Patent Application
20230332087
The present disclosure generally relates to systems, devices, and methods for performing electrochemical control and monitoring of bacterial gene expression to a precisely assigned level, and may be used, for example, for controlling the production of a protein of interest.
The concept of communication, or the transmission of information, builds the basis of the interconnected modern world we live in today. While current electronic devices are capable of freely exchanging information through electron transfer and electromagnetic waves, building smart systems that can connect both biological processes and electronic systems remains a challenging task due to their disparate communication modalities. Recently, redox-active molecules, due to their dual nature, were introduced as a novel medium to connect biological systems and electronics (1-4). No stranger to biology, redox-active molecules are present in the core of biological systems (e.g., NADH) serving as a bridge between electron transfer and molecular communication (5,6). When electrons flow between them, causing their distinct redox state to change, the redox molecules can also freely move in and out of the cell initiating a molecular dialogue. In addition, these redox activities can be easily and precisely controlled through routine electrochemical instrumentation, thus realizing programmed, automated regulation, which is believed to be one of the key functions in next generation biohybrid devices.
Employing peroxide (H2O2) as the electrochemical conduit, an electrogenetic system that enables information exchange between a living cell-embedded bioelectronics interface and an engineered microbial network was developed 3,4. Similarly, an electrically controlled, CRISPR-mediated toolkit for transcriptional regulation was built on the original “electrogenetics” archetype (1), adding an array of CRISPR functions to allow more genetically-focused intracellular control (2). Thus, the development of novel systems that can connect both biological processes and electronic systems is greatly needed.
The present disclosure relates to a system for achieving, for example, real-time electrochemical control and monitoring of bacterial gene expression (the control and monitoring can occur while the gene expression is occurring) to a precisely assigned level, and for example controlling the production of a gene product of interest. The provided integrated system comprises four components: (i) a biological signal detection: gene-of-interest (GOI) expression level can be detected by using optical (fluorescence, luminescence) or electrochemical methods; (ii) a signal processing: converting biological signals into the digital signals which would be further processed by different algorithms for a particular output method (e.g., voltage, current, optical) to achieve feedback control of gene expression or remote control separate biological systems; (iii) a 3D-printed, ITO-based multi-chamber cell culture platform: This can be connected to the detection unit to receive the control signal from the signal processor, and generate the biological actuation signal i.e. H2O2; and (iv) an engineered bacteria: The engineered E. coli can be induced by the biological actuation signal generated from the device to express the GOI.
As a first component, a custom biological signal detection system is provided for precisely reading the fluorescence level of the cells under test, and in such a way that the raw optical signals are converted to electrical signals.
As a second component, a signal processing unit is provided. The photomultiplier tube converts the incident fluorescence signal from the cells into a proportional voltage by means of an integrated transimpedance amplifier. This analog voltage is then digitized by a high resolution ADC. This is fed into a processor unit (currently a PC, but later will be a FPGA or other microprocessor). The processor then determines whether the level of the fluorescence signal is high enough to require production of H2O2 (or some other action). If so, the processor then enables its onboard potentiostat to control the cell culture device to produce it. The cells' response to this chemical production is then monitored by the fluorescence reader in #1, thereby enabling closed loop control. This processing unit is generalizable to any manner of input and output methods.
As a third component, a 3D-printed, ITO-based multi-chamber cell culture platform is provided. Although conventional gold working electrodes offer great stability and electron delivery capability, the strong reflection could hinder the optic measurement and the detection of targeted fluorescent proteins expressed from the cell deposited on the electrode. To overcome this issue, we have developed an indium tin oxide (ITO)-based multi-chamber electrochemistry platform that aims for the high-throughput stimulation/measurement of a targeted microenvironment and cells using one-step 3D printing (
As a fourth component, the present disclosure provides cells where the transcription of a gene-of-interest is regulated by electrochemical signaling and provides methods for real-time electrochemical control and monitoring of gene expression to a precisely assigned level, for example controlling the production of a gene product of interest. In an embodiment, the cells are recombinant cells into which expression vectors designed for expression of a gene-of-interest, or a reporter gene, under the transcriptional control of electrochemical signals have been introduced. Such cells include bacterial as well as eukaryotic cells. The introduced nucleic acid may be present independently of the genome of the host cell or in the state of being incorporated into the genome of the host cell.
In one aspect, an engineered bacteria harboring an eCRISPR system is provided. A peroxide-inducible electrochemical CRISPR (eCRISPR) system for controlling genetic expression was developed by rewiring E. coli's native oxyRS regulon for combating oxidative stress from hydrogen peroxide. The guide RNA (gRNA) expression Is placed under the control of oxyS promoter and peroxide sensor, OxyR. To achieve CRISPR activation, the transcribed gRNA would form a complex with transcription factor ω subunit-fused deactivated Cas9 (dCas9ω) and activate the gene-of-interest (GOI; GFPmut2). Increasing levels of peroxide result in increased levels of green fluorescence, indicating that the expression of the GOI was activated. These peroxide-inducible CRISPRa bacteria were co-deposited with PEG-SH to form a film as described herein. After electrodeposition, 200 μL of LB and PBS mixture is added into the sample well, on top of the deposited film. Voltage (−0.8 V) is then applied to the films for various times from 5-30 minutes. After 4 hours of incubation at 37° C., the device is placed under a confocal microscope for a parallel observation. With increasing charge applied, both the percentage of E. coli expressing GFPmut2 and the intensity of green fluorescence had gone up, which agreed with observations with suspension culture. The increasing fluorescence may also be measured through the electronic device disclosed herein and the output signal can be further processed by a signal processing unit's algorithms to control the potentiostat for voltage output. This will in turn influence the production of peroxide and change the GOI's expression level, thus completing the bio-electronic control loop.
Another aspect of the present disclosure pertains to nucleic acids encoding the gene-of-interest, or a reporter gene, wherein expression of said gene-of-interest or reporter gene is mediated by electrochemical signaling. Nucleic acids may also include those encoding for Cas9 protein (inhibitor (CRISPRi) or activator (CRISPRa)) and a gRNA of interest. Such nucleic acids may be introduced into a variety of different expression vectors, including for example, bacterial, and eukaryotic expression vectors for expression of the gene-of-interest, or reporter gene.
In still another aspect, a method is provided of preparing a gene product of interest in a cell wherein expression of the gene product of interest is under the control of electrochemical signaling. The preparation method comprises (i) culturing the cells as described herein having expression of a gene-of-interest mediated by electrochemical signaling; (ii) providing an electrochemical signal; and (iii) allowing for expression of the gene product of interest. In a further step, the gene product of interest is purified from the cell culture.
The present disclosure provides systems and methods for controlling and monitoring electrically induced gene expression. As will be described by an example below, electric voltage may be applied to generate peroxide for a peroxide-mediated, electrically inducible CRISPR transcriptional activation (CRISPRa) system, which may be monitored by using plasmid pMC-GFP in which the expression of reporter gene gfpmut2 is upregulated by the CRISPR components. In such an example, the resultant fluorescence would be indicative of the gene-of-interest (“GOI”) expression level. Increasing levels of peroxide result in increased levels of green fluorescence, indicating that the expression of the GOI was activated. The fluorescence may be detected by a transducer which converts optical signals to electrical signals to indicate the GOI expression level. The electrical signals may be processed by a computing system to implement closed-loop control of the applied electric voltage to regulate the peroxide generation and, thereby, regulate the GOI expression level. While the present disclosure primarily describes this example, the example is merely illustrative of an electrically controllable gene expression system. It is intended that other electrically controllable gene expression systems and methods would be within the scope of the present disclosure.
The present disclosure provides an example of a system for achieving real-time electrochemical control and monitoring of bacterial gene expression to a precisely assigned level, for example controlling the production of a gene product of interest. The provided example of an integrated system includes four components:
Referring now to
The output of the detector filter is coupled to the photomultiplier tube. The photomultiplier tube converts the fluorescence signal from the biological sample into a proportional voltage using, e.g., an integrated transimpedance amplifier. This analog voltage is then digitized by the ADC, which may be a high-resolution ADC, to provide an electrical signal indicative of the GOI expression level. In embodiments, the detection system is fiber-coupled to reduce noise and simplify alignment. In embodiments, the digital samples provided by the ADC may be communicated to and stored in an electronic storage device (not shown), which may be local to the detection system or remote from the detection system. Altogether, the fiber probe, the optional detector filter, the photomultiplier, and the ADC, may be referred to as a measurement sensor, and the outputs of the ADC may be referred to as sensor measurements.
In
The fluorescence reader may include a 470 nm excitation light source and driver (Thorlabs #M470F4 and #DC2200) that sends light to a fiber-coupled filter mount (Thorlabs #FOFMS) via a 1000 μm diameter core multimode fiber (Thorlabs #FT1000EMT-CUSTOM). An OD6 fluorescence filter (Edmund Optics #67-027) can be used to set the excitation band. This light is then directed onto the test target via a fiber coupled reflection probe (Thorlabs #RP22). The emission from the target is collected by the other fibers in this bundle and directed to a second fiber coupled filter mount (Thorlabs #FOFMS) which contained two OD6 emission filters (Edmund Optics #67-030). The filtered light in turn passed through another fiber (Thorlabs #FT1000EMT-CUSTOM) to a fluorescence enhanced PMT (Thorlabs #PMT2101) where it can be converted to an electrical signal that travels to the Analogue to Digital Converter (ADC) on a FPGA potentiostat board. A 3-axis motion platform having three stepper motor kits (Thorlabs #KMTS50E) connected together with a base plate (Thorlabs #MTS50A-Z8), XY plate (Thorlabs #MTS50B-Z8) and right-angle plate (Thorlabs #MTS50C-Z8) can be used to allow x, y, and z-axis movement of the probe.
The system illustrated and described in connection with
A FPGA-potentiostat implementation is described as follows.
A potentiostat through fabrication and assembly of a FPGA board is described. The core component is a Spartan-7 module (Opal Kelly #XEM7305) and breakout board (Opal Kelly #BRK7305). Power can be supplied via an ultralow-noise linear power supply (Acopian #DBS-50). While many components can be used on the potentiostat board, the three primary components of interest included (a) a femtoampere input bias current op-amps (Analog Devices #ADA4530), (b) a 16-bit multichannel Digital-to-analog converter (DAC) (Analog Devices #AD5765), and a 18-bit multichannel ADC (Texas Instruments #ADS8698).
Referring again to
In the example of
Regarding the example 3D printed ITO-based multi-chamber electrochemistry platform,
Fabrication of the Biohybrid Electronic Device
The custom-designed housing was printed with a 3D printer and was then attached to the ITO-coated glass slide (Sigma) as the working electrode using the same resin and UV-curing (company). The Ag/AgCl reference electrode (Pine Research) can be inserted from the side well into the central well containing the salt bridge. Agarose salt bridge, consisting of 0.1% agarose in 1M KCl, was heated and added into the central well. After the agarose solidified, 1M KCl was added to submerge the salt bridge. A separate custom connector was also printed (with a 3D printer), and a Pt wire was attached as the counter electrode. With the device fully assembled, the counter electrode was immersed in the 1M KCl solution present in the central salt bridge well.
Electrode Chip Fabrication
Gold patterned electrodes from Platypus Technologies and Pine Research may be used. Steps for fabricating the custom patterned gold electrode on silicon wafers may be as follows: First, metal deposition was performed on standard 4-inch silicon wafers using a Denton thermal evaporator (Denton Vacuum), with metal deposition rates of 2-3 Å s-1. Specifically, a 50 nm chromium adhesion layer was evaporated, followed by 100 nm gold. Next, photolithography utilized direct writing of photoresist via a DWL66fs laser writer (Heidelberg Instruments), guided by a laser exposure map designed in AutoCAD (Autodesk). Photoresist spin-coating and development steps were performed using an EVG120 automated resist processing system (EV Group). The patterned wafer was post-processed by etching, photoresist stripping and cutting individual electrodes with a DAD dicing saw (DISCO). The patterned gold electrode purchased from Pine research was made by screen-printing gold on a ceramic base, with a 2 mm2 diameter gold working electrode, a printed Ag/AgCl reference electrode, and a printed gold reference electrode.
The description and illustration of
An algorithm was built to control the ‘actuation checkpoint’ bio-electrochemical platform. After receiving the initiation message from the local bioelectronic system, the algorithm then commands a potentiostat at the remote location to run a pre-set program (specifically, apply −0.8 V on WE1 for 600 s, followed by 0 V on WE2 for 120s). It then compares the value of the output current to that of the user-defined current threshold: if the output current does not exceed the threshold, a message is sent to the local system to administer electroinduction right after the upcoming fluorescence measurement (for a user-defined duration or charge); otherwise, the algorithm sends a SMS message to alert the users. If ‘Y’ is received from the users for termination, a termination message will be sent to the local system to initiate photobleaching right after the upcoming fluorescence measurement. If ‘N’ is received from the users, the ‘actuation checkpoint’ proceeds to run the potentiostat program once more.
More details of the algorithm are as follows.
Fluorescence was taken at a fixed time interval (15 min), and the slope was defined as the difference between two neighboring fluorescence measurements.
Slope Sn=F(n+1)−Fn
The algorithm stores and updates the value of the maximum slope (Smax) to serve as a reference point for the progression of the fluorescence level. To determine the rate of change in fluorescence levels, we took the ratio between the current slope and Smax as described by (3).
Slope Ratio R(n-1)=Sn÷Smax
If two consecutive ratios fall below the user-defined ratio limit, the algorithm considers the ratio threshold met and will initiate electro-induction by sending a command to the potentiostat. Variables such as the duration of voltage application or the total charge applied can be set in the GUI program. The Smax will return to 0 and a new cycle will start subsequently.
The present disclosure provides a cellular system for controlling the activation, inhibition, or multiplexed control of a variety of different targets (e.g., genes-of-interest) wherein said control is mediated by electrochemical signaling within the cell and/or between a population of cells. In an embodiment, said system can be used to regulate the expression of a gene-of-interest, or for monitoring gene expression, within said cell. In an embodiment of the invention, the cell may be an engineered eukaryotic or engineered prokaryotic cell. In a specific embodiment, the cell is an engineered bacterial cell.
In one aspect, a cellular system is provided wherein cells of the system comprise constructs that place the activation or repression of gene expression, e.g., of a gene-of-interest, or a reporter gene, under the control of electrochemical signaling. Said control of expression may be mediated through the inclusion of cis-regulatory elements, such as promoters, enhancers, and silencers, which are regions of non-coding DNA, which regulate the transcription of nearby genes. For example, the gene-of-interest, or reporter gene may be cloned adjacent to a promoter that activates gene expression in the presence of an electrochemical signal. In another embodiment, the gene-of-interest, or reporter gene construct, may be cloned adjacent to a silencer that inhibits gene expression in the presence of an electrochemical signal. In specific embodiments, said cis-regulatory elements include, but are not limited to, those that are regulated by electrochemical signaling such as redox-based cell signaling.
In another aspect, the cells of the system may express trans-regulatory factors under the control of electrochemical signaling that regulate or modify the expression of distant genes by binding with the gene's target sequences, e.g., cis-regulatory elements. In such an instance, the activation or inhibition of expression of the trans-regulatory factor can result in mediation of expression of the gene-of-interest or reporter gene. In an embodiment, the trans-regulatory factor may be an inhibitor or activator of gene expression. In another embodiment, the activity of the trans-regulatory factor may be placed under the control of electrochemical signaling. In a specific embodiment, the trans-regulatory factor may be a CRISPR guide RNA (gRNA) the expression of which is mediated by electrochemical signaling. Said expressed gRNA is designed to form a complex with an engineered Cas9 transcription factor (inhibitor or activator) that is, for example, constitutively expressed within the cell. Once formed, the gRNA/Cas9 complex will then bind to the promoter region of the gene-of-interest and modulate expression of said gene.
Such a promoter/regulator cellular system includes, for example, the bacterial oxyRS regulon that is used naturally to combat oxidative stress from hydrogen peroxide. In such an instance, expression of a gene-of-interest, may be placed under the control of the oxyS promoter and the peroxide sensor, OxyR.
In an embodiment, an eCRISPR cellular system is provided for controlling the activation, inhibition, or multiplexed control of a variety of different targets, e.g., genes-of-interest. Engineered CRISPR systems contain two components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (Cas9 protein). Said Cas9 protein, for use in the presently disclosed eCRISPR cellular system, include those Cas9 proteins devoid of enzyme activity that have been engineered to act as transcription factors. Said Cas9 transcription factors may act as activators (CRISPRa) or inhibitors (CRISPRi) of gene expression. In one aspect, the Cas9 transcription activator is a transcription factor ω subunit-fused deactivated Cas9 (dCas9ω). In another aspect, the Cas9 transcription inhibitor is a repurposed dCas9ω for inhibition. The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas9-binding and a user-defined ˜20 nucleotide spacer that defines the genomic target to be modified. For example, the nucleotide spacer may be designed to bind to the promoter of a gene of interest. Components of the eCRISPR system disclosed herein, e.g., Cas9 transcription factors and gRNAs, are well known in the art.
In an embodiment, the expression of the gRNA is under the control of electrochemical signaling. Thus, one can change the genomic target of the Cas9 protein, and thus expression of the target, by simply changing the nucleotide spacer sequences present in the gRNA. Accordingly, the expression of any gene-of-interest can be targeted through specific selection of a gRNA spacer sequence that will associate with Cas9 and then with the promoter of a gene-of-interest.
In a specific embodiment, expression of a gene-of-interest is mediated by the co-expression, in the cell, of a transcription factor ω subunit-fused deactivated Cas9 (dCas9ω) and a gRNA. In one aspect, a peroxide-inducible electrochemical CRISPR (eCRISPR) system for controlling genetic expression is provided by reprogramming E. coli's native oxyRS regulon for combating oxidative stress from hydrogen peroxide. In such an instance, CRISPR guide RNA (gRNA) expression is placed under the control of the oxyS promoter and peroxide sensor, OxyR. To achieve CRISPR mediated activation, the transcribed gRNA forms a complex with the co-expressed transcription factor ω subunit-fused deactivated Cas9 (dCas9ω) and activates expression of the gene-of-interest.
In an embodiment, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) may be used to either inhibit or activate gene expression through co-expression with a gRNA of choice (depending on the target gene-of-interest). Each of the technologies utilizes a nuclease-deactivated Cas9 that binds to the target genomic region and results in RNA-directed transcriptional control of the target region. In the case of CRISPRi, dCas9 may be coupled to a transcriptional repressor domain that can effectively silence expression of one or more endogenous genes.
In an embodiment, a system is provided wherein the electrochemical mediated signaling of gene expression can be monitored through use of a gene-of-interest encoding for a tag-labeled protein, as described herein. In this case, in the presence of an electrochemical signal, the amounts of detectable label will increase (activation) or decrease (inhibition) depending on the presence of electrochemical mediated signaling. For example, as described herein, increasing levels of peroxide may result in increased levels of the detectable label, indicating that the expression of the gene-of-interest was activated. In an embodiment, the label is a fluorescent tag. In yet another aspect, the florescent label may act as a further inducer of electrochemical signaling thereby providing a signaling loop.
In yet another embodiment, the present disclosure provides recombinant cells wherein expression of a gene-of-interest is mediated electrochemically. For example, said cells may include those into which expression vectors designed for expression of a gene-of-interest, or a reporter gene, under the transcriptional control of electrochemical signals have been introduced. Such cells include bacterial as well as eukaryotic cells. The introduced nucleic acid may be present independently of the genome of the host cell or in the state of being incorporated into the genome of the host cell.
The provided recombinant cells comprise a recombinant construct that places the activation or repression of gene expression, e.g., of a gene-of-interest, or a reporter gene, under the control of electrochemical signaling. Said control of expression may be mediated through the inclusion in the construct of cis-regulatory elements, such as promoters, enhancers, and silencers, which are regions of non-coding DNA, which regulate the transcription of nearby genes into the recombinant constructs.
In an embodiment, the recombinant construct further comprises one or more genes of interest that may be expressed under electrochemical control. Such genes of interest include those encoding a wide range of therapeutic products for use in medical genetics and biomedicine. Therapeutic proteins as defined herein are peptides or proteins Which are beneficial for the treatment of any inherited or acquired disease or which improves the condition of an individual. Particularly, therapeutic proteins are those agents that can modify and repair genetic errors, destroy cancer cells or pathogen infected cells, treat immune system disorders, treat metabolic or endocrine disorders, among other functions. Therefore therapeutic proteins can be used for various purposes including treatment of various diseases like e.g. infectious diseases, neoplasms (e.g. cancer or tumor diseases), diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, independently if they are inherited or acquired. tissue, diseases of the musculoskeletal system and connective tissue. In one aspect, the recombinant cells contain a construct that directs the co-expression of additional proteins or nucleic acids necessary for electrochemical signaling. Such proteins may include, for example, Cas9 transcription proteins and/or gRNAs.
In a specific, recombinant cells are provided that place control of endogenous gene expression under the control of the eCRISPR system as disclosed herein. Said recombinant cells comprise co-expression of a Cas9 protein, inhibitor or activator transcription factor, and a target guide RNA for targeting of the endogenous gene expression. In an embodiment, the recombinant cells may express multiple Cas9 proteins, inhibitors and activators, as well as multiple gRNAs. Where multiple layers of gene expression are utilized, multiplexed control of gene expression may be achieved allowing for electrochemical signaling quorum sensing (QS) and communication between cells.
In one aspect, the recombinant cell comprises E. coli 's native oxyRS regulon for combating oxidative stress from hydrogen peroxide. In such an instance, CRISPR guide RNA (gRNA) expression is under the control of the oxyS promoter and the peroxide sensor OxyR. To activate CRISPR mediated gene expression, the gRNA forms a complex with constitutively-expressed dCas9ω (CRISPRa) to activate transcription of the gene-of-interest. In such an embodiment the guide RNA is designed to bind to a promoter of interest. In another embodiment, for inhibition of gene expression, the dCas9ω may be reengineered to CRISPR inhibition of gene expression.
Another aspect of the present disclosure pertains to nucleic acids encoding the gene-of-interest, or a reporter gene, wherein expression of said gene-of-interest or reporter gene is mediated by electrochemical signaling. Nucleic acid constructs may also include those encoding for Cas9 protein (inhibitor or activator transcription factors) and gRNAs of interest. Such nucleic acids may be introduced into a variety of different expression vectors, including for example, bacterial, and eukaryotic expression vectors for expression of the gene-of-interest, or reporter gene.
When the preparation method is through recombinant DNA technology, the expression vector may be a nucleic acid in the form of a plasmid, a cosmid, a phagemid, a phage, a viral vector or the like. Depending on the host microorganism, an appropriate vector may be purchased among commercially available vectors or may be used after being purchased and modified. For example, when Escherichia coli is used as the host microorganism, pUC19, pSTV28, pBBR1MCS, pBluscriptII, pBAD, pTrc99A, pET, pACYC184, pBR322, pJE101, pJE102, pJE103, etc. may be used. The expression vector may further include a selectable marker gene. The selectable marker gene is a gene encoding a trait that enables selection of a host microorganism containing such a marker gene and is generally an antibiotic resistance gene. For expression vector construction including recombinant DNA technology, reference may be made to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2001), F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley amp; Sons, Inc. (1994), and Marston, F (1987) DNA Cloning Techniques) and the like. All of the documents cited in the present specification are incorporated by reference in their entirety.
Methods of transforming the expression vectors into a host cell are also known in the art, and any of the known methods may be selected and used. For example, when the host cell is prokaryotic cells such as Escherichia coli, the transformation may be carried out through a CaCl2 method, a Hanahan method, an electroporation method, a calcium phosphate precipitation method, or the like, and when the host cell is eukaryotic cells such as yeast or mammalian cells, a microinjection method, a calcium phosphate precipitation method, an electroporation method, a liposome-mediated transfection method, a DEAE-dextran treatment method, a gene bombardment method, or the like may be used. Regarding details of the transformation method, reference may be made to (Cohen, S. N. et al., Proc. Natl. Acad. Sci. USA, 9:2110-2114 (1973); Hanahan, D., J. Mol. Biol., 166:557-580 (1983); Dower, W. J. et al., Nucleic. Acids Res., 16:6127-6145 (1988); Capecchi, M. R., Cell, 22:479 (19800; Graham, F. L. et al., Virology, 52:456 (1973); Neumann, E. et al., EMBO J., 1:841 (1982); Wong, T. K. et al., Gene, 10:87 (1980); Gopal, Mol. Cell Biol., 5:1188-1190 (1985); Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990); Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980); and Luchansky et al Mol. Microbiol. 2, 637-646 (1988), etc.)
The host cell that may be used for transformation in the method of the present disclosure may be prokaryotic or eukaryotic cells. As the prokaryotic cells, any gram-positive bacteria and gram-negative bacteria may be used. In a specific embodiment, Escherichia coli is used. In order to optimize expression and maintain the functions of the gene product of interest in Escherichia coli, the cell may have impaired protease activity.
The host cell transformed above is cultured, thus producing the gene product of interest. The culture of the transformed host cell may be performed through any method known in the art. As the medium used for cell culture, any of a natural medium and a synthetic medium may be used, so long as it contains a carbon source, a nitrogen source, a trace element, etc. which may be efficiently used by the transformed host cell. When animal cells are used as host cells, Eagle's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432 (1959)0, α-MEM (Stanner, C. P. et al., Nat. New Biol. 230:52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923 (1978)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8:396 (1959)) or the like may be used. Regarding details of the medium, see, for example, R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York.
In still another aspect, a method is provided of preparing a gene product of interest in a cell wherein expression of the gene product of interest is under the control of electrochemical signaling. The preparation method comprises (i) culturing the recombinant cells as described herein; (ii) providing an electrochemical signal; and (iii) allowing for expression of the gene product of interest. In a further step, the gene product of interest is purified from the cell.
Potassium chloride (KCl), H2O2 (30%), phosphate buffered saline (PBS), potassium phosphate monobasic, potassium phosphate dibasic, potassium hexachloroiridate(III) (K3IrCl6, Ir), and gelatin from porcine skin (gel strength≈175 g Bloom, Type A, G2625) were purchased from Millipore-Sigma. All antibiotics (ampicillin, kanamycin and chloramphenicol) were purchased from Millipore-Sigma. Lysogeny broth (LB) and agarose were from Fisher Scientific. Fluorescent beads and Pierce® horseradish peroxidase (HRP) were purchased from Thermo Fisher Scientific. 4-arm PEG-SH (MW 5000) were from JenKem USA. 1,1′-Ferrocene dimethanol (FcN) was from Santa Cruz Biology. AI-1 (N-3-oxo-dodecanoyl-L-Homoserine lactone) was from Cayman Chemicals. Ir, FcN, and AI-1 were initially prepared as 20 mM, 1 M, and 1 mM DMSO stocks, respectively.
Unless otherwise indicated, cells were grown overnight in LB at 37° C., 250 r.p.m. shaking, inoculated at OD600=0.1 in LB media the following day, and grown until the indicated cell density (optical density at 600 nm, OD600). Optical density was measured using an UV-Vis Spectrophotometer (Beckman Coulter).
All bacterial strains and constructs used in this study were listed in Table 1. All enzymes, competent cells and reagents were from New England Biolabs and used according to provided protocols. Q5 polymerase and primers in Table 3 (gene sequences in Table 2) were used for PCR reactions. DpnI digestion, polynucleotide kinase phosphorylation, T4 ligations, Gibson assembly and E. coli chemical transformation were performed using New England Biolabs product protocols. DNA clean-up (Zymo Research), gel extraction (Zymo Research) and plasmid preparation kits (Qiagen) were performed using provided protocols. Synthetic gene fragment containing the multiplex crRNAs was purchased from Thermofisher Scientific. Synthetic gene fragment containing the 108 spacer and gRNA scaffold was purchased from Integrated DNA Technologies (IDT). Synthetic gene fragment containing tracrRNA was purchased from Integrated DNA Technologies (IDT).
Electrodeposition of E. coli and PEG-SH
E. coli grown to mid-log phase were harvested via centrifugation at 3000 rcf for 10 minutes, then resuspended in 1×PBS to 2× the desired OD. To prepare the 2×PEG-SH solution, 100 mg/mL of PEG-SH were dissolved in phosphate buffer (PB) containing 10 mM FcN. Prior to electrodeposition, the two solutions were mixed at a 1:1 ratio. We then performed chronoamperometry, poised at 0.8 V for 30s, to initiate oxidation of the thiol group for crosslinking. The endpoint charge was recorded for each run. Excess cell/PEG-SH solution was then removed, and the generated film was carefully washed with PBS to remove any unbound cell/PEG-SH.
Composite fluorescence images were obtained using a ZEISS LSM700 confocal microscope
Overnight culture of DH5α-sfGFP was harvested via centrifugation (3000 rcf, 15 min) and resuspended in PBS to OD600=12/mL. 2×PEG-SH solution was prepared as previously stated and was mixed 1:1 with the E. coli solution. 100 μL of cell/PEG-SH mixture were loaded into the wells of our ITO-based electronic device and electrodeposition (0.8 V) was performed for the indicated duration. After decanting the excess solution and thorough washing, a ZEISS LSM700 confocal microscope was used to obtain Z-stack images of the generated film.
Electrochemical peroxide generation was performed in the biohybrid electronic device with its setup as described above. 150 μL of 20% LB mixed with 80% PBS (20% LB) was added into each well (surface area=(x) mm2). The working electrode solution was undisturbed (or, where indicated, stirred via blowing O2 into the wells). The electrodes were connected to a potentiostat (either 700-series CH Instruments or a custom FPGA-based potentiostat). Chronoamperometry, poised at −0.8 V for the indicated duration, was performed to generate hydrogen peroxide. The endpoint charge was recorded for each run.
To quantify the generated peroxide, we used the Pierce® quantitative peroxide assay kit (aqueous) (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the working reagent was prepared by mixing one volume of Reagent A with 100 volumes of Reagent B, with at least 200 μl prepared for each sample to be assayed. Ten volumes of the working reagent were added to one volume of sample (typically 200 μl working reagent to 20 μl sample) in a well of a clear-bottomed 96-well plate. The reaction was mixed and incubated for 15-20 min, after which a Spark® microplate reader (Tecan) was used to measure the absorbance at 595 nm. Sample peroxide concentration was calculated by comparison with a standard curve (dilutions of 30% peroxide) performed the same day.
Electroinduction experiments were performed in the custom biohybrid electronic device. Deposition of the cell/PEG-SH film was performed as described in previous sections. After thorough washing to remove excess cell/PEG-SH solution, 150 μL of 20% LB was added to each well as the culture media. Peroxide was generated via chronoamperometry as described in sections above, with voltage application for a specified duration (e.g., 1800 s). The biohybrid device was then moved to an incubator (30 or 37° C., as indicated) for incubation. For all automated experiments, the biohybrid device remained in a custom-made environmental chamber inside the Biospark system with temperature (34° C.) and humidity (≥80%) control. 0.4 ft3/h (scfh) of oxygen was supplied during electroinduction for media perturbation and oxygen supply to generate sufficient peroxide.
Samples (i.e., the media immersing the film) were removed at indicated time intervals and sterile filtered for downstream bioassays. For coculture experiments, AI-1 responsive strain (NEB10β+pLasR_S129T-GFPmut3) that were grown to mid-log phase were inoculated to an OD600 of 0.025 in 20% LB, then added into the wells containing the generated ‘artificial biofilm’ for electroinduction.
Unless stated otherwise, a Spark® plate reader (Tecan) was used to measure GFP fluorescence with excitation/emission wavelengths of 488/520 nm. For dynamic gene expression control experiments, GFP fluorescence was measured using our custom-built Biospark platform, as described in sections below.
AI-1 quantification was performed by bioluminescence assay. AI-1 reporter cells JLD271 pAL105 were grown overnight in LB at 37° C. and 250 r.p.m. shaking with the appropriate antibiotics. The following day, AI-1 solutions (0-84 nM) for the standard calibration curve were prepared in 20% LB. The reporter cells were diluted 500-fold in LB with the appropriate antibiotics. For every experimental replicate, 90 μl of diluted reporter cells and 10 μl of the standard AI-1 solutions were added into the wells of a white-bottom 96-well plate (Corning). Experimental conditioned media samples were prepared similarly after sterile filtering and diluting between two- and thousand-fold to maintain a linear assay range. Microplate with reporter cells and conditioned media samples were incubated at 30° C. and 250 r.p.m. shaking in a Tecan® microplate reader, and its luminescence was measured by the plate reader every 30 minutes for 3-5 hours. The AHL concentration of each sample was calculated using the standard curve.
NB101 harboring the plasmids that allow CRISPR activation of GFP (pSC-O108+pdCas9ω+pMC-GFP) were grown at 37° C. and 250 r.p.m in LB overnight. The following day, overnight cultures were diluted to OD600=0.1 and grown at 37° C. and 250 r.p.m until reaching mid-log phase. Peroxide stock solution (10 mM) were then spiked into the cultures to induce the expression of sgRNA. After incubation, samples were collected via centrifugation. Total and microRNA were extracted using a miRNeasy Kit (Qiagen) and quantified using a Nanodrop (Thermo Scientific). RT-qPCR was then carried out using the Power SYBR™ Green RNA-to-CT™ 1-Step Kit (Applied Biosystems) and a Quantstudio 7 Flex Real-time PCR system (Applied Biosystems).
Relative AI-2 levels were determined using the V. harveyi reporter BB170 bioluminescence assay11 with slight modifications. AI-2 reporter cells BB170 were grown overnight in AB media at 30° C. and 250 r.p.m shaking with appropriate antibiotics. The following day, overnight BB170 culture was diluted 5000-fold in AB media. For every experimental replicate, 180 μl of diluted BB170 culture and 20 μl of conditioned media were added into the wells of a white-bottom 96-well plate (Corning). The microplate was then incubated at 30° C. and 250 r.p.m. shaking in a Tecan® microplate reader, and its luminescence was monitored by the plate reader every 30 minutes after 3-hour incubation. AI-2 activity was calculated by dividing the RLU produced by the reporter after addition of conditioned media by the RLU of the reporter when growth medium alone was added.
Electrodeposition of HRP/Gelatin Hydrogel and Electrochemical Detection of H2O2
The protocol for HRP/gelatin deposition and electrochemical detection of peroxide as described by Li et al. was used with slight modifications7. Deposition of HRP/gelatin was performed on a custom patterned electrode generated through laser-cutting the ITO-coated glass slide into two separate interweaving working electrodes. This zig-zag pattern was chosen to ensure the generated peroxide be at the vicinity of the deposited HRP/gelatin hydrogel for detection. A custom 3D-printed housing with one central well exposing the working electrodes and two side openings for the insertion of Ag/AgCl reference electrodes was then attached to the patterned ITO electrode (
Cloning of Peroxide-Inducible gRNA Expression Plasmids (Supplementary)
Plasmid pSC-O108 (Table 1) for peroxide-inducible expression of sgRNA sg108 was made via PCR amplification and Gibson assembly. First, pSC-108gRNA2 was removed of soxR, soxRS promoter region, and sgRNA sg108 by restriction digestion with ClaI and BamHI. Gene fragment containing oxyR and oxyS promoter (Table 2) was amplified from plasmid pOxy-LacZlaa3 with primers SW01 and SW02 (Table 3). After DpnI treatment, fragments were ligated by Gibson Assembly. Next, gene fragment containing spacer 108 and gRNA scaffold was then PCR amplified with primers SW03 and SW04 and inserted into the linearized intermediate product via primers SW05 and SW06 to generate the construct pSC-O108.
Plasmid pSC-LuxS1 for peroxide-inducible expression of sgRNA LuxS1 was made via site-directed mutagenesis. Spacer 108 (Table 2) in plasmid pSC-O108 was swapped with LuxS1 (Supplementary Table 2) using primers 7 and 8.
Plasmid pSC-sg108+LuxS1 for peroxide-inducible expression of crRNAs spacer 108 and LuxS1 was made via PCR linearization, restriction digestion, and T4 ligation. pSC-O108 was initially PCR linearized using primers SW09 and SW10 to remove the spacer 108 and the gRNA scaffold, as well as adding restriction sites BamHI and XhoI. Gene fragment containing crRNAs spacer 108 and LuxS1 (Table 2) was inserted into linearized pSC-O108 backbone via restriction digestion and T4 ligation. After this, the gene fragment containing the tracrRNA (Table 2) was inserted into the product generated from the previous step via restriction digestion and T4 ligation to generate the final construct.
Cloning of pLuxS1
Plasmid pLuxS1 for constitutive expression of sgRNA LuxS1 was constructed via site-directed mutagenesis using primers 11 and 12 to swap the spacer 108 in plasmid pS108gRNA2 to LuxS1.
Cloning of pMC-lasI-LAA
Plasmid pMC-lasI-LAA was constructed via PCR amplification and Gibson assembly. pMC-GFP2 was linearized and removed of GFPmut2 by primers SW13 and SW14. Primers SW15 and SW16 were used to amplify and add the LAA ssRA tag to lasI. lasI with the added LAA tag was then inserted to the linearized backbone via Gibson assembly.
Cloning of pOxy-sfGFP-AAV (Supplementary)
Plasmid pOxy-sfGFP-AAV was constructed via site-directed mutagenesis using primers 17 and 18 to add the AAV ssRA tag to pOxy-sfGFP24.
Controllable Hydrogel Deposition for Immobilizing E. coli to Integrate with Biohybrid Device.
To better integrate biological systems that were mostly cultured in a suspension aqueous state with electronic devices, inspiration was taken from bacterial biofilms found in nature and created an ‘artificial biofilm’ to immobilize electrogenetic E. coli onto an electrode. Due to the diffusion-limiting nature of peroxide, this also greatly benefits the signal transfer by localizing the cells at the bioelectronic interface3. The ‘artificial biofilm’ was generated through electrodepositing the electrogenetic bacteria with thiolated polyethylene glycol (PEG-SH) to form a cell-containing hydrogel. Specifically, cells were mixed with PEG-SH monomer in a solution containing a redox mediator ferrocene (FcN) to facilitate the oxidation of thiol groups in PEG-SH to form disulfide bonds (
We also created an ITO-based electronic device to serve as the experimental platform in the study. Following a typical 3-electrode setup, ITO-coated glass was chosen as the working electrode (WE) due to its transparency for optical observations and measurements. A 3D-printed housing was attached to the ITO electrode to generate separate wells with similar dimensions to a conventional 96-well plate. The device's ability to generate peroxide via electronic input was tested. Although previous studies reported that peroxide can be generated by applying voltages ranging from −0.3 to −0.9 V (versus Ag/AgCl) to partially reduce oxygen8 (according to reaction O2+2H++2e↔H2O2), we found that biasing the ITO electrode to −0.8 V produced the highest level of peroxide (
oxyRS-Based Electrogenetics CRISPR Activation
In hopes of expanding the oxyRS-based electrogenetics toolbox, a versatile CRISPR transcriptional regulation system was developed to allow activation, repression, and multiplexed regulation of genes at the transcription level. Here, a peroxide-mediated, electrically-inducible CRISPR transcriptional activation (CRISPRa) system was created by rewiring the previously developed soxRS-based electrogenetic CRISPR (eCRISPR)2 and placing the single guide RNA (sgRNA) under the control of oxyS promoter (
Having shown that one can electrically induce CRISPRa in the biohybrid device, testing was done to demonstrate information routing between electronic to biological signaling modalities by initiating a specific QS communication (i.e., las AI-1 QS system from Pseudomonas aeruginosa) that is not natively found in E. coli through peroxide-mediated eCRISPR. A new reporter plasmid (pMC-lasI-LAA) was constructed by replacing gfpmut2 with the AI-1 producer lasI (fused with a ssRA tag), allowing CRISPRa control of AI-1 production. This new reporter plasmid, along with the other CRISPRa components (dCas9ω and pSC-O108) were transformed into NB101 cells. These populations were later referred to as CRISPRa lasI cells. Prior to electro-induction tests, CRISPRa lasI cells were also confirmed to be peroxide-inducible in liquid culture (
While it was shown that by enabling peroxide-mediated eCRISPR activation of QS can open a new a line of communication to other microbial or even mammalian populations9,10, CRISPR transcription regulation offers additional functions such as gene repression and multiplexed control for multi-locus transcription regulation. Here, these functions were explored and applied to enable electrically-controlled ‘multilingual’ QS communication. In particular, the aim was to create an engineered E. coli that switches its ‘spoken language’ (i.e., QS signaling) from its ‘mother tongue’ (luxS-based AI-2) to a ‘foreign language’ (las AI-1 from P. aeruginosa) upon receiving the electronic signal. Unlike the las system, luxS/AI-2 QS system is found natively in many bacteria, Gram positive and negative alike, including the experiment chassis E. coli. To prohibit E. coli from secreting AI-2, a sgRNA LuxS1 was designed that is homologous to the AI-2 producer gene luxS and repurposed dCas9ω for CRISPR inhibition (CRISPRi) (
Having shown successful eCRISPRi of luxS, it was then decided to harness the multiplexed nature of CRISPR and combine CRISPRa of lasI and CRISPRi of luxS to create a ‘bilingual’ strain. Though many strategies for multiplexed gRNA expression have been previously explored15, a method derived from the native CRISPR-Cas system was chosen. By flanking an array of gRNAs with ‘direct repeats’ (DR), which are repetitive sequences required for crRNA processing, the tandem gRNAs can be processed by RNase III in a tracrRNA-dependent manner, like how crRNAs are processed in Type II CRISPR systems16-18. Based on this approach, plasmid pSC-sg108+LuxS1 was constructed that comprised two key parts for inducible expression of multiple gRNAs: (1) gRNAs sg108 and LuxS1 were flanked with DR sequences and together were placed downstream of the oxyS promoter for peroxide-inducible control and (2) tracrRNA, required for gRNA processing, was individually expressed under a synthetic constitutive promoter. Next, pSC-sg108+LuxS1, with pdCas9ω and pMC-lasI-LAA were then transformed into NB101 cells to generate the ‘bilingual’ strain (
Automated Dynamic Control of eCRISPRa Activity
Inspired by the discovery of which of the oxyRS-based eCRISPR transcription regulation toolbox could be dynamically controlled, it was aimed to investigate and employ the temporal nature of this system to build a fully automated bioelectronic platform for electrogenetics control. First, a simpler construct (pOxy-sfGFP-AAV) was created that was stripped of the CRISPR components but retaining its ability to respond to peroxide, for studying the dynamics of the oxyRS induction system in suspension culture (
While electrogenetics provides an approach to seamlessly integrate signal transmission from electronic devices to biological systems, a pathway allowing communication from bio to electronics is also needed to form a closed network for fully automated control. Anoptical signaling (in our case, fluorescence) was chosen to bridge this gap since (1) fluorescence proteins have long been serving as proxy for gene expression19,20, (2) fluorescence output allows for real-time measurements, and (3) optical signals are easily translated into electronic signal. Therefore, a bioelectronic system named ‘Biospark’ was constructed that consists of a fluorescence detection module for gene expression measurements, a built-in potentiostat for sending electronic commands, and a custom environmental chamber for cell culture (
By connecting everyday electronic devices to the internet and forming a collective network like the Internet of Things (IoT)21, each member within can collect data and respond intelligently to users, thereby benefitting daily lives. The local bioelectronic device can be integrated into a network allowing multidirectional communication between ‘living’ systems, and realize remote or even remote feedback control of a dynamic biological system.
In this work, a simple, controllable method was developed for assembling live cells into ‘artificial biofilms’ directly on the electrode to ensure efficient information flow at the bio-electronic interface and eliminate transport-limited heterogeneous responses in suspended biological systems23,24. This approach does not require genetic engineering; thus relieving the extra metabolic burden from the entrapped engineered cells for more effective use of resources on critical functions25, or instead opening more possibility for immobilizing any living organisms without the need for inserting non-native genetic circuitries. Moreover, cell/PEG-SH co-deposition is shown to be scalable and spatially programmable, inviting future opportunities for cell grafting on complex formats like three-dimensional, miniaturized, arrayed electrodes; or on soft, flexible, bio-compatible materials, for wearable, ingestible or other portable system26,27.
The repertoire of oxyRS-based electrogenetics was then expanded by engineering peroxide-mediated eCRISPR, enabling not only upregulation, but inhibition, and multiplexed control of a variety of genetic targets. These include two crucial QS-related genes that allow the relay of electrochemical signals to a broader yet selective audience of microbial populations through QS communication. Prevalent in nature, QS signaling is an ideal candidate for dispatching the highly-localized electrochemical cue to other ex situ biological populations with none or minimal genetic rewiring9,28, and achieving coordination within a natural or synthetic consortium4,29.
Next, the transient nature of oxyRS-based electrogenetics was studied and this feature was harnessed for dynamic control of eCRISPR activity. A first integrated electrogenetic system was presented, including both custom-made hardware and software, for fully automated control of eCRISPR-mediated expression. Here, biological responses were coupled with light to facilitate their transition into electronic signals in real time. Though noted that optogenetics, the use of light-sensitive proteins for regulation of various biological functions, has been a blooming field for interfacing biology and cybernetics30,31, the disclosed multimodal approach that incorporated electrogenetics induction eliminated the need of an optical input in situations where light is not penetrable, such as commercial bioreactors with high-densities of cells. Furthermore, eCRISPR also provides one with an adaptable platform for simultaneous control of genes other than fluorescent proteins via multiplexed transcriptional regulation. It is also envisioned that the addition of artificial intelligence such as machine learning techniques for combining the data obtained in this study, for example, correlative dose-response relationships between charge inputs and outputs with gene expression, to generate an optimized model allowing smarter precision control.
Finally, the integration of bioelectronic systems with the internet was demonstrated to form a complete electrogenetic framework. By establishing this network of Bio-Nano Things, real-time, feedback control of eCRISPR activity was realized regardless of the constraint in physical distances. An immediate application of this framework would be for online monitoring and feedback control of cells inside bioreactors for smart biomanufacturing32. Nevertheless, since all electrogenetics components in this study (that is, peroxide signaling, CRISPR techniques, and QS communication) are either native to or can be easily ported to most biological systems, it is foreseen that the Bio-Nano Things network could be employed as the foundation of a plethora of intelligent systems or devices33. These include self-regulated, real-time reporting drug delivery biomedical devices for in situ production of a therapeutic34,35, smart agriculture systems for monitored rhizosphere microbiome manipulation36, or “sense-and-clean” strategies for battling environmental pollution to actively sense and remove contaminants37, together serving as the blueprint for a more connected world in the future.
E. coli Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15
E. coli DH5α attTn7::mTn7ΦsfGFP
E. coli ZK126 ΔrpoZ
E. coli K-12 ΔlacX74 sdiA271::Cam
V. harveyi luxN::Tn5
The following are hereby incorporated by reference herein in their entirety.
This application claims the benefit of and priority to U.S. Provisional Application No. 63/363,004 filed on Apr. 14, 2022, which hereby is incorporated herein by reference in its entirety.
This invention was made with government support under CBET1805274, ECCS1807604, and ECCS1926793 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63363004 | Apr 2022 | US |
