Patent Application
20240035059
The present invention relates to heat-repressible split-T7 polymerases comprising temperature-sensitive domains of Tlpa protein fused with split T7 RNA polymerase (T7RNAP) to introduce thermal control into widely used T7 RNA polymerase, creating a heat-repressible Thermal-T7RNAP system. The invention further provides polynucleotides encoding the heat-repressible split-T7 polymerases, and methods of thermal control of bioproduction.
Existing heat-repressible systems either function at too low temperatures (<30° C.) for optimal enzymatic bioprocesses to occur, suffer from low fold inductions and long delays or lack orthogonal control.
The heat-repressible systems generally fall into two main categories, RNA based or transcriptional regulations. RNA based thermometers typically suffer from long delay in responses and small fold inductions (<10-fold) [Hoynes-O'Connor, A. et al., Nucleic Acids Res 43:6166-6179 (2015)]. In the second category, there are very limited heat-repressible systems based solely on transcriptional regulation. The closest example was the coupling of an inverter with the heat-inducible transcription repressor cl434 to enable a low temperature input (25° C.) be converted into high expressions [Yang, Z. et al., Nucleic Acids Research 47(21):e137 (2019)]. By introducing additional complexity into the thermal system, it is likely to result in the circuit being intricately tied to the host's metabolism and become unstable as it is hyper-sensitized to global cellular effects [Segall-Shapiro, T. H. et al., Mol Syst Biol 10:742 (2014)].
To address these issues, a compact and orthogonal thermal system is of great significance to minimize the burden to the host. Among the numerous phage-based polymerases, the T7 RNA polymerase (T7RNAP) is a popular variant of orthogonal RNAP in the field of biotechnology [Wang, W. et al., Biotechnol Adv. 36:2129-2137 (2018)]. The polymerase can effectively decouple transcription from the host and recognize specifically its cognate T7 promoter [Segall-Shapiro, T. H. et al., Mol Syst Biol 10:742 (2014)]. These advantages have been recognized early and led to efforts in making T7RNAP system thermal controllable which mainly involved placing the T7RNAP gene under heat-inducible operons [Wang, Z. W. et al., Biotechnology Progress 20:1352-1358 (2004); Chao, Y. P. et al., Appl Microbiol Biotechnol 58:446-453 (2002)]. Fundamentally many protein interactions are modular in nature and it is a common strategy for native proteins to be fused with foreign domains, to convert a physical or chemical signal into a change in physical interactions between the fusion pairs [Grunber, T. and Serrano, L. Nucleic Acids Res 38:2663-2675 (2010)]. The fusion of light sensitive protein domains with the split N-terminal and C-terminal T7RNAP fragments represents the recent work in providing a means of using physical cue to control the activity of the polymerase directly, [Han, T. et al. ACS Synth Biol 6:357-366 (2017); Baumschlager, A. et al., ACS Synth Biol 6:2157-2167 (2017)]. Along the same vein, it is likely to introduce direct thermal control into T7RNAP by its fusion with temperature-sensitive domains. An early attempt to introduce temperature-sensitive intein into T7RNAP gene [Liang, R. et al., J Microbiol Methods 68:497-506 (2007)] was compounded with a number of limitations. Firstly, the system was activated at a low temperature of 18° C., which makes many bioprocesses non-optimal. Secondly, temperature-mediated splicing is slow and takes several hours [Shah, N. H. and Muir, T. W., Chem Sci 5:446-461 (2014)]. Thirdly, the splicing requires extensive design before achieving optimum activity. Lastly, the ‘permanent nature’ of this post-translational modification has limited its applicability.
There is a need for improved methods and constructs for heat-repressible production of recombinant proteins or small molecules.
While many studies reported on the development of heat-inducible systems [Piraner, D. I. et al., Nat Chem Biol 13:75-80 (2017); Rodrigues, J. L. and Rodrigues, L. R. Trends Biotechnol 36:186-198 (2018)], very few heat-repressible systems have been developed. Nevertheless, low temperature in general remains useful for improving protein solubility, increasing its stability by promoting proper folding and reduce the detrimental effects from toxic stress proteins [Qing, G. et al., Nat Biotechnol 22:887-882 (2004); Sorensen, H. O and Mortensen, K. K., Microb Cell Fact 4:1 (2005)]. However, earlier reported systems switch on at very low temperature (e.g., <30° C.). The milder temperatures can also lead to more efficient bio-catalysis processes and increases the yield of unstable heat-labile proteins [Singh, R. et al., 3 Biotech 6174-174 (2016)]. Known heat-inducible systems and their characteristics are listed in Table 1, in comparison to the heat-repressible thermal T7RNAP system of the invention.
Accordingly, an alternative mode of thermal regulation was developed by providing a reversible and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs) which fuses temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30-42° C.
According to a first aspect of the invention, there is provided an isolated heat-repressible Split-T7 polymerase fusion protein, comprising:
In some embodiments, the coiled-coil domain is selected from the group comprising TlpA polypeptide, M class C proteins from group A streptococci, such as Arp4 and Sir22, and Hv1/VSOP voltage-gated H+ channel protein.
In some embodiments, the N-terminal fragment of T7 RNA polymerase and the C-terminal fragment of T7 RNA polymerase are derived by splitting the T7 RNA polymerase polypeptide at amino acid position 563/564 of the mature peptide sequence.
In some embodiments, the T7RNAP comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence is set forth in SEQ ID NO: 1 and/or the TlpA coiled-coil comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3.
In some embodiments, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase comprises one or more domains, X1, X2, X3, X4 and X5, encoded by polynucleotide sequences having at least 70%, at least 80%, at least 90% or 100% identity with sequences selected from the group comprising X1 (SEQ ID NO: 14), X2 (SEQ ID NO: 15), X3 (SEQ ID NO: 16), X4 (SEQ ID NO: 17) and X5 (SEQ ID NO: 18) or combinations thereof.
In some embodiments, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase comprises a X1 domain at the N-terminal end and a X4 domain at the C-terminal end of the coiled-coil domain.
In some embodiments, the polynucleotide sequence set forth in X1 (SEQ ID NO: 14) comprises a G/A substitution at position 52 and/or the polynucleotide sequence set forth in X4 (SEQ ID NO: 17) comprises a T/A substitution at position 4.
In some embodiments, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase comprises a plurality of X5 domains.
In some embodiments, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase comprises, from N-terminal to C-terminal, domains X1, X5, X5 and either X4 or X5. Preferably, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 12.
In some embodiments, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase consists of, from N-terminal to C-terminal, domains X1, X2, X3 and either X4 or X5. Preferably, the coiled-coil domain fused to the C-terminal fragment of T7 RNA polymerase is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 13.
In some embodiments, as a result of varying combinations of coiled-coil domains X1, X2, X3, X4 and/or X5, the active temperature range can be tuned.
In some embodiments, the Split-T7 polymerase is active at temperatures in the range of about 30° C. to about 39° C. and is thermally repressed above 39° C.; preferably repressed at 37° C. and above.
According to a second aspect of the invention, there is provided an isolated nucleic acid molecule capable of expressing the fusion protein of any embodiment of the first aspect.
According to a third aspect of the invention, there is provided a plasmid or vector comprising the nucleic acid molecule of the second aspect.
According to a fourth aspect of the invention, there is provided a host cell comprising the nucleic acid molecule of the second aspect and/or the plasmid or vector of the third aspect, and a gene encoding a product of interest operably linked to a T7 promoter.
According to a fifth aspect of the invention, there is provided a composition comprising the host cell of the fourth aspect.
According to a fifth aspect of the invention, there is provided a method of regulating the relative proportions of two or more cell populations within a co-culture, comprising:
In some embodiments, the respective growth regulatory genes are the same.
In some embodiments, the respective growth regulatory genes slow down cellular growth, such as by limiting glucose uptake by expressing a SgrS sRNA which functions to degrade ptsG mRNA that encodes for a glucose transporter, IICBGlc.
According to a sixth aspect of the invention, there is provided a kit comprising:
In some embodiments, the kit further comprises one or more ribonucleoside triphosphates.
According to a seventh aspect of the invention, there is provided a method of synthesizing an RNA molecule comprising:
In some embodiments, the incubating is done at a temperature of less than 40° C., such as in a range of 30° C. to 39° C., preferably 37° C. or less, such as in a range of 30° C. to 37° C.
Certain terms employed in the specification, examples and appended claims are collected here for convenience.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.
Bibliographic references mentioned in the present specification are for convenience listed at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.
All plasmids were designed in silico using Benchling (Benchling, Inc. San Francisco, CA, USA). Individual gene fragments and primers were synthesized from Integrated DNA Technologies (Integrated Device Technology, Inc. San Rose, CA, USA). The polymerase chain reaction (PCR) products were amplified using Q5 High-Fidelity DNA polymerase (New England Biolabs, MA, USA) with strict accordance to the manufacturer's protocols. PCR products were analysed by gel electrophoresis using 1% agarose gel and purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany). The DNA concentrations of the gel-purified samples were quantified with Nanodrop™ Onec (Thermo Fisher Scientific, MA, USA). Gibson assembly was subsequently performed using the NEBuilder HiFi DNA assembly (New England Biolabs, MA, USA) with strict accordance to the manufacturer's protocols. Subsequent assembly products were chemically transformed into E. coli K-12 strain NEB DH-10 Beta (New England Biolabs, MA, USA) unless stated otherwise. Colonies that grown on the LB-antibiotic plate were picked and inoculated into fresh LB-antibiotic medium at 37° C. to prepare overnight culture for plasmids extraction using QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany). The plasmids were then sent for DNA sequencing (1st BASE, Singapore). The sequencing results were subsequently aligned with the digital template and analysed on Benchling platform.
pBbE6K (JBEI Part ID: JPUB 000054, colE1 ori, Kanr), pBbE8K (JBEI Part ID: JPUB 000036, colE1ori, Kanr), pBbA6C (JBEI Part ID: JPUB 000056, p15A ori, Cmr), pBbA8C (JBEI Part ID: JPUB 000038, p15A ori, Cmr) and pNO4, which was a gift from Jeffrey Tabor (pSC101 ori, Ampr) (Addgene plasmid #101066; n2tdotnet/addgene:101066; RRID:Addgene_101066), were used as the backbones in the plasmid constructions whenever necessary. Constitutive promoters J23101 (BBa_J23101) and gabDP2 (BBa_K3252022), pT7 promoter (BBa_R0085), double terminator 15T (BBa_B0015), rrnBT1 terminator (BBa_B0010), T7 terminator (BBa_K731721), ribosome binding site (rbs34) (BBa_B0034), green fluorescence protein gene GFPmut3b (BBa_E0040), red fluorescence protein gene DsRed (BBa_K2782004), sugar transport related sRNA gene (SgrS) (BBa_K581005) and the T7 RNA polymerase gene (BBa_12032) were obtained from iGEM Registry of Standard Biological Parts (iGEM Foundation, Cambridge, MA, USA) (http://partsregistry.org) and used in plasmid constructions whenever necessary. The T7 RNA polymerase gene was split at the 563 (S)/564th (E) location, to form the N-terminal T7 protein unit and C-terminal T7 protein unit, as guided by previous work when attempting to find the most optimum split sites [Baumschlager et al., ACS Synth Biol 6:2157-2167 (2017); Han et al., ACS Synth Biol 6:357-366 (2017)]. The GGSGG linker was obtained from a previous study (Baumschlager et al., ACS Synth Biol 6:2157-216 (2017)]. The full length 371 amino acids (a.a) long Tlpa36 gene (referred herein as Tlpa) and its pTlpa promoter were derived from the paper (Piraner et al., Nat Chem Biol 13:75-80 (2017)], and synthesised as a gene fragment. The list of promoters, RBS and relevant gene sequences are listed in Table 2.
QDRYDSLTLALESERSLRQQHDVEMAQLKERLAAAEENTRQREE
RYQEQRTVLQDALNAEQAQHINTREDQQKRLEQISAEANARTEEL
KSERDKVNTLLTRLESQENALASERQQHLATRETLQQRLEQAIADT
QARAGEIALERDRVSSLTARLESQEKASSEQLVRMGSEIASLTERC
TQLENQRDDARLETMGEKETVAALRGEAEALKRQNQSLMAALSG
NKQTGGQNA
The plasmids PGFP reporter and PGFP reporter(2) were generated by inserting PCR amplified sequence (pT7-rbs34-GFP) into backbone pBbE6k and pNO4 respectively. The plasmid PgabDP2-NT7Tlpa-GFP was generated by inserting PCR amplified sequences (promoter gabDP2-rbsDefault-NT7-linker-Tlpa coil) into PGFP reporter with multiple PCR steps. Similarly, Plasmids PJ23101-NT7Tlpa-GFP was generated by inserting PCR amplified sequences of promoter J23101. The plasmid PJ23101-CT7Tlpa was generated by inserting the PCR amplified sequence (J23101-rbs34-Tlpa coil-linker-CT7) into backbone pBbA8c. The plasmid PJ23101-rbs34-T7full was generated by inserting PCR amplified sequence (promoter J23101-rbs34-T7RNAP) into backbone pBbA8c. The plasmids PJ23101-NT7-GFP and PJ23101-CT7 were generated by PCR-mediated excision of the Tlpa coiled-coil sequences from the original plasmids PJ23101-NT7Tlpa-GFP and PJ23101-CT7Tlpa respectively. The plasmids PJ23101-NT7-GFP and PJ23101-CT7 were generated by PCR-mediated excision of the Tlpa coiled-coil sequences from the original plasmids PJ23101-NT7TIpa-GFP and PJ23101-CT7Tlpa respectively. While, PJ23101-NT7Tlpa-J23101-CT7Tlpa was generated by inserting the PCR amplified sequence (J23101-rbs34-Tlpa coil-linker-CT7) originally from PJ23101-CT7Tlpa into backbone PJ23101-NT7Tlpa-GFP. The plasmids Pplac-NT7Tlpa and Pplac-NT7Tlpa* were generated by inserting the PCR amplified sequences (rbs34-NT7-Tlpa coil) and sequence (rbs34-NT7-Tlpa truncated coil) into backbone pBbA6c respectively. PpBad-CT7 mutatedTlpa and PpBad-CT7mutatedTlpa* were generated by inserting PCR amplified sequences (rbs34-Tlpa coil-linker-CT7R632S) and sequence (rbs34-Tlpa truncated coil-linker-CT7R632S) in the backbone pBbE8k respectively. Each of the plasmids PgabDP2-NT7Tlpa-GFP and PJ23101-NT7Tlpa-GFP was co-transformed with PJ23101-CT7Tlpa to form Thermal-T7RNAP(v1 and v2) systems. PJ23101-NT7Tlpa-J23101-CT7Tlpa was co-transformed with PGFP reporter to form Thermal-T7RNAP(v3). PJ23101-rbs34-T7full was co-transformed with PGFP reporter to form the full length T7-WT control. PJ23101-NT7Tlpa-GFP and PJ23101-CT7Tlpa was co-transformed to form the Split-T7 control. Pplac-NT7Tlpa, PpBad-CT7mutatedTlpa and PGFP reporter(2) were tri-transformed to form [NT7-Tlpa+CT7(R632S)-Tlpa] system. While, Pplac-NT7Tlpa*, PpBad-CT7mutatedTlpa* and PGFP reporter(2) were tri-transformed to form the [NT7-Tlpa*+CT7(R632S)-Tlpa*] system. Next, the plasmids PJ23100-Tlpa and PpTlpa-RFP were generated by inserting PCR amplified sequences (promoter J23100-rbs34-Tlpa36) and (promoter pTlpa-rbs34-RFP) into backbones pBbA8c and pBbE6k respectively. Correspondingly, the plasmids PJ23100-Tlpa and PpTlpa-RFP were co-transformed to form the Tlpa system. In the growth inhibition experiments, the plasmid PgabDP2-NT7Tlpa-pT7-sgrs-GFPreporter was generated by inserting PCR amplified sequence (rbs34-SgrS-15T-J23101-rbs34-GFP-rrnBT1) into backbone PgabDP2-NT7Tlpa-GFP with multiple PCR steps. Its control plasmid PgabDP2-NT7Tlpa-GFPreporter(control) was generated by PCR-mediated excision of the sequence (promoter pT7-rbs34-GFP-15T) from the backbone PgabDP2-NT7Tlpa-GFP and the subsequent insertion of sequence (promoter J23101-rbs34-GFP-rrnBT1) into the same backbone. Both of the plasmids PgabDP2-NT7Tlpa-pT7-sgrs-GFPreporter and PgabDP2-NT7Tlpa-GFPreporter(control) were each co-transformed with PJ23101-CT7Tlpa in chemical competent E.coli K-12 MG1655 cells to form the ThermalT7RNAP-SgrS system and the ThermalT7RNAP-SgrS (control) system respectively. Next, the plasmid PTlpa-sgrs-RFPreporter was generated by inserting PCR amplified sequence (SgrS-15T-J23101-rbs34) into backbone PpTlpa-RFP. Its control plasmid PpTlpa-sgrs-RFPreporter(control) was generated by PCR-mediated excision of the promoter sequence pTlpa from the backbone PpTlpa-RFP and the subsequent insertion of new promoter J23101 into the same backbone. Both of the plasmids PpTlpa-sgrs-RFPreporter and PpTlpa-sgrs-RFPreporter(control) were each co-transformed with PJ23100-Tlpa in MG1655 cells to form the Tlpa-SgrS system and the Tlpa-SgrS (control) system respectively.
All chemicals were purchased from Sigma Aldrich (Sigma, MO, USA), unless stated otherwise. All glycerol stocks were prepared by mixing 500 μL overnight culture with 500 μL of sterilized 100% glycerol. Seed cultures from glycerol stocks were inoculated 20 hours overnight in 5 mL Invitrogen Luria Broth Base LB medium (ThermoFischer, USA) and supplemented with Kanamycin sulfate (Merck, Germany) (50 μg/mL), Chloramphenicol (25 μg/mL) and Ampicillin (100 μg/mL) whenever necessary and incubated in a mini NB-205 shaking incubator (BioTek, USA) at 40° C., 225 rpm.
To run the thermal regulation assay, 100 μL of overnight cultures were added to 5 mL fresh pre-warmed LB and grown for 90 mins at 40° C. at 225 rpm. Subsequently, the refreshed cell cultures were dispensed into the wells of the 96-Well Non-Skirted PCR plate (Thermo Scientific, USA) at 100 μL. Final concentrations of 1 mM of IPTG (FirstBase, Singapore) and 0.2% L(+) arabinose were added to some cell cultures which required chemical induction. Once 96-Well PCR plate had been loaded, the plate was sealed fully with adhesive film, and left to incubate inside the thermal cycler T100 (Bio-Rad Laboratories, Hercules, CA, USA). Each row was programmed at the same temperature using the thermal ‘gradient’ function to create the desired temperature range from 30 to 42° C. After the 18 hours incubation, the cell culture in each individual well was transferred into the 96-well microplate using a multi-channel pipette. The corresponding fluorescence intensities and optical density readings were read in the H1 Synergy (BioTek, USA) at the following settings: GFP gain: 75, GFP: excitation 485 nm, emission 528 nm; OD: 600 nm.
For continuous kinetic experiments, the refreshed cell cultures were dispensed into the 96-well microplate at 300 μL each. The plate was covered with the plate lid and time series optical density and fluorescence were obtained at an interval of 10 minutes for a total duration of 12 hours and configured at double orbital shaking speed of 282 rpm continuously. Depending on the need of the studies, the temperature profiles were configured differently (at a fixed temperature of 30° C./37° C. or transitioning between the two temperatures at intervals of 2/3 hours) in the microplate reader. Prior to the experiment, the system was pre-incubated at 40° C. to ensure tight repressibility.
Error-prone PCR was performed on the coiled-coil domains of the NT7 and CT7 protein fragments on plasmids PgabDP2-NT7Tlpa-GFP and PJ23101-CT7Tlpa of the Thermal-T7RNAP(v1) system, using the GeneMorph II random mutagenesis kit (Agilent, USA). The generated PCR products were inserted back into the respective backbones using Gibson Assembly. The resultant mutant libraries were co-transformed with its un-mutated counterpart plasmid PgabDP2-NT7Tlpa-GFP or PJ23101-CT7Tlpa respectively in DH-10 Beta cells in LB Agar. Approximately 10 rounds of mutagenesis were conducted to generate sufficient variants. After incubating overnight at 30° C., each individual colony was isolated using the Rotor HDA colony picker (Singer instruments, UK). Using the colony picker, each colony was replicative plated onto three identical 384-format agar plates. These three identical agar plates were each grown overnight at 30° C., 37° C. and 40° C. to screen for colonies with different activation and repressed temperatures. The next day, each identical colony under different temperatures was imaged in the colony picker upon illuminating with blue epifluorescence. Data processing was conducted to normalize the captured green fluorescence intensities with the corresponding size of individual colony (measured in mm2) and each colony was sorted and ranked by the respective fold changes in fluorescence intensities between 30° C./37° C. and 30° C./40° C. Approximately 1700 colonies were screened in the library and 130 CT7 mutant colonies were shortlisted and quantified in the thermal regulation assay as described before. The thermal induction profiles generated were each fitted with a Hill's equation and correspondingly the activity and performance are quantified with various ranking indexes such as fold change between 10% and 90% of fluorescence intensities, the steepness of transition and half-activation temperatures. The sequences of the mutants are provided in Table 3.
Seed cultures of the GFP cells (ThermalT7RNAP-SgrS system) and the RFP cells (Tlpa-SgrS system), along with their control cells, were cultured 20 hours overnight in 5 ml of LB medium with the necessary antibiotics at their repressive temperature of 40° C. and 30° C. respectively. 50 μL of overnight cultures were added to 5 mL fresh pre-warmed M9 medium and grown for 90 mins at their repressive temperatures at 225 rpm. The M9 medium was prepared by adding 5×M9 salts, with final concentrations of 0.2% casamino acids, 100 μM CaCl2, 2 mM MgSO4 and 0.2% glucose into distilled water. The refreshed cell cultures were individually corrected to reach growth density of OD=0.1, before being dispensed in the 96-well microplate at their respective cell proportions into each well to form various co-cultures. The microplates were conducted in a continuous kinetic fashion at the required temperatures following the same microplate settings as described before.
A mechanistic model represented in the form of ordinary differential equations (ODEs) was formulated to describe the kinetics of the thermal-repressible split-T7RNAP fusion protein and used to examine the different gene circuit configurations (data not shown). The same model was also employed to gain insight into the dynamic behaviors of the 3-hour and 2-hour interval OFF-ON thermal duty cycles. To better quantify transcriptional kinetics, a simple model was adopted from Motta-Mena et al. [Motta-Mena et al., Nature Chemical Biology 10:196-202 (2014)] to derive the activation (τON) and deactivation (τOFF) time constants from the protein synthesis rate profiles.
Further, growth models were developed based upon monoculture data to predict the combined growth profiles and individual growth profiles of the cell populations within the co-cultures, seeded at different initial cell proportions and temperatures (data not shown). As an extension, a phenomenological model was developed (data not shown) to correlate the cell growth with the reporting fluorescence.
To facilitate the model development process, several ‘modules’ (consisting of promoter-rbs-GFP) were characterised using microplate reader and their time-series profiles were fed into the BioModel Selection System (BMSS) [Yeoh et al., ACS Synthetic Biology 8:1484-1497 (2019)] to identify an appropriate representative ODE model to be used for full model construction. These model parameters derived from the BMSS were estimated using a two-step optimization: differential evolution global optimizer followed by a constrained Nelder-Mead local optimizer (githubdotcom/EngBioNUS/BMSSlib). The same optimization technique was employed for parameter inference in most of the developed models including the models used in predicting the co-culture dynamics.
To better capture the uncertainties of model parameter estimates, Bayesian parameter inference method was also deployed to infer the probability distributions of some parameters estimates as opposed to the singleton estimated parameters values (data not shown). This inference approach is closely relevance to the Bayesian interpretation of probability, in which it depends on prior knowledge or beliefs, evidence, and likelihood to infer the posterior distributions of the parameter's estimates. The Metropolis-Hastings algorithm of the Markov chain Monte Carlo (MCMC) method was implemented in the parameterization process [Yildirim, Bayesian inference: Metropolis-hastings samplings (2012)], which enables one to sample from distribution without having to compute all the high dimensional integrals that demands huge computational efforts. A normal distribution is assumed for all the priors with half of the individual mean values were assigned to the individual standard deviations.
All data were shown as mean±S.D (n=3). All samples were prepared in technical triplicates; Statistical significance was determined by performing a two-sample unpaired t-test using Microsoft Excel (Microsoft, USA), a prior F-test was conducted to reveal equal variance or unequal variance of the samples in comparison. Blanking was included in each experiment whereby the auto-fluorescence reading of the medium was recorded. The GFP/OD600nm reading was calculated as fluorescence of (GFPsample−GFPblank)/OD600nm at each time point.
It was hypothesized that direct thermal control over the T7RNAP can be established (
The performance of the Thermal-T7RNAP(v1) system was studied using the thermal regulation assay. Within a biological relevant temperature range (30-42° C.) that is suitable for cell growth and for many biocatalytic processes, the thermal system exhibited a dynamic range of 31-fold between the permissive (30° C.) and restrictive (42° C.) states and displayed sharp thermal transition centred at 37.5° C. within a narrow functional range of 5° C. (
aThe Hill equation is given as
where Kinh indicates the maximum repression capacity; Km and n represent the half-activation temperature and the hill coefficient respectively.
In comparison, the fold-difference between 30° C. and 42° C. for the Split-T7 system (
The inventors also investigated the suitability of various lengths of the Tlpa's coiled-coil acting as fusion partners for the NT7 and CT7 protein fragments. The coiled-coil consists mainly of heptad repeats and separated into two distinctive coiled-coil regions: the longer N-terminal based 164-residues coil and the shorter C-terminal based 114-residues coil (
Next, when compared to Thermal-T7RNAP(v1), the full length T7-WT control exhibited 5 times lower maximal expression and no visible fold-difference (
In the process, the expression levels and the ratios were varied between the individual NT7-Tlpa coil and CT7-Tlpa coil proteins (
In general, the model predicted the fold-change of Thermal-T7RNAP(v3) fluorescence expression at 30° C. and 42° C. The relative expression levels of Thermal-T7RNAP(v3) and Thermal-T7RNAP(v1) (
The high expression levels of Thermal-T7RNAP(v3) at 30° C. could be due to better balance between the amount of NT7 and CT7 protein fragments as observed in the model (
Typically, in bioproduction, there is a need to induce the expression of the enzymes.
In part, the Thermal-T7RNAP system offers two key advantages: First, the decrease in temperature is already a conventional practice to enhance the product stability and yield [Qing et al., Nat Biotechnol 22:877-882 (2004)], and secondly, it offers a direct method of regulating enzyme expressions without the need of chemical inducers [Valdez-Cruz, N. A., et al., Microb Cell Fact, 9, 1-16 (2010)]. The Thermal-T7RNAP was utilised to thermally control the expression of two enzymes, feruloyl-CoA synthetase (fcs) and feruloyl-CoA hydratase (ech) located in the same operon involved in the vanillin bio-conversion pathway at 30° C. (
A key feature of an ideal thermal-switchable system is the ease of tuning the gene expression levels by means of adjustment to the temperature protocols. The dynamic study has two main objectives—firstly, to study whether temperature-controlled (re)activations of existing and de novo polymerases can generate reversible gene expression following different cooling and heating regimes and secondly, to leverage on the developed mechanistic model to offer quantitative insights into the inherent kinetics of the Thermal-T7RNAP.
The effects of administering a patterned 3-hour (h)-3 h-2 h-2 h OFF-ON (40-30° C.) thermal duty cycle was investigated, through examining the GFP expressions (
Next, the performance of Thermal-T7RNAP(v1) and Thermal-T7RNAP(v3) systems was tested when the temperatures were altered between 37° C. and 30° C. in the patterned 3 h-3 h-2 h-2 h thermal duty cycle (
The activation and deactivation kinetics of the systems were determined under the temperature cycling between permissible ON temperature (30° C.) and restrictive OFF temperature (40° C.). While in vitro measurements can directly probe into the coiling/uncoiling of alpha-helix structures [Naik, R. R., et al., Biosensors and Bioelectronics 16: 1051-1057 (2001)] in assisting split-polymerase reconstitution/sequestration but precise and reliable measurements of Thermal-T7RNAP initiating transcription are nonetheless undermined by reporter mRNA instability and slow reporter protein turnover [Motta-Mena, L. B., et al., Nature Chemical Biology 10: 196-202 (2014)]. As an alternative, a mathematical model from earlier work was adopted, which has been previously used to provide information of transcriptional kinetics based on activation/deactivation measurements [Motta-Mena, L. B., et al., Nature Chemical Biology 10: 196-202 (2014)], to obtain similar transcriptional kinetics information of the Thermal-T7RNAP systems during activation (30° C.) and deactivation phases (40° C.). From the model, under the specific temperature regime, the activation (τON) and deactivation (τOFF) time constants represent the respective time taken to attain half-activation of the maximum steady synthesis rate and attain exponential decay to 1/e of its initial rate, as derived from the synthesis rate profiles of the 3 h-3 h-2 h-2 h OFF-ON cycle (
The dynamic performance of Thermal-T7RNAP(v3) system was the examined under a temperature range commonly used in bioproduction (30-37° C.). The intent is to study the induction time response and the performance under different thermal duty cycles (
A mechanistic model was developed for Thermal-T7RNAP (v3) to gain quantitative insights of its thermal-switching behavior and enable prediction of the thermal performance under different thermal duty cycles. The inventors leveraged the earlier developed mechanistic model (not shown) in predicting the dynamic behaviour of the 3-hour OFF-ON cycle. Generally, when the system was switched to the ON state (
Using the synthesis rate profiles of 3-hour and 2-hour OFF-ON cycles, the τTON and τOFF time constants were also derived when temperatures alternated between 37° C. and 30° C. A least-squared error analysis [Motta-Mena et al., Nature Chem Bio 10:196-202 (2014)] was performed to examine the best combination of τON and τOFF time constants while cross-referencing with experimental data from both thermal cycles (data not shown). Under this set of temperature condition and time interval patterns, the analysis showed that the estimated time constants of Thermal-T7RNAP(v3) fall within a narrow range (τON:18-23 min and τOFF:14-29 min) with a small margin of error (2−3e−6).
Taken together, the results suggest that the Thermal-T7RNAP systems exhibited responsive and reversible ON/OFF kinetics following dynamic periods of cooling and heating.
Different microbial applications require unique temperature ranges to achieve optimal activities while maintaining the system's performance. To tune the performance of the Thermal-T7RNAP and create mutants with different thermal characteristics, the coiled-coil domain of the NT7 and CT7 fusion proteins was mutated (
Using the high throughput system, over 1700 colonies were screened and many exhibited constitutive expressions regardless of temperature change while others exhibited loss of expression (
In the analysis of the mutation sites within the CT7 protein fragments, due to the effects of random mutations, we observed the selected mutants (Mut 1 and Mut 2) contained varying truncated lengths of the coiled-coil domains which were attached to their respective split polymerase units (
Specifically, it was discovered that the X1 and X4 domains which are located at either ends of the coiled-coil were present in both the wild-type and the mutated fusion proteins. It is likely that these preserved regions are highly essential to allow proper folding and guidance of the split polymerase units. The further left shifting of the temperature transition (measured in Km) for Mut 1 can be likely accounted for by the short but prominent X5 domains that were highly repeated and interspersed throughout the coiled-coil region (
The ability of simultaneously thermally activating and inhibiting expressions in different cell populations can be a powerful tool in biotechnology. As a proof of concept, the heat-repressible Thermal-T7RNAP system was coupled to control GFP expression in one cell and the heat-inducible Tlpa system to control RFP expression in another cell (
The use of thermal biosensors in controlling cell distribution within microbial community is still not well undertaken. While existing study leveraged optimal temperature ranges to enable native microbial species to co-exist [(Krieger et al., Biotechnology and Bioengineering 118:1-12 (2021)], there was a lack of capability in active control of the individual population. To address the need, the inventors developed temperature-based genetic circuits which forms thermal logic to enable thermal modulation of the growth of two engineered E. coli populations within a co-culture (
Independent characterisation of the ThermalT7RNAP-SgrS and the Tlpa-SgrS systems was first conducted to study the growth inhibition profiles at different temperatures (
Next, both GFPThermalT7RNAP-SgrS and RFPTlpa-sgrS cells were co-cultured at similar initial ODs and mixed at equal proportion (G50%+R50%) to demonstrate that the proportion of the cells can be actively controlled using the thermal genetic circuits (i.e., the final cell distributions can be varied). The growth model developed from the monoculture was used to predict the individual growth profiles of the two cell populations within the co-culture. The growth of the two cell populations was presumed to be constrained by the shared carbon source. Their combined growth was thus computed as the sum of the individual cell populations. As evidenced, the predicted combined growth profiles of the co-culture (G50%+R50%) at different temperatures agreed well with the experimental results (
While fluorescence-reporting is commonly used to estimate the individual cell growths within a co-culture [Nikolic N. et al., BMC Microbiology 13:258 (2013)], it was observed that the correlations between the reporting fluorescence and OD values were different even for cells with similar genetic makeup. This indicates that the use of fluorescence reporting level to estimate the individual cell growths within a co-culture may not be a good representative metric. Hence, for better representation, the inventors derived the final cell distributions of the co-cultures under different temperatures from both the experimentally measured fluorescence and the model-predicted individual growth profiles (
To reveal changes in cell proportions over time, the OD ratios were computed through dividing the predicted ODs of the GFP cells with the RFP cells (
Next, the effect of various initial cell proportions in the final cell distributions within the co-cultures was determined (
To expand the tunability of thermal modulation in co-culture, the inventors leveraged the discovered Thermal-T7RNAP CT7 mutants (Mut 1 and Mut 2) to regulate the SgrS expression in the GFP cells (
In the original G50%+R50% co-culture, the GFPThermalT7RNAP-SgrS cells were cultured together with the RFPTlpa-SgrS cells at equal proportion. Results show that at 30° C., the GFPThermalT7RNAP-SgrS cells exhibited the desired growth inhibition as portrayed by the 63% decrease in fluorescence intensity from the GFP control cells (
Similarly, we predicted the growth profiles of the mutant co-cultures at different initial cell proportions (
In the thermogenetic toolkit, existing heat-repressible systems are limited in parts [(Hoynes-O'Connor A. et al., Nucleic Acids Res 43:6166-6179 (2015); Liang R. et al., J Microbiol Methods 68:497-506 (2007); Qing G. et al., Nat Biotechnol 22:877-882 (2004); Yang Z. et al., Nucleic Acids Res 47:e137 (2019)] and restricted to thermal sensing mechanisms which rely mainly on RNA or transcription factors; and face potential issues such as functioning at low temperatures (<30° C.) which are non-optimal for cell growth and bio-catalytic processes, have wide temperature transitions, suffer delays or complex in design. To address the limitations and provide an alternative mode of thermal regulation, the present invention provides novel heat-repressible Thermal-T7 RNA polymerase (Thermal-T7RNAP) systems by fusing Tlpa coiled-coil domain with split-T7RNAP to introduce direct temperature control into the polymerase. The Examples show that the polymerase activity is well repressed at high temperatures (>40° C.) and highly suggests the NT7 and CT7 protein fragments can be sequestered by the temperature-sensitive coiled-coil domain at high temperatures (
One unique feature of the Thermal-T7RNAP system is the ability to undergo sharp two-way thermal switching, which is likely attributed to the highly sensitive temperature-dependent coiling and uncoiling of the coiled-coil domain (
Krieger, A. G., Zhang, J., and Lin, X. N. (2021). Temperature regulation as a tool to program synthetic microbial community composition. Biotechnology and bioengineering 118, 1-12. https://doi.org/10.1002/bit.27662.
Piraner, D. I., Wu, Y., and Shapiro, M. G. (2019). Modular Thermal Control of Protein Dimerization. ACS Synthetic Biology 8, 2256-2262. 10.1021/acssynbio.9b00275.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202012000X | Dec 2020 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050744 | 12/1/2021 | WO |
