stable over many cycles of passage, such as more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 60, more than 70, more than 80, more than 90, or, more than 100 cycles.
In some embodiments, one or more of the genes associated with the products and methods disclosed herein is expressed in a recombinant expression vector, including genetic circuits that can be used to control expression of one or more output proteins of the synthetic biology host organism. As used herein, the terms “protein” and “polypeptide” are used interchangeably and thus the term polypeptide may be used to refer to a full-length polypeptide and may also be used to refer to a fragment of a full-length polypeptide.
Examples of such proteins expressed by synthetic biology host organisms in a co-culture include enzymes that can be used to provide various functionalities to the bacterial cellulose produced by the bacteria in the co-culture or to modify the physical properties of the bacterial cellulose produced by the bacteria in the co-culture. Examples of such enzymes include β-lactam hydrolyzing enzymes, such as β-lactamases; alpha-galactosidases, such as S. cerevisiae Mel1; laccases, such as laccases from Myceliophthora thermophila (MtLcc1) or from Coriolopsis troggi (CtLcc1); lactases; glucose isomerases; lipases; cellulases; cellobiohydrolases, such as CBH1 and CBH2; endoglucanases, such as EGL2; 13-glucosidases, such as BGL1; and lytic polysaccharide monooxygenases, such as LPMO. Other proteins expressed by synthetic biology host organisms can include G-protein coupled receptors (GPCRs); optical dimerizer proteins, such as the CRY2/CIB transcription system; and detectable proteins, such as fluorescent proteins and colorimetric proteins. Proteins that are homologous to the above enzymes and proteins also can be used.
In some embodiments, the proteins such as enzymes are linked to a cellulose binding protein, such as a cellulose-binding domain (CBD) to provide a specific, stable binding interaction between the enzyme and cellulose. In some embodiments, the CBD is fused to the C-terminus of the protein, such as an enzyme. In some embodiments, the CBD is CBDcex, the 112 amino acid region from the C-terminus of the Cex exoglucanase from Cellulomonas fimi.
To secrete such enzymes into the co-culture media for binding to bacterial cellulose and incorporation into the pellicle, variants of each protein can be made that include a suitable secretion signal peptide such that the protein is exported from the synthetic biology host cell. For example, if a native secretion signal peptide is present in the protein, it can be used and tested for suitability in the synthetic biology host cell. Alternatively, a secretion signal peptide from the synthetic biology host cell itself can be used, such as the S. cerevisiae MFα signal peptide.
As used herein with respect to cells, cultures, co-cultures, pellicles, polypeptides, proteins, or fragments thereof, “isolated” means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified such as by chromatography or electrophoresis. Isolated proteins or polypeptides may be, but need not be, substantially pure. The term “substantially pure” means that the proteins or polypeptides are essentially free of other substances with which they may be found in production, nature, or in vivo systems to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be obtained naturally or produced using methods described herein and may be purified with techniques well known in the art. Because an isolated protein, for example, may be admixed with other components in a preparation, the protein may comprise only a small percentage by weight of the preparation. The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e. isolated from other proteins.
As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes.
A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
As used herein, a coding sequence and regulatory sequences are said to be “operably joined” (or “operably linked”) when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
When the nucleic acid molecule is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors used herein may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (or RNA). That heterologous DNA (or RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA (or RNA) in the host cell. For example, heterologous expression of genes associated with the disclosed methods and products, such as for production of enzymes in a synthetic biology host cell such as S. cerevisiae, is demonstrated herein.
A nucleic acid molecule as used in the products and methods disclosed herein can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule may also be accomplished by integrating the nucleic acid molecule into the genome.
In some embodiments one or more genes used in the products and methods disclosed herein is expressed recombinantly in a bacterial cell. Bacterial cells can be cultured in media of any type (rich or minimal) and any composition. As would be understood by one of ordinary skill in the art, a variety of types of media can be compatible with the products and methods disclosed herein. However, as shown herein, certain types of media or components of media (e.g., carbon source) provide superior results for the products and methods disclosed herein. The selected medium can be supplemented with various additional components. Some non-limiting examples of supplemental components include glucose or other carbon sources (such as described elsewhere herein), antibiotics, IPTG for gene induction, ATCC Trace Mineral Supplement, and glycolate. Similarly, other aspects of the medium, and growth conditions of the cells used in the products and methods disclosed herein may be optimized through routine experimentation. In some embodiments, factors such as choice of media, media supplements, and temperature can influence growth of organisms in the co-cultures described herein, or production levels of bacterial cellulose or of enzymes or other proteins expressed in organisms in the co-cultures. In some embodiments the concentration and amount of a supplemental component may be optimized. In some embodiments, how often the media is supplemented with one or more supplemental components, and the amount of time that the organisms are co-cultured is optimized.
The term “linked” or “linkage” refers to an association of two entities, for example, of two molecules such as two proteins, or a protein and a reactive handle, or a protein and an agent, e.g., a detectable label. The association can be, for example, via a direct or indirect (e.g., via a linker) covalent linkage or via non-covalent interactions. In some embodiments, the association is covalent. In some embodiments, two molecules are linked via a linker connecting both molecules. For example, in some embodiments where two proteins are linked to each other to form a fusion protein, the two proteins may be linked via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein.
The term “detectable label” refers to a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the molecule, e.g., a protein or peptide, or other entity, to which the label is attached. Labels can be directly attached or can be attached via a linker. It will be appreciated that the label may be attached to or incorporated into a molecule, for example, a protein, polypeptide, or other entity, at any position. In general, a detectable label can fall into any one (or more) of five classes: I) a label which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 18F, 31F, 32F, 35S, 67Ga, 76Br, 99mTc (T-99m) 111In, 123I, 125I, 131I, 153Gd, 169Yb and 186Re; II) a label which contains an immune moiety, which may be antibodies or antigens, which may be bound to enzymes (e.g., such as horseradish peroxidase); III) a label which is a colored, luminescent, phosphorescent, or fluorescent moieties (e.g., such as the fluorescent label fluorescein-isothiocyanate (FITC); IV) a label which has one or more photo affinity moieties; and V) a label which is a ligand for one or more known binding partners (e.g., biotin-streptavidin, FK506-FKBP). In certain embodiments, a label comprises a radioactive isotope, preferably an isotope which emits detectable particles, such as beta particles. In certain embodiments, the label comprises a fluorescent moiety. In certain embodiments, the label is the fluorescent label fluorescein-isothiocyanate (FITC). In certain embodiments, the label comprises a ligand moiety with one or more known binding partners. In certain embodiments, the label comprises biotin, which may be detected using a streptavidin conjugate (e.g., fluorescent streptavidin conjugates such as Streptavidin ALEXA FLUOR® 568 conjugate (SA-568) and Streptavidin ALEXA FLUOR® 800 conjugate (SA-800), Invitrogen). In some embodiments, a label is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussia luciferase). It will be appreciated that, in certain embodiments, a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal. Non-limiting examples of fluorescent proteins include GFP and derivatives thereof, proteins comprising fluorophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins. Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See, e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein: properties, applications, and protocols Methods of biochemical analysis, v. 47 Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, D M, et al., Physiol Rev. 90(3):1103-63, 2010, for discussion of GFP and numerous other fluorescent or luminescent proteins. In some embodiments, a label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.
The term “homologous,” as used herein, is an art-understood term that refers to nucleic acids or polypeptides that are highly related at the level of nucleotide or amino acid sequence. Nucleic acids or polypeptides that are homologous to each other are termed “homologs” or “homologues.” Homology between two sequences can be determined by sequence alignment methods known to those of skill in the art. Two sequences are considered to be homologous if they are at least about 50-60% identical, e.g., share identical residues (e.g., amino acid residues) in at least about 50-60% of all residues comprised in one or the other sequence, at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical, for at least one stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 amino acids, or over the full sequence of one or both of the molecules being compared. The “percent identity” of two nucleic acid or two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleic acid or protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain sequences homologous to the nucleic acid or protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
The products and methods disclosed herein are further illustrated by the following Examples, which in no way should be construed as further limiting. It should be understood that these Examples, while indicating preferred embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the products and methods disclosed herein, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the products and methods disclosed herein to various uses and conditions.
The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, particularly for the teachings referenced herein.
Strains, constructs and DNA assembly: Strains and DNA constructs used in this study include Kr, BY4741, yCelMix, yCBH1, yCBH2, yBGL1, yEGL2, yLPMO, yXTH3, yTT-GFP, yRR-GFP, yTR-GFP, yRT-GFP, yNC, yNS, yNSΔSED1. The plasmids constructed in this study were constructed using standard cloning techniques. Oligonucleotides were obtained from IDT. Restriction endonucleases, Phusion-HF DNA polymerase and T7 DNA ligase were obtained from NEB. Unless stated, the plasmids were transformed into E. coli turbo (NEB) for amplification and verification before transforming into S. cerevisiae for protein expression and secretion. The constructs were verified by restriction enzyme digestion and Sanger sequencing (Source Bioscience).
S. cerevisiae constructs for strains yCG01, yCG02, yCG04 and yCG05 were cloned using the yeast toolkit (YTK) system developed by the Dueber lab27. The YTK system uses Golden Gate assembly to combine pre-assembled, defined parts into single gene cassettes and multi-gene cassettes. The final positions of pre-assembled parts within constructs are determined by the sequences of 4 bp overhangs created by digestion with type IIS restriction enzymes (BsaI or BsmBI). Users can therefore pick and choose from pre-assembled promoter, terminator and protein-coding parts to create expression cassettes. The combination of parts used to create strains yCG01, yCG02, yCG04 and yCG05 were cloned into a pre-assembled backbone plasmid, pYTK096, containing genetic elements enabling cloning in E. coli and later integrative transformation into the URA3 locus in S. cerevisiae. Type 2, 3 and 4 parts were cloned into the pre-assembled backbone. To create more complex fusion proteins, additional subparts were used (e.g., 3a and 3b parts). New parts were codon optimized for S. cerevisiae expression, synthesized commercially by GeneArt or IDT and cloned into the YTK system entry vector, pYTK001, for storage and verification. The other parts were taken from the YTK. Golden gate assembly reactions were performed as described in Lee et al.27. Other strains, including Sc GFP, yWS890 and yGPY093, were similarly constructed using the YTK system and kindly provided by the Ellis lab.
Culture conditions and media: Yeast extract peptone dextrose (YPD) and yeast extract peptone sucrose (YPS) media were prepared with 10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose or sucrose. Synthetic complete (SC) dropout media were prepared with 1.4 g/L yeast synthetic dropout medium supplements, 6.8 g/L yeast nitrogen base without amino acids and 20 g/L glucose. Depending on the specified selection, SC media were supplemented with stock solutions of one or more of uracil (final concentration 2 g/L), tryptophan (final concentration 50 mg/L), histidine (final concentration 50 mg/L) and leucine (final concentration 0.1 g/L). Hestrin-Schramm (HS) media were prepared with 5 g/L yeast extract, 5 g/L peptone, 2.7 g/L Na2HPO4, 1.5 g/L citric acid and 20 g/L glucose or sucrose. Where needed, media were supplemented with 20 g/L bacteriological agar. Partway through this study, a switch was made between sources of peptone for co-culture medium preparation from peptone from casein, to peptone from soybean. It was noted that peptone from soybean resulted in higher and more consistent pellicle yields.
E. coli was grown in LB medium at 37° C., supplemented with appropriate antibiotics at the following concentrations: chloramphenicol 34 μg/mL, kanamycin 50 μg/mL. For biomass accumulation, K. rhaeticus was grown at 30° C. in yeast extract peptone dextrose (YPD) medium supplemented with 34 μg/mL chloramphenicol and 1% (v/v) cellulase from T. reseii (Sigma Aldrich, C2730). It was found that the growth of K. rhaeticus liquid cultures was significantly more reliable when inoculated from glycerol stock, rather than from colonies. Therefore, unless otherwise indicated, the K. rhaeticus cultures were inoculated from glycerol stocks. S. cerevisiae was grown at 30° C. in rich YPD medium or selective, SC medium lacking the appropriate supplements, each supplemented with 50 μg/mL kanamycin.
Co-culture condition screen: Triplicate samples of K. rhaeticus Kr RFP were inoculated from glycerol stocks into 5 mL YPD medium supplemented with cellulase (1% v/v) and grown in shaking conditions for 3 days. Triplicate samples of S. cerevisiae Sc GFP were inoculated from plates into 5 mL YPD medium and grown in shaking conditions for 24 hours. To prepare screens, K. rhaeticus and S. cerevisiae were inoculated into 2 mL volumes of YPD, YPS, HS-glucose or HS-sucrose media in 24-well cell culture plates. K. rhaeticus cultures were diluted 1/50 into fresh media. S. cerevisiae cultures were inoculated over a range of dilutions: 1/100, 1/1000, 1/10,000, 1/100,000 and 1/1,000,000. To enable pellicle formation, plates were incubated for 4 days under static conditions at 30° C. After 4 days of incubation, cultures were photographed under identical conditions. Where present, pellicle layers were removed from the culture surface and photographed.
Standard co-culture protocol: Triplicate samples of K. rhaeticus were inoculated from glycerol stocks into 5 mL YPD medium supplemented with cellulase (1% v/v) and grown in shaking conditions for 3 days. Triplicate samples of S. cerevisiae were inoculated from plates into 5 mL YPD medium and grown in shaking conditions for 24 hours. To enable inoculation of co-cultures with equivalent cell densities of different samples, OD600 measurements were made and used to normalize pre-culture densities. K. rhaeticus pre-cultures were centrifuged at 3220×g for 10 min and cell pellets resuspended in sufficient volume of YPS medium to result in a final OD600 of 2.5. S. cerevisiae pre-cultures were diluted in YPS medium to a final OD600 of 0.01. To prepare final co-cultures, resuspended K. rhaeticus samples were diluted 1/50 and pre-diluted S. cerevisiae samples were diluted 1/100 into fresh YPS medium. In instances where strains were inoculated into various different final media, K. rhaeticus pellets were resuspended in PBS buffer and S. cerevisiae cultures were pre-diluted in PBS buffer. To prepare OptiPrep-containing co-cultures, OptiPrep (D1556, Sigma-Aldrich) was added to YPS media to a final concentration of 45% (v/v). Co-cultures were grown in 55 mm petri dishes (15 mL) or 12 well cell culture plates (4 mL). Co-cultures were incubated for 3 days at 30° C. under static conditions. For even pellicle formation, culture vessels should not be disturbed during the incubation period.
Determining BC pellicle yields: To determine the yields of BC pellicles, pellicle layers were removed from the surfaces of cultures and dried using the ‘sandwich method’. Here, pellicles were sandwiched between two sheets of greaseproof paper and then further sandwiched between multiple sheets of absorbent paper and finally placed under a heavy weighted object. After 24 hours, fresh sheets of absorbent paper were added and pellicles were then left for an additional 24 hours. Pellicles dried in this way were then weighed to determine pellicle yields. Pellicles were not treated with NaOH to lyse and remove cells embedded within the BC matrix.
This method was used to follow the yields of pellicle formation over time. Here, multiple co-cultures were prepared in triplicate using Sc GFP and Kr RFP strains and the standard co-culture procedure. Co-cultures were grown in 12 well plate format. At indicated time points, pellicle layers were removed to be dried and weighed.
This method was also used to compare the pellicle yield between K. rhaeticus mono-culture and co-culture with S. cerevisiae. Here, mono-cultures of K. rhaeticus (Kr RFP) were prepared in YPD medium and in co-culture with S. cerevisiae (Sc GFP) in YPS medium, using the standard co-culture protocol. After 3 days of incubation at 30° C., pellicle layers were removed to be dried and weighed.
Co-culture passage: To test whether co-cultures could be passaged, initial co-cultures between S. cerevisiae (Sc GFP) and K. rhaeticus (Kr RFP) were prepared in triplicate in 15 mL YPS cultures using the standard co-culture protocol. After 3 days incubation at 30° C., photographs were taken of the resultant cultures. To initiate new rounds of growth, pellicle layers were removed and the liquid below mixed by aspiration and diluted 1/100 into fresh samples of 15 mL YPS. This process was repeated over 16 rounds.
To confirm that the initial strain of GFP-expressing S. cerevisiae (Sc GFP) was maintained during passage, samples were plated at the end of each round. Samples from both the liquid below the pellicle and the pellicle layer itself were plated at various dilutions onto YPD-kanamycin plates. To enable plating, pellicles were digested by shaking gently for 16 hours at 4° C. in 15 mL of PBS buffer with 2% (v/v) cellulase from T. reseii (Sigma Aldrich, C2730). After 48 hours of incubation at 30° C., plates were imaged for GFP fluorescence. Dilutions were selected which enabled visualization of single colonies. Initially plates were imaged using a Fujifilm FLA-5000 Fluorescent Image Analyzer. However, due to equipment malfunction, later plates were photographed under a transluminator.
Determining cell distribution in co-cultures: Cell distributions were determined by plating samples of cells onto solid media and counting the resultant colonies. Pellicle samples were first rinsed by inverting ten times in 15 mL PBS and then digested by shaking gently for 16 hours at 4° C. in 15 mL of PBS buffer with 2% (v/v) cellulase from T. reseii (Sigma Aldrich, C2730). Samples were diluted at various levels into PBS. For S. cerevisiae cell counts, samples were plated onto YPD-kanamycin media. For K. rhaeticus cell counts, samples were plated onto SC media lacking the four supplements essential for S. cerevisiae growth (histidine, leucine, tryptophan and uracil). In these instances, Kr RFP and Sc GFP strains were used. Therefore, to ensure the colonies counted were the target strains, plates were scanned for fluorescence using a Fujifilm FLA-5000 Fluorescent Image Analyzer. Plate cell counts were used to calculate the original colony forming units (cfu) per unit volume for liquid samples. However, since the exact volumes of pellicle were not measured prior to degradation, it was not possible to calculate the exact cell counts in cfu per unit volume. To enable a rough approximation of the cell counts per unit volume, pellicle volumes were estimated at fixed levels and these values were used to calculate estimated cfu per unit volume. For 15 mL cultures, pellicle volumes were estimated at 4 mL and for 4 mL cultures in 12 well plates pellicle volumes were estimated at 1 mL.
To compare cell counts from mono-cultures and co-cultures of K. rhaeticus and S. cerevisiae, pre-cultures of K. rhaeticus Kr RFP were pelleted and resuspended in PBS buffer and pre-cultures of S. cerevisiae Sc GFP were diluted in PBS buffer, according to the standard co-culture procedure. Various co-cultures and mono-cultures were then prepared in different media in 15 mL volumes. After 3 days incubation at 30° C., pellicle and liquid samples were prepared, diluted and plated for cell counts.
To determine the reproducibility of co-culture cell counts, co-cultures were prepared according to the standard co-culture protocol in 15 mL cultures on three separate occasions. After 3 days incubation at 30° C., pellicle and liquid samples were prepared, diluted and plated for cell counts.
To determine cell counts in BC balls, balls were degraded by gently mixing for 16 hours at 4° C. in 1 mL of PBS buffer with 2% (v/v) cellulase from T. reseii (Sigma Aldrich, C2730). Degraded ball samples were diluted and plated for cell counts as above. To estimate the cell counts of S. cerevisiae in cfu per unit volume, a ball diameter of 3 mm was assumed.
Fluorescence microscopy: Images of pellicles were prepared using a 20× objective lens mounted on a Nikon Eclipse Ti inverted microscope. Slices of pellicle were mounted on slides with the bottom face of the pellicle facing downwards. To visualize samples, a phase filter (Ph1) was used to enhance contrast. GFP fluorescence images were taken using 480 nm excitation and 535 nm emission wavelengths. The NIS-elements microscope imaging software was used for initial image capture and ImageJ was used for downstream image analysis and stacking of GFP and brightfield images.
Invertase supplementation experiment: Co-cultures and K. rhaeticus Kr RFP mono-cultures were prepared in YPS medium according to the standard co-culture procedure. Recombinant, purified S. cerevisiae invertase (Sigma-Aldrich, 19274) was resuspended in 100 mM citrate buffer, pH 4.5 to create a stock solution at a final concentration of 5 U/μL. This stock solution was diluted into YPS medium for a range of final invertase concentrations: 50 mU/mL (10−2), 5 mU/mL (10−3), 0.5 mU/mL (104), 50 μU/mL (10−5). After 3 days growth at 30° C., cultures and, where present, pellicles were imaged.
Supernatant nitrocefin assay: For culture supernatant assays, WT BY4741, yCG04 and yCG05 S. cerevisiae strains were grown in triplicate overnight in YPD liquid medium with shaking. As used in the studies presented herein, the WT BY4741 strain used was MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0, from the Dharmacon yeast collection. After 16 hours growth, liquid cultures were back-diluted to final OD600=0.01 in 5 mL fresh YPS medium and grown for 24 hours with shaking. The resultant cultures were centrifuged at 3220×g for 10 min and the supernatant fractions harvested. Supernatant samples were pipetted in 50 μL volumes into the wells of a 96 well plate. The colorimetric substrate, nitrocefin (484400, Merck-Millipore), was resuspended in DMSO to create a 10 mg/mL working stock. This stock was diluted to 50 μg/mL in nitrocefin assay buffer (50 mM sodium phosphate, 1 mM EDTA, pH 7.4). To start the reaction, 50 μL of nitrocefin at 50 μg/mL was added to each of the samples simultaneously and the absorbance at 490 nm was measured over time. Active β-lactamase converts nitrocefin to a red substrate, increasing the absorbance of light at 490 nm. Therefore, to calculate the relative β-lactamase activity in samples, the rate of change in the absorbance of light at 490 nm was determined. Specifically, the product formation rates were calculated from the gradient over the linear region of a graph plotting fluorescence AU over time.
Pellicle nitrocefin assays: For initial pellicle assays, WT BY4741, yCG04 and yCG05 S. cerevisiae strains were co-cultured with K. rhaeticus (Kr RFP) in triplicate, according to the standard co-culture protocol. Following 3 days growth, pellicles were removed and washed in 15 mL PBS buffer for 30 min with shaking at 150 rpm. Square pieces of pellicle, measuring 5 mm×5 mm, were then cut using a scalpel. The remainder of the pellicle was dried using the sandwich method. Once dried, pellicles were again cut to produce 5 mm×5 mm pieces. Dried pellicle pieces were rehydrated by adding 25 μL of PBS buffer and incubating for 30 min. Assays for both wet and dried samples were run by adding 10 μL of nitrocefin, diluted to 1 mg/mL in PBS buffer, to each of the pellicle pieces simultaneously. Initial assays were performed at room temperature. Photographs were taken of pellicles over the course of 35 min to follow the color change. To provide a quantitative measure of color change, the ImageJ (NIH) image analysis software was used. Images were first split into individual color channels. Since yellow-to-red color change is caused by an increase in the absorbance of green light wavelengths, the green channel was selected. To quantify the yellow-to-red color change, the green channel intensity was then measured from greyscale-inverted images of pellicle slices over time. Since preliminary results showed that WT pellicles exhibited no color change, the signal from WT pellicles was used as a baseline value to correct for background levels of green channel intensity.
To determine absolute levels of β-lactamase activity in wet and dried pellicles, a similar protocol was used to create standard curves. Standard curves were prepared using a commercial E. coli β-lactamase enzyme (ENZ-351, ProSpec). First, pellicles grown with WT BY4741 S. cerevisiae were washed in nitrocefin assay buffer (50 mM sodium phosphate, 1 mM EDTA, pH 7.4). Pieces measuring 5 mm×5 mm were cut and weighed to enable determination of the approximate volume of liquid within the pellicle. The remainder of the pellicles were dried using the sandwich method. Once dried, 5 mm×5 mm pieces of pellicle were cut for dried pellicle standard curves. Dried pellicle pieces were rehydrated by adding 20 μL of nitrocefin assay buffer. Pre-diluted standard β-lactamase samples were then added to pellicle pieces in 5 μL volumes and allowed to diffuse throughout the BC for 30 min. To initiate the reaction, 5 μL aliquots of nitrocefin, diluted to 2 mg/mL in nitrocefin assay buffer, were added to each of the pellicle pieces simultaneously. Samples were incubated at 25° C. and photographs taken over the course of the reaction. Again, ImageJ was used to quantify the yellow-to-red color change at given time points. Time points were chosen to maximize the dynamic range, without reaching saturation. For wet pellicles, it was necessary to use the measured weight of pellicle slices to determine the actual final concentration of the standard β-lactamase. Standard curves using fresh wet pellicles, dried pellicles and dried pellicles stored for 1 month or 6 months at room temperature were prepared according to this method. For long-term storage, dried pellicles were stored in petri dishes at room temperature and protected from light.
Alongside standard curves, pellicles grown with yCG05 S. cerevisiae were analyzed using an identical protocol. To enable cross comparison with standard curves, negative samples (pellicles from co-cultures with WT S. cerevisiae) and positive samples (pellicles from co-cultures with WT S. cerevisiae to which a known amount of β-lactamase standard had been added) were run with samples. For samples to which no standard β-lactamase was added, 5 μL of nitrocefin assay buffer was added to maintain equal final liquid volumes. Photographs taken at identical time points were then used with standard curves to calculate absolute values of β-lactamase activity. Again, ImageJ was used to quantify the yellow-to-red color change. For wet pellicles, it was necessary to use the measured weight of pellicle slices to determine the actual final concentration of enzyme. Again, fresh wet pellicles, dried pellicles and dried pellicles stored for 1 month at room temperature were assayed according to this method.
β-lactamase activity retention assay: To determine the retention of β-lactamase within BC following multiple rounds of washes, nitrocefin assays were performed. Pieces measuring 5 mm×5 mm were cut from dried pellicles grown with yCG04 and yCG05. The pellicle pieces were rehydrated by incubating in 1 mL of PBS buffer. Pieces were subjected to a variable number of wash steps, where pellicle pieces were incubated in 4 mL PBS buffer at 25° C. and 150 rpm for 30 min. After washing, pellicles were assayed for β-lactamase activity. Negative samples (pellicles from co-cultures with WT S. cerevisiae) and positive samples (pellicles from co-cultures with WT S. cerevisiae to which a known amount of β-lactamase standard had been added) were run alongside the samples. For samples to which no standard β-lactamase was added, 5 μL of PBS buffer was added to maintain equal final liquid volumes. As before, assays were initiated by adding 5 μL of nitrocefin, diluted to 2 mg/mL in PBS buffer, to each of the pellicle pieces simultaneously. Since the number of samples that can be run in parallel is limited, samples were run in batches based on the number of washes. Again, ImageJ was used to quantify the yellow-to-red color change at given time points. To enable cross-comparison between different assay runs, negative samples were used to subtract background signals and positive samples were used to normalize signals. To ensure that yellow-to-red color change values were within a range in which there is a linear relationship between β-lactamase activity and the yellow-to-red color change signal, a standard curve was run. The standard curve (r2=0.9571) confirmed that detected yellow-to-red color change values fell within the linear range.
X-α-gal α-galactosidase assays: A stock solution of X-α-galactosidase (Sigma-Aldrich, 16555) was prepared in DMSO at a concentration of 40 mg/mL. For plate assays, 100 μL of X-α-gal were spread on plates prior to cell plating and images taken after 3 days growth at 30° C. For pellicle assays, pellicles grown with K. rhaeticus Kr RFP were harvested after 3 days growth following the standard co-culture procedure. Pellicles were then washed in 15 mL 100 mM citrate buffer, pH 4.5 for 30 min with shaking at 150 rpm. Square pieces of pellicle, measuring 5 mm×5 mm, were then cut using a scalpel. The remainder of the pellicle was dried using the sandwich method. Once dried, pellicles were again cut to produce 5 mm×5 mm pieces. Dried pellicle pieces were rehydrated by adding 25 μL of 100 mM citrate buffer, pH 4.5 and incubating for 30 min. Assays for both wet and dried samples were run by adding 2.5 μL of X-α-gal stock solution and incubating at 25° C. Images were taken over the course of several hours.
ABTS laccase activity assays: Stock solutions were prepared of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Sigma-Aldrich, A1888) at a final concentration of 0.1M and copper sulphate at a final concentration of 1M. Laccases are copper-containing enzymes, requiring supplementation of copper for culture and assay conditions. For plate assays, 125 μL of 0.1M ABTS and 25 μL of 1M CuSO4 were spread on plates prior to cell plating and images taken after 3 days growth at 30° C. For pellicle assays, pellicles grown with K. rhaeticus Kr RFP were harvested after 3 days growth following the standard co-culture procedure. The only modification was the addition of 1 mM CuSO4 to the culture medium of both S. cerevisiae pre-cultures and co-cultures. Pellicles were then washed in 15 mL 100 mM citrate buffer, 1 mM CuSO4, pH 4.5 for 30 min with shaking at 150 rpm. Square pieces of pellicle, measuring 5 mm×5 mm, were then cut using a scalpel. The remainder of the pellicle was dried using the sandwich method. Once dried, pellicles were again cut to produce 5 mm×5 mm pieces. Dried pellicle pieces were rehydrated by adding 25 μL of 100 mM citrate buffer, 1 mM CuSO4, pH 4.5 and incubating for 30 min. Assays for both wet and dried samples were run by adding 5 μL of ABTS stock solution and incubating at 25° C. Images were taken over the course of several hours.
Assaying GFP secretion into supernatant and pellicles: As preliminary test of ability of yCG01 and yCG02 to secrete GFP, individual colonies were grown in 5 mL YPD medium for 48 hours. As a negative control strain, non-fluorescent yCG04 was used. After 48 hours growth, cultures were centrifuged at 3220×g for 10 min and supernatant samples imaged for fluorescence under a transluminator.
To test whether S. cerevisiae could secrete detectable levels of GFP into BC materials, co-cultures were prepared in triplicate according to the standard co-culture protocol using WT BY4741, yCG01 and yCG02 strains. Co-cultures were grown in 4 mL volumes in 12 well plates. Since GFP secretion yields were anticipated to be low, co-cultures were allowed to grow for either 7 days or 14 days before imaging. After incubation, pellicles were washed by incubating for 30 min in 15 mL PBS buffer. Washed pellicles were then imaged for GFP using Fujifilm FLA-5000 Fluorescent Image Analyzer. Images were analyzed and modified for presentation using ImageJ (NIH). Specifically, brightness and contrast were adjusted, equally for all samples, to the point at which the background fluorescence of pellicles grown with WT S. cerevisiae was just visible.
Scanning electron microscopy (SEM): Pellicles were grown for 3 days following the co-culture procedure and washed with deionized water 3 times (shaking at 70 rpm at 4° C. for 12 hours per wash) to remove residue YPS or OptiPrep. Washed pellicles were then free-dried with a lyophilizer for at least 48 hours before coated with a gold sputter. Images were taken with a JEOL 6010LA benchtop scanning electron microscope.
Brunauer-Emmett-Teller (BET) surface area analysis: Free-dried pellicles were cut into 5 mm×5 mm piece and placed in sample tube for 1 hour degas at 423 K using a Micromeritics (Atlanta, Ga.) ASAP 2020 analyzer. BET surface area and pore size were then determined with N2 adsorption at 77 K using Brunauer-Emmett-Teller and Barrett-Joyner-Halenda analyses on the same machine.
Preparing and assaying sense-and-response pellicles: In GPY093 transcription from the BED-inducible promoter is controlled by a synthetic transcription factor (Z3EV) consisting of three domains: the Zif268 DNA-binding domain, the human estrogen receptor (hER) ligand binding domain, and the transcriptional activation domain of viral protein 16 (VP16AD)45. When present, β-estadiol binds to the hER ligand binding domain of Z3EV, releasing it from its basal sequestration in the cytosol and enabling it to translocate into the nucleus. Once in the nucleus, the Zif268 domain binds cognate DNA sequences in engineered promoters and the VP16AD domain activates transcription of downstream genes. As a preliminary test of S. cerevisiae sense-and-response in BC pellicles, co-cultures were prepared in triplicate according to the standard co-culture protocol using WT BY4741 and GPY093 strains. Co-cultures were inoculated into 4 mL YPS-OptiPrep medium in 12 well cell culture plates. After 3 days of growth, pellicles were removed and washed by incubating at 25° C. with shaking at 150 rpm in 15 mL PBS. Pellicles were then placed in fresh 15 mL of YPD medium in the presence or absence of 5 nM β-estradiol (E8875, Sigma-Aldrich) and incubated for 24 hours at 30° C. and 150 rpm. Large quantities of cells had ‘escaped’ from biosensor pellicles, making the medium surrounding the pellicles turbid. Therefore, to remove loosely-associated cells, pellicles were washed twice by incubating at for 30 min at 25° C. and 150 rpm in 15 mL of PBS buffer. Finally, pellicles were imaged simultaneously for GFP fluorescence under a transluminator.
Similarly, dried biosensor pellicles were prepared were prepared in triplicate according to the standard co-culture protocol using WT BY4741 and yGPY093 or WT BY4741 and yWS890 strains. Co-cultures were inoculated into 4 mL YPS-OptiPrep medium in 12 well cell culture plates. After 3 days of growth, pellicles were dried using the ‘sandwich method’. Dried pellicles were then placed in fresh 15 mL of YPD medium in the presence or absence of 5 nM β-estradiol or 50 nM S. cerevisiae α-mating factor (RP01002, GenScript) and incubated 24 hours at 30° C. To more closely match the potential use of biosensors in an on-site detection setting, pellicles were incubated without agitation in this and subsequent experiments. Static growth was chosen to more closely mimic a potential in-the-field application. Since static growth result in far less growth in the surrounding liquid, pellicles were only briefly washed by inverting ten times in 15 mL PBS buffer. Finally, pellicles were imaged side-by-side for GFP fluorescence under a transluminator. To test for stability after long-term storage, pellicles were stored for 4 months at room temperature stored in petri dishes protected from light. These pellicles were cut in half prior to induction, which was performed as above.
The BED-inducible CtLcc1-secreting strain yCG23 was initially screened for laccase induction using a plate-based ABTS assay. Transformants of yCG23 were re-streaked in triplicate on SC URA− plates supplemented with 125 μL of 0.1 M ABTS and 25 μL of 1 M CuSO4. After 3 days of incubation at 30° C. colonies were imaged. Co-cultures between K. rhaeticus Kr RFP and yCG01 or yCG23 were then prepared in triplicate in 12-well plate format, using YPS-OptiPrep medium supplemented with 1 mM CuSO4. After 3 days growth, pellicles were harvested and were washed by incubating for 30 min at 25° C. and 150 rpm in 15 mL of 100 mM citrate buffer, 1 mM CuSO4, pH 4.5. Pellicles were then inoculated into 15 mL of fresh YPD supplemented with 1 mM CuSO4 in the presence or absence of 5 nM β-estradiol and incubated at 30° C. for 24 hours statically. After incubation, pellicles were washed by incubating for 30 min at 25° C. and 150 rpm in 15 mL of 100 mM citrate buffer, 1 mM CuSO4, pH 4.5. Pellicles were then placed in a 12-well plate and 75 μL of 0.1 M ABTS added to each well to assay for laccase activity. Pellicles were incubated at 25° C. and imaged after 72 hours.
In other embodiments of the ABTS assay, the plate was sealed with breathe-easy, which should allow gas exchange, since oxygen is needed for the reaction. A faint green color was detected after 48 hours and 72 hours, at which time, the film was removed and only a few hours later the intensity of the color was much, much stronger.
Determining the viability of S. cerevisiae in dried BC pellicles: Co-cultures were prepared in triplicate according to the standard co-culture protocol using Sc GFP and Kr RFP. Co-cultures were inoculated into 4 mL YPS-OptiPrep medium in 12 well cell culture plates. Counts of viable S. cerevisiae cells within wet and dried pellicles were determined as described previously. Dried pellicles were also stored for 1 month at room temperature, and then degraded and plated onto YPD medium. Since one of the triplicate samples produced no colonies, estimated cell counts within pellicles could not be calculated. However, images of the three plates showed that viable cells were indeed recovered from the other two samples.
Total cellulase activity assay: Yeast strains BY4741 and yCelMix were grown overnight in YPS in triplicate with shaking. After 16 hours growth, liquid cultures were back-diluted to final OD600=0.1 in 5 mL fresh YPS medium with 2 mM L-ascorbic acid (A7506, Sigma-Aldrich) and grown for 24 hours with shaking. The resultant cultures were centrifuged at 3220×g for 10 min and the supernatant fractions harvested. Supernatant samples were pipetted in 50 μL volumes into the wells of a 96 well plate. The EnzChek® Cellulase Substrate (E33953, Thermo-Fisher) was resuspended in 50% DMSO and diluted 5-fold in 100 mM sodium acetate (pH 5.0). To start the reaction, 50 μL of cellulase substrate was added to the supernatant and let incubated for 30 minutes in dark at room temperature. To build an enzyme activity standard curve, the cellulase from T. reesi (C2730, Sigma-Aldrich) was used to prepare a serial dilution in YPS medium and mixed with the substrate at 1:1 ratio. Blue fluorescence (360/460) was detected using a plate reader (Synergy H1, BioTek) after 30 minutes incubation in dark at room temperature. The data from enzyme standards was fit to an exponential model, a*exp(b*x)+c*exp(d*x) in MATLAB. This model was then used to calculate the total cellulase activity of the supernatant from yCelMix (using supernatant from BY4741 as a blank control).
Pellicle tensile test: Co-cultures were set up in 40 mL YPS+OptiPrep (plus 2 mM L-ascorbic acid) and grown in square plates (100 mm×15 mm) for 2 days at 30° C. Pellicles were then washed in deionized water 3 times (shaking at 70 rpm at 4° C. for 12 hours per wash) and dried using the sandwich method described previously but with an extended 3 days drying to ensure water removal. Dried pellicles were cut into 60 mm*10 mm stripes and their thickness were measured with a micrometer. Tensile test was performed with a Zwick mechanical tester (BTC-ExMacro 0.001, Roell) following the ASTM D882 protocol at 1 mm/min speed.
Pellicle rheology analysis: The rheological properties of washed pellicles were characterized on a rheometer (AR2000, TA Instruments) with a 25 mm ETC aluminum plate (1 mm gap). The strain sweep measurements were taken from 0.01% to 100% strain amplitude at a constant frequency of 1 rad/s while frequency sweep measurements were taken from 0.1 rad/s to 100 rad/s at a constant strain amplitude of 1%. Samples were kept fully-hydrated with deionized water at 25° C. on a Peltier thermoelectric plate.
Light-inducible circuit promoter characterization: Yeast strains were grown overnight in YPD in triplicate with shaking. After 16 hours growth, liquid cultures were back-diluted to final OD600=0.2 in 100 μL fresh YPD and pipetted into the wells of two 96 well plates (duplicates). One of the two plates was wrapped in black aluminum foil as a dark control. Both plates were placed under a LED lamp at 30° C. for 4 hours. Green fluorescence was then measured with a plate reader.
Light-inducible luciferase assay: Yeast strains were grown overnight in YPD in triplicate with shaking. After 16 hours growth, liquid cultures were back-diluted to final OD600=0.2 in 15 μL fresh YPS and pipetted into the wells of two 96 well plates (duplicates for light and dark conditions, as previously described). Plates were placed under a LED lamp at 30° C. for 4 hours. Substrate in buffer from Nano-Glo® Luciferase Assay System (N1120, Promega) were added to the culture at 1:1 ratio at the end of incubation. After incubation in dark for 5 minutes, bioluminescence of the samples was measured with a plate reader.
Light-inducible pellicle response assay: Co-cultures were set up using yeast strains BY4741, yNC, and yNS along with wildtype K. rhaeticus in 10 mL YPS+OptiPrep. For long term exposure experiment, 60 mm petri dishes were prepared as duplicates, one was wrapped in black aluminum foil while the other one was not. The plates were placed under a LED lamp at 30° C. for 3 days. After the incubation, pellicles were flipped so the bottom side was facing up, and transferred onto YPD agar plates. 500 μL of Nano-Glo mix was applied onto the pellicles evenly through the entire surface. After incubation in dark for 10 minutes, bioluminescence of the samples was detected with a ChemiDoc Touch imager (BioRad). For short term exposure experiment (masking), co-cultures were grown in dark at 30° C. for 3 days. Pellicles were flipped so the bottom side was facing up, and transferred onto YPD agar plates. A mask made of black aluminum foil with carved pattern in the center was placed on top of the pellicles. Plates were placed under a LED lamp and incubated at 30° C. for 4 hours. Mask was then removed and 500 μL of Nano-Glo mix was applied onto the pellicles evenly through the entire surface. After incubation in dark for 10 minutes, bioluminescence of the samples was detected with a ChemiDoc Touch imager.
Light-patterning on pellicles: Co-cultures were grown in 100 mm square plates protected from light as previously described. Pellicles were rinsed in PBS, flipped, placed on YPD agar, and placed in an incubator with a projector mounted on top. After incubation under the projected pattern (with no lid to prevent water condensation) at 30° C. for 12 hours or more, 3 mL of Nano-Glo mix was applied onto the pellicles evenly through the entire surface. Bioluminescence images was detected with a ChemiDoc Touch imager after 30 minutes incubation in dark.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.
This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 62/889,837, filed Aug. 21, 2019, and entitled “Growing Programmable Enzyme-Functionalized and Sense-and-Response Bacterial Cellulose Living Materials with Engineered Microbial Co-Cultures,” which is incorporated herein by reference in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/047330 | 8/21/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62889837 | Aug 2019 | US |
