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1. GFP Secretion
Design: The first experiment carried out was to select a viable secretion peptide for our protein. The signal peptide mfa was chosen as it is a well-characterized tag in S.cerevisiae. The next step was to apply the functional mfa tag to GFP to test the functionality of the tag within our system.
Build: An mfa signal peptide ordered from IDT was inserted into pGT-H using Gibson assembly, alongside a 6xHis tagged yeGFP gene obtained from pTK-0127, forming pGmGFP-H. pGmGFP-H was transformed into BY4741 to test secretion of GFP mediated by mfa.
Test: pGmGFP-H was inoculated from glycerol stock into YPD-HygB and left overnight. The next day, the culture was induced by galactose and left overnight.
The next day, cells were spun down in a centrifuge at 4K rpm for 5 minutes, the supernatant was removed and the cells were resuspended in equivalent volume.
Figure 1: Image of the media supernatant, resuspended cells and blank control media under gel visualizer blue light, media and supernatant are clearly fluorescent compared to the control media.
The supernatant and resuspended cells had their fluorescence measured at Ex:485, Em:528, corresponding to the emission spectrum of GFP, and normalized against the blank media.
Figure 2: GFP absorbance of the supernatant and cells of both induced and uninduced pGmFaGFP-H (BY4741), after normalization to blank media. Induced cells secreted approximately 42% of the GFP, mean±standard deviation(n=3) while uninduced cells produced and secreted significantly less GFP than induced cells.
Learn: Figure 2 demonstrates that 42% of the GFP was secreted, meaning that while secretion was taking place, majority was retained in the cell. To confirm that the GFP was secreted into the medium, the supernatant was concentrated with a size exclusion filter, and an SDS-PAGE followed by western blot carried out to visualize the 6xHIS tag (Figure 3). Western blot demonstrated a band at around 27kDa, agreeing with the expected size of GFP.
Figure 3: Western blot of the media isolated from the supernatant of the cells, and incubated with mAb-6xHIS. The band at 27kDa corresponds to the expected molecular weight of GFP.
2. Lactoferrin Production
Design: Initially, lactoferrin was chosen as an effective biopesticide, as powdery mildew was identified as the primary pest of interest during dialogue with the stakeholders, and in many other countries, a solution of diluted milk is usually applied to hamper the growth of powdery mildew. Several studies have pinpointed the presence of the iron binding protein lactoferrin as the key compound responsible for it’s anti-fungal activity, . The next step was to apply the functional mFa tag to the lactoferrin to visualize and confirm protein production and secretion. To simulate the inducible nature of the end-goal system, GAL1 promoter would be used to control production of lactoferrin.
Build: Human lactoferrin tagged at the C-terminus with a 6xHis-tag given to us by Dr Ho Chun Loong was first cloned into the plasmid pGT-H using Gibson assembly, producing the plasmid pGhLF-H. Gibson assembly was then used to attach the mFa signal peptide to the N-terminus of the protein, producing the plasmid pGmLf-H. pGmLf-H was then transformed into BY4741 to test inducible secretion of lactoferrin.
Figure 4: Coomassie blue stain of protein precipitates. Samples were loaded in the following order: Ladder, 80 kDa positive control, pGmLf-H, WT BY4741 negative control.
Test: pGmLF-H (BY4741) was culture in YPD overnight, and then transferred to YPGR medium and left to grow for 3 days. Media was then isolated and concentrated with TCA precipitation, and a western blot run on the results.
Figure 5: Western blot stain using anti-His mAb conjugated with HRP. Samples were loaded in the following order: Ladder, pGmLf-H, 80 kDa positive control, WT BY4741 negative control.
Learn: Lactoferrin production in S. cerevisiae was not successful. No detectable amounts of lactoferrin were produced even with multiple optimization attempts. Due to the limited time available, we decided that it would not be prudent to continue attempting to produce recombinant lactoferrin. Instead, it was decided that the best course of action was to search for another suitable antimicrobial peptide candidate that had novel biopesticide applications and had literature to support the successful expression of the candidate in S. cerevisiae.
Failure to produce recombinant lactoferrin in S. cerevisiae.
Due to limited time, a decision was made to abandon lactoferrin production in favour of another suitable AMP that had literature supporting its expressability in S. cerevisiae whilst having novel biopesticide applications.
3. HBD Production
Design: Due to problems associated with expressing recombinant lactoferrin in S. cerevisiae, an alternative antimicrobial peptide(AMPs) was required as a candidate biopesticide. After extensive research into literature and consulting with companies exploring the use of AMPs for agriculture use, Beta-defensins were chosen. Vaciome, a Singaporean synbio startup focusing on AMPs for livestock protection, agreed that in theory, defensins could function as robust antifungals. Jonathan Bester, CEO of Vaciome, strongly believed this project had strong potential to impact the future of crop protection and generously donated to a Human Beta Defensin 2 (HBD2) plasmid optimized for P.pastoris.
Build: The HBD2 fragment was obtained by amplifying the HBD2 gene from the P. pastoris plasmid donated by Vaciome. Using Gibson Assembly, GFP in pGmGFP-H plasmid was replaced with the HBD2 gene, with a 6xHis-tag fused to the C-terminus. The new construct was named pGmHBD2-H, and was transformed into BY4741 to test the expression, secretion, and functionality of HBD2 via galactose induction.
Test: pGmHBD2-H was inoculated from glycerol stock in YPD-HygB and incubated overnight. Culture media was swapped to YPGR to initiate galactose induction. After 48 hours, the culture was spun down to isolate the media supernatant and the cell pellet. The media was concentrated by 100x and purified using centrifugal Ni-NTA columns. Cell pellet was washed and lysed, after which it was centrifuged, and the lysate supernatant was retained. SDS-Page was performed on the media, media concentrate, purified media, and media concentrate, as well as unpurified and His-tag purified cell lysates.
Figure 6: Coomasie blue staining of the media and cell lysate containing induced pGmHBD2-H. 8kDa band corresponding to size of human beta defensin 2 was observed in all lanes except the wildtype negative control.
Purified samples of both the cell lysate and the supernatant of the culture showed an 8kDa band corresponding to the size of human beta defensin 2 which was absent from the wild type control (Figure 6), demonstrating successful production of HBD2.
To test the functionality of the HBD2 produced, we performed a MIC assay using the agar diffusion method against E. coli to validate the HBD2 function with the MIC data from Vaciome.
Figure 7: MIC carried out with samples isolated from pGmHBD2-H. Top left: Flow through from His column purification, Top right: 1mg/ml Ampicillin positive control, Bottom left: Unconcentrated media, Bottom right: 100x concentrated media from His purification.
Learn: The total protein expression of HBD2 was subpar in comparison to the MIC data that Vaciome produced using HBD2 expressed from P. pastoris culture. A small zone of inhibition was produced only when we concentrated our media isolate by 100x, in contrast, Vaciome’s P. pastoris media isolate had enough HBD2 to produce a zone of inhibition when used neat. A possible explanation is that S. cerevisiae was not as efficient in expression and secretion of HBD2 as compared to P. pastoris.
Due to the time constraints of this competition, we were unable to shift our entire optogenetic circuitry into P. pastoris, however, in future work we hope to do so in order to further improve on our current system using the DBTL framework.
- Crisp, P., Wicks, T.J., Troup, G. et al. Mode of action of milk and whey in the control of grapevine powdery mildew. Australasian Plant Pathology 35, 487–493 (2006).
- Buziashvili, A., Cherednichenko, L., Kropyvko, S. et al. Obtaining Transgenic Potato Plants Expressing the Human Lactoferrin Gene and Analysis of Their Resistance to Phytopathogens. Cytol. Genet. 54, 179–188 (2020).
- Soyer F, Keman D, Eroğlu E, Türe H. Synergistic antimicrobial effects of activated lactoferrin and rosemary extract in vitro and potential application in meat storage. J Food Sci Technol 57(12),4395-4403(2020).
- CL Ho , IY Hwang , K Loh and MW Chang. Matrix-immobilized yeast for large-scale production of recombinant human lactoferrin, MedChemComm 6, 486-491(2015).
Design: Flocculation is encoded by the gene FLO1 found in the S.cerevisiae genome. Literature asserts that flocculation, while present in most brewing strains of yeast, is non-functional in lab strains. This difference stems from a defunct regulatory gene FLO8 rather than a non-functional Flo1 protein. Thus, the FLO1 gene was directly amplified from the genome and its expression studied under a well characterized, inducible promoter GAL1p.
Build: PCR was used to amplify the FLO1 gene, and Gibson assembly was used to replace lactoferrin in pGmLf-H, producing the plasmids pGFT-H. pGFT was then transformed into BY4741 to test inducible onset of flocculation.
Test: pGFT-H (BY4741) was inoculated in YPD-HygB overnight, and the next day culture was spun down and the media was replaced with either fresh YPD-HygB (Uninduced) or YPGR-HygB (Induced). Cultures were then left overnight to observe the qualitative effect of flocculation induction.
Figure 8: Galactose induced flocculation as observed in a flask (left) and with contents transferred to a falcon tube (right). YPGR indicates the strain with flocculation induced by growing the culture with galactose as the sole carbon source, while YPD culture indicates a strain that is grown with glucose as the sole carbon source, thus remaining uninduced.
Flocculation was successfully demonstrated (Figure 8), with pGFT-H (BY4741) cultured in a galactose medium showing clear separation into a solid phase consisting of cell pellets and a liquid supernatant phase, while pGFT-H (BY4741), the uninduced culture, remains mixed in a cloudy suspension.
To further examine the effect of flocculation cells that were induced for 3 hours were spread onto a slide and examined with microscopy.
Figure 9: Uninduced pGFT-H(BY4741)(A) and induced pGFT-H(BY4741)(B) under microscopy at 20X magnification.
As expected, when the FLO1 gene is expressed, cells will clump together in dense masses, which promotes sinking as well as aggregation into large, visible pellets.
The next step was to quantify the effect of flocculation. To achieve this, a modified version of a flocculation index(FI) was used, which measures the change in the OD of the supernatant in a flocculated culture directly after agitation, and 30 minutes after static incubation. This is meant to capture the rate of flocculation mediated sedimentation due to the aggregation of yeast cells. To measure the strength of flocculation relative to induction, the ratio of FIinduced to FIuninduced at a specified time point is then measured.
To adjust the protocol for use with the strong and rapid flocculation observed under the GAL1 promoter, the resolution was increased by measuring the OD of the supernatant just after 2 minutes of static incubation. FI measurements were taken every hour for 4 hours.
Figure 10: Plot of flocculation index against time for both strain induced with YPGR and strain culture in YPD. Flocculation fold at 4 hours is 3.33 times.Mean±standard deviation(n=3)
Figure 10 demonstrates that over time, the FI of induced pGFT-H (BY4741) increases whilst FI of uninduced pGFT-H (BY4741) remains the same, such at around 4 hours there is a 3.33 times difference between the induced and uninduced flocculating strain.
- Watari, J., Takata, Y., Ogawa, M., Sahara, H., Koshino, S., Onnela, M.-L., Airaksinen, U., Jaatinen, R., Penttilä, M. and Keränen, S. Molecular cloning and analysis of the yeast flocculation gene FLO1. Yeast, 10, 211-225(1994).
- Haoping, L., Cora, A.S and Gerald, R. F. Saccharomyces cerevisiae S288C Has a Mutation in FL08, a Gene Required for Filamentous Growth . GENETICS, 144(3),967-978(1996)
- Salinas, F., Rojas, V., Delgado, V., López, J., Agosin, E., Larrondo, L.F. Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast. mBio. 9(4):e00626-18(2018)
Blue Light Activation
Testing a preliminary single construct blue light system
Design: The blue light sensitive EL222 system used in yeast proposed by Benzinger and Mustafa was used as a preliminary test to construct a rudimentary light sensitive circuit. EL222 fused to a nuclear localization sequence and a VP16 activation domain dimerizes in blue light and localizes to the nucleus, binding to the C120 motif CATGGACTAAAGGCTA. To ensure minimal components for the system were functional, a low copy episomal plasmid containing constitutive expression of EL222, and a truncated CYC1 promoter with a single C120 site controlling a fluorescent protein was designed.
Build: A DNA fragment containing the C120-CYC promoter, as well as a constitutive expression cassette for NLS-VP16-EL222 was ordered from IDT, and Gibson assembly was used to assemble it into a plasmid with the mKO orange fluorescent protein terminated by LSC2 terminator, producing the plasmid pC120-mKO-EL-U. The plasmid was then transformed into BY4741 to test for EL222 mediated blue light induction of mKO.
Test: pC120-mKO-EL-U (BY4741) was compared to the wildtype BY4741 as a negative control. Cells were cultured overnight, and the next day two cultures inoculated in 25ml YNB-URA media to OD600~1.2, and then cultured in a shaking incubator at 30 degrees Celsius, 220rpm in either blue light or darkness, with wildtype BY4741 undergoing an identical protocol. After 6 hours, the cells were washed and the level of mKO fluorescence (Ex:515, Em:560) was measured and normalized to the OD of the culture to ascertain the level of mKO expression under the C120-CYC promoter.
Other than the absolute induction fold, it was also important to characterize the effect of dose-dependent activation as well as the possible metabolic burden that the circuit may impose. As such, the experiment was replicated with the same induction protocol, but instead of an endpoint measurement the OD600 and mKO expression was measured hourly to plot the expression and growth curve in darkness, 100% blue light or using a 50%, half-hour-on half hour-off cycle.
Figure 11: Normalized mKO fluorescence of the wildtype in dark and blue light, as well as pC120-mKO-EL-U(BY4741) in dark and blue light after 6 hours. Results shown are mean±standard deviation(n=3).
Figure 12: Normalized expression of BY4741 transformed with pC120-mKO-EL-U over time, in either darkness, blue light, or half an hour cycles of blue light and darkness. Results shown are the mean±standard deviation(n=3).
Figure 13: Growth curve of BY4741 transformed with pC120-mKO-EL-U over time, in either darkness, blue light, or half an hour cycles of blue light and darkness Results shown are the mean±standard deviation(n=3).
From Figure 12, it can be seen that the mKO expression of the system in blue light significantly diverges from the expression in darkness. In addition, cells exposed to a 50% light cycle, with half an hour in blue light and half an hour in darkness, showed moderate expression in between 100% dark or light, demonstrating the dose-dependent nature of the blue light induction. The growth curve of all 3 cultures shows minimal deviation, demonstrating that minimal burden is imposed on the cell due to blue light induction.
Learn: Figure 11 shows that when induced, pC120-mKO-EL-U (BY4741) shows high amounts of mKO expression compared to the uninduced culture and the wildtype strain. Thus, we can conclude that the EL222-system can be utilized to construct a light sensitive genetic circuit. However, even in the dark pC120-mKO-EL-U exhibits mKO expression significantly higher than the wildtype strain, implying that expression is highly leaky.
- Benzinger D, Khammash M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat Commun. 9(1):3521, (2018).
Blue Light Induced Flocculation
Comparing blue light induced and galactose induced flocculation
Design: With the functionality of the blue light circuit confirmed, the next step was to couple flocculation to this basic circuit to assess how well it would work within our system.
Build: Using Gibson assembly, the mKO gene in pC120-mKO-EL-U was replaced with the FLO1 gene from pGFT-H, creating pC120-Flo-EL-U. pC120-Flo-EL-U was then transformed into BY4741 to test blue light induced flocculation.
Test: Cells were cultured overnight, and the next day two cultures inoculated in 25ml YNB-URA media to OD600~1.2, and then cultured in a shaking incubator at 30 degrees Celsius, 220rpm in either blue light or darkness. The FI of the culture was measured in accordance to the earlier method, every hour for 4 hours.
Figure 14: Plot of flocculation index against time for both pC120-Flo-EL-U(BY4741) strain induced with light and strain culture in the dark. Flocculation fold at 4 hours is 1.37 times. Results shown are mean±standard deviation(n=3).
Learn: It is apparent that the leakiness of the blue light promoter results in a greatly reduced flocculation fold at the 4 hour mark (Figure 14), largely in part to an increased flocculating behaviour even in the uninduced culture kept in the dark. This is undesirable, as it indicates substantial amounts of Flo1 protein is being produced consititutively, which may compete with the secretory pathways needed to secrete HBD2.
Figure 15: Blue light induced flocculation of pC120-Flo-EL-U (BY4741) (Top right), and uninduced culture (Top left), galactose induced flocculation of pGFT-H (BY4741) (Bottom right), and uninduced culture (Bottom left), after 6 hours (Left side) and 24 hours (Right side)
Upon visual inspection (Figure 15), it was also noted that flocculation did continue to occur despite the flocculation index of the induced pC120-Flo-EL-U (BY4741) culture decreasing after 2 hours. At 6 hours, cell pellets could be observed in suspension and at 24 hours a large accumulated pellet was formed at the bottom. It was hypothesized while the Flocculation index could assess early stages of flocculation, beyond a certain point of flocculation, there would be minimal cells left in the medium. Thus, the initial OD600 of the supernatant would not capture completely flocculated cells that are constitutively sunk at the bottom of the culture, and thus the flocculation index may underestimate the rate of flocculation for cultures that flocculate very rapidly. A new protocol is thus needed to capture end-point data beyond this influx point.
It was also noted that while galactose induced flocculation was more extensive than blue light induced flocculation after 24 hours, blue light induced flocculation had a more rapid onset and produced visible pellets faster within the first 6 hours.
An alternative protocol was developed to capture the end point, after 24 hours of flocculation, cultures would be left to stand for 2 minutes, 15 minutes and 30 minutes, and the the OD600 of the supernatant was measured at each time point to capture the total flocculation at the end of the 24 hours. Afterwards the entire culture would be centrifuged, and the dry mass measured. The ratio of the OD600 to dry mass would then represent the proportion of total cells that remained unflocculated, while capturing the permanently sunked cells as part of the total cell count. We coined this as an ‘un-flocculated index’ to assess the extent of total cell mass that remains in suspension after flocculation, and should be more accurately correlated to the extent of flocculation relative to biomass.
Figure 16: Data of the OD600 of flocculated cultures over 30 minutes, cultures that were induced in either light or galactose medium sedimented faster than their uninduced counter parts consistently. Results shown are mean±standard deviation(n=3).
Figure 16 demonstrates the final sedimentation of the cultures after 24 hours of culturing. Both cultures with induced flocculation showed lower OD600 overall, but as with the initial flocculation index, it was difficult to accurate assess the strength of pGFT-H (BY4741) YPGR as most of the cells were completely flocculated, and thus the rate of flocculation was negligible,
Figure 17: Un-flocculated index, comparing induction driven flocculation in pGFT-H (BY4741) and pC120-Flo-EL222-U (BY4741) to the uninduced cultures. Results shown are mean±standard deviation(n=3)..
Our un-flocculated index (Figure 17) shows better correlation with the visual inspection of the flocculated cultures. The induced pGFT-H (BY4741) showed a much lower proportion of unflocculated cells compared to the uninduced culture, and pC120-Flo-EL222-U(BY4741) kept it light demonstrated less unflocculated cells compared to dark, but the cells in the dark still demonstrated highly leaky flocculation. It is also salient from Figure 16 and 17 that the blue light flocculation is not yet as extensive as the galactose induced flocculation.
While blue light flocculation was successfully achieved, we aimed to achieve tighter control and greater turnaround time, and thus set about improving the blue light EL222 system.
Improving the Blue Light System
Increasing Induction Fold, Decreasing Leakiness
Design: The blue light EL222 system was of insufficient quality to pragmatically support tight control of flocculation, and thus additional engineering was required beyond this basic system. According to the model developed for expression through the EL222 blue light system, excessive EL222, even in the dark, could dimerize spontaneously and result in increased leakiness. Hence, to reduce unwanted EL222, the copy number of the EL222 expression cassette was reduced by integrating it into the genome.
Build: The integrative plasmid EL222_integrate was developed by using Gibson assembly to insert the EL222 constitutive expression cassette into a plasmid designed to release an integrative cassette upon digestion with EcoRI. This cassette was designed to insert itself into YPRCd15, a region of non-coding DNA in BY4741 that was well characterized for genome integration and expression. pEL222_integrate was then digested and integrated into BY4741, forming the strain BY474B with EL222 expression stably integrated into the genome.
To test expression from this new strain, a plasmid devoid of EL222 had to be constructed. To this end the expression cassette containing C120-CYCp-mKO-LSC2t was cloned into pGmLfT-H using Gibson Assembly to form the plasmid pGLCM-H. pGLCM-H was then transformed into blue light backend-containing strain BY474B to test genome integrated EL222 expression.
Test: pGLCM-H (BY474B) was tested in an identical manner to previous inductions, with an overnight culture inoculated to OD600~1.2 in 25ml of YPD-HygB, and left in either blue light or darkness, and expression of mKO measured using fluorescence normalized to OD600. Both the absolute induction fold after 6 hours, expression over time and growth curves were measured.
The extent of flocculation is a function of time, and differences in flocculation trends were observable when comparing flocculation within 6 hours and 24 hours. To accurately compare the blue light system’s utility for controlling the FLO1 gene in comparison to the GAL1 promoter, both 6 hour and 24 hour timepoints were measured.
Figure 18: Normalized fluorescence of pGLCM-H(BY474B) over time, in darkness, 50% blue light duty cycle and 100% blue light duty cycle. 50% duty cycle was carried out in half an hour intervals. Results shown are mean±standard deviation(n=3).
Figure 19: OD600 of pGLCM-H(BY474B) over time, in darkness, 50% blue light duty cycle and 100% blue light duty cycle. 50% duty cycle was carried out in half an hour intervals. Results shown are mean±standard deviation(n=3).
Learn: In Figure 19 it can be seen how over time, expression levels in dark and blue light diverge significantly. Using a 50% duty cycle for blue light also showed reduced expression compared to the 100% blue light, demonstrating changes in the patterns of blue light illumination can be used to control the expression of pGLCM-H(BY474B). The OD600 across time showed that the strain that was exposed to 100% light and 50% light grew in a similar fashion to the strain growing in dark, indicating that blue light inducible expression in pGLCM-H(BY474B) has minimal impact on the cell’s metabolism and growth.
Figure 20: Induction of pC120-mKO-EL-U(BY4741) in blue light or darkness versus pGLCM-H(BY474B). Results shown are mean±standard deviation(n=3).
After measuring the induction fold, it was determined that this system had reduced leakiness compared to pC120-mKO-EL-U by about half, although the overall expression had been reduced by a similar factor (Figure 21). This correlates with the the prediction made by our developed model.
Test: To assess the impact of this reduction in total expression, it was decided to compare blue-light induced expression from pGLCM-H(BY474B) to the expression driven by galactose induction. In these experiments, BY4741 with a URA-selective plasmid containing GAL1 induced mKO was used as a positive control, cultured in YNB-URA with either glucose(uninduced) or galactose(induced) as a carbon source.
Figure 21: mKO expression of induced and uninduced GAL1p, and pGLCM(BY474B) after 6 hours. Results shown are mean±standard deviation(n=3).
Figure 22: mKO expression of induced and uninduced GAL1p, and pGLCM(BY474B) after 24 hours.Results shown are mean±standard deviation(n=3).
Learn: Figure 21 and 22 demonstrated that pGLCM-H (BY474B) had similar normalized expression of mKO to the galactose induced construct in the first 6 hours, but within 24 hours the galactose induced expression rapidly increased to more than 500x of the blue light induced expression. It was also noted that normalized expression of mKO from pGLCM-H (BY474B) was lower at 24 hours than after 6 hours. This indicates that protein degradation and cell growth had outpaced the production of protein from the C120-CYC promoter, resulting in a low yield over time, while the GAL1p controlled mKO expression was able to sustain and accumulate over the entire time period.
Thus, it was decided that further engineering was needed to increase the activity of the C120-CYC promoter, while at the same time, reducing leakiness.
- Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, Wang L, Shen Y, Luo Y, Zheng Y, Lin J, Chen GQ, Wu Q, Cai Y, Dai J. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 43(13):e88, (2015).
Dual Repression Modules
Blue Light Dual Repression Circuit
Design: First and foremost, the blue light model developed by our modelling teamshowed that the most efficient way to increase the activity of the promoter, was to increase the number of binding sites available for EL222. Hence, to increase the overall activation of the promoter, we planned to add additional C120 repeats upstream of the TATA box.
However, modelling for promoter activation kinetics also showed that simply increasing the activity of activation elements is likely to be accompanied by an increase in leakiness. Presented with the dilemma of increasing overall expression while preventing increased leakiness, an abstracted construct of a modular promoter was developed. This modular promoter would consist of a core promoter and an upstream activation module that would increase activity in the presence of blue light, similar to the construction of the C120-CYC basic promoter. However, this design includes an additional repression module, that is meant to be repressed in the in darkness to suppress leakiness. In the presence of blue light, this module should be de-repressed, allowing the activated promoter to function as per normal. Thus, it would require two repression modules, one to directly repress the promoter of interest, and a second blue light activated repressor to repress the primary repression module.
With this design, it was hoped that strong blue light activation modules with rapid activation kinetics could be developed without the pitfalls of increased leakiness
Build: To implement a blue light activated module, 2 additional C120 repeats were added upstream to the C120-CYC promoter using Golden Gate Assembly. The bacterial repressor protein LacI, which regulates the lac operon, has been show to be active in S.cerevisiae , and synthetic repressible promoters have been constructed by placing DNA-binding sequences for orthogonal repressor proteins downstream of the TATA box in native S.cerevisiae promoters. Thus for the primary repression module, the published lacO sequence was thus inserted 23 base pairs downstream of the TATA box of the CYC1 promoter, to avoid possible interference with the assembly of the pre-initiation complex. As the secondary repressor module was planned to be a trans-activating element, it was left as a black box, and testing on this isolated synthetic promoter was done first. The entire synthetic promoter was inserted into pGLCM-H to produce the plasmid pGL3CM-H. pGL3CM-H was transformed into strain BY474B for testing of the synthetic construct.
Test: Induction fold of pGL3CM-H (BY474B) was tested across 6 hours and 24 hours and compared to pGLCM-H (BY474B) and the galactose induced mKO. As with previous blue light systems, the expression and growth over time in the presence of 100% blue light, 50% blue light and darkness was plotted.
Figure 23: Induction of pGL3CM-H(BY474B) over 6 hours, in 100% blue light, 50% blue light duty cycles and darkness. 50% duty cycle was carried out in half an hour intervals. Results shown are mean±standard deviation(n=3).
Figure 24: OD600 of pGL3CM-H(BY474B) over 6 hours, in 100% blue light, 50% blue light duty cycles and darkness. 50% duty cycle was carried out in half an hour intervals. Results shown are mean±standard deviation(n=3).
Learn: Figure 24 demonstrates that the expression of mKO over time in different duty cycles in pGL3CM-H (BY474B) mirrors that of pGLCM-H (BY474B), expression in blue light steadily increases over time, whereas expression in the dark decreases over time, while expression in 50% blue light duty cycle shows a slower increase than 100% blue light, confirming that blue light induced expression from pGL3CM-H (BY474B) can be modulated by changing the dosage of light. It should also be noted that compared to the single repeat C120-CYC promoter in pGLCM-H (BY474B), the fluorescence level in darkness does not decrease as rapidly, remaining above the 50% light cycle until the third hour, which may correspond with increased leakiness. As with pGLCM-H (BY474B), growth over time is not significantly affected by blue light induction, and represents minimal metabolic burden of the induction system in pGL3CM-H (BY474B).
When compared to expression of pGLCM-H (BY474B) within 6 hours, as predicted, induction fold increased, but leakiness simultaneously increased(Figure 26). However, over 24 hours, while expression maintained better than pGLCM-H (BY474B), it had still decreased compared to induction at 6 hours, and hence was still not comparable to the amount produced by the galactose induced expression. Thus, further increase in expression needs to be engineered.
Figure 25: Induction fold of pGL3CM-H(BY474B) in response to 6 hours of blue light induction, compared to induction of pGLCM(BY474B), and 6 hours of galactose induced expression of mKO. Results shown are mean±standard deviation(n=3).
Figure 26: Induction fold of pGL3CM-H(BY474B) in response to 24 hours of blue light induction, compared to induction of pGLCM(BY474B), and 24 hours of galactose induced expression of mKO. Results shown are mean±standard deviation(n=3).
Design: To test the primary repression module of the synthetic promoter independent of the secondary, blue light inverter module, the protein LacI would be expressed constitutively to assess if it could reliably reduce the leakiness of the promoter.
Build: A yeast codon optimized LacI was synthesized from IDT, and Gibson Assembly was used to integrate the LacI fragment into a plasmid containing the constitutive S.cerevisiae promoter PGK1p and the terminator RPP2Bt, with a URA selection marker and a low copy CEN/ARS, forming pConLac. pConLac was co-transformed into pGL3CM-H (BY474B) and selected for with both uracil auxotrophy and hygromycin, forming the strain pGL3CM-H/pConLac (BY474B).
Test: Blue light induction protocol was tested on pGL3CM-H/pConLac (BY474B), IPTG was used to temporarily simulate the derepression module, however, anticipating that derepression of the promoter may require additional time, circuit was tested over a period of 48 hours, with measurements taken at 4, 24 and 48 hours.
Figure 27: pGL3CM-H/pConLac (BY474B) cultured in darkness, blue light, darkness with 5mM IPTG and blue light with 5mM IPTG for 4 hours, 24 hours and 48 hours. Results shown are mean±standard deviation(n=3).
Learn: Figure 28 demonstrates that when LacI is constitutively active, it effectively represses the synthetic promoter 3C120-CYC-LacO in both light and darkness. When LacI was disabled with 5mM IPTG, blue light exposure activated the circuit, whereas when IPTG was added without blue light, moderate amounts of expression was detected. This confirms that LacI repressed leaky activity of the synthetic promoter that would otherwise be present without blue light induction.
Design: Secondary repression module had to invert a blue light signal to repress an otherwise active promoter, such that it could be used to suppress LacI expression in blue light. Similar to the design of the synthetic promoter, the region downstream of the TATA box in PGK1p was designated as a repression zone(Figure 29) and several iterations of a repressible promoter was attempted.
Figure 28: Abstracted repressor plasmid, with a repressor module inserted within the PGK1p promoter downstream of the TATA box, controlling expression of the fluorescent protein mTurquoise.
In E.coli, blue light repressible promoters have been constructed by using a C120 binding site in close proxitimity to the TATA box and expressing EL222 constitutively. In blue light, EL222 dimerizes and binds to the C120 site, sterically inhibiting the assembly of the pre-initiation complex. The same approach was used here, where 3 C120 sites were inserted downstream of the TATA box.
Build: 3xC120 motif was PCR amplified from pGL3CM-H and inserted into the repressor plasmid backbone downstream of the TATA box of a PGK1 promoter controlling the expression of mTurquoise using Gibson Assembly, forming pRepress-1. pRepress-1 was transformed into BY474B to form the strain pRepress-1 (BY474B), as well as BY4741 to form the strain pRepress (BY4741).
Test: pRepress-1 (BY474B) was cultured in light and dark for 24 hours, while a strain constitutively expressing mTurquoise under an unaltered PGK1p promoter was cultured in darkness for 24 hours.
Figure 29: RFU of pRepress-1 (BY474B) in light and darkness compared to pRepress-1 transformed into BY4741, and a constitutive, unaltered PGK1 promoter. Results shown are mean±standard deviation(n=3).
Learn: There was minimal difference between the florescence of the illuminated and dark cultures, while the unaltered promoter showed high levels of expression. Two possible explanations exist, which is the unspecific binding of EL222 in darkness permanently represses the promoter, or the insertion of the sequence itself had disrupted a key site in the promoter.
To verify why the promoter did not show activity in dark as expected, the pRepress-1 plasmid was transformed into BY4741 and tested, where it also showed minimal activity. Since BY4741 does not contain the EL222 gene, it could be confirmed that the lack of activity was a result of alterations made specifically to the promoter and not due to EL222 binding. It was thus hypothesized that the fragment inserted, which was 100bp large, resulted in too large a distance between the TATA box and the transcription start site, resulting in minimal transcription of the desired protein.
Design: Thus, the next iteration of the system aimed to add a smaller repression sequence. As success was experienced inserting a single lacO site downstream of the C120-CYC promoter, the 3xC120 repression sites were reduced to a single site downstream of the PGK1 promoter TATA box.
Build: Overlap PCR and Gibson assembly were used to insert a single C120 site 25 base pairs downstream of the TATA box of the PGK1 promoter controlling mTurquoise, producing the plasmid pRepress-2. pRepress-2 was transformed into BY474B to form the strain pRepress-2 (BY474B).
Test: pRepress-2 was cultured in darkness and blue light for 24 hours, and the fluorescence normalized to OD600.
Figure 30: RFU for the pRepress-2(BY474B) with and without blue light compared to constitutive expression of mTurquoise under unedited PGK1 promoter. Results shown are mean±standard deviation(n=3).
Learn: pRepress-2 did not show any differential expression in darkness or light, however, it did show comparable expression to the constitutive promoter, indicating that the insertion of 20bp at the chosen site did not disable the activity of the promoter, and thus moving forward, this site will be used for the insertion of repression sites.
Design: Designs were remade for a third iteration of the blue light repression circuit. Because directly using EL222 to hinder the promoter did not work, an inverter circuit was constructed such that repression of the promoter was reliant on EL222 driven expression. To achieve this, a TetO site was inserted into the identified repression site 25 base pairs downstream of the TATA box in the PGK1 promoter, and a secondary cassette containing the part BBa_C0040 coding for a tetR protein under the control of the initial C120-CYC promoter was conjugated to it. It was hypothesized that under blue light exposure, C120-CYC would activate the production of TetR, which would then repress the PGK1 promoter.
Build: Overlap PCR was used to assemble the expression cassette C120-CYC-TetR-ADHt, and Gibson assembly was used to add this cassette alongside the TetO sequence into the blank repression plasmid, forming the plasmid pTetRepress. pTetRepress was transformed into the strain BY474B for testing.
Test: pTetRepress (BY474B) was cultured in darkness and blue light for 24 hours, and the fluorescence normalized to OD600.
Figure 31: Expression of mKO by pTetRepress(BY474B) cultured in darkness and blue light compared to the constitutive, unedited PGK1p promoter. Results shown are mean±standard deviation(n=3).
Learn: Unfortunately, the third iteration of the secondary repressor module was still non-functional. Although it retained it’s baseline expression, exposure to light did not result in the expect reduction in expression. Further testing on the PGK1-tetO promoter, and the C120-CYC driven expression of tetR will be needed to fully troubleshoot this system.
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- Jayaraman, P., Devarajan, K., Chua, T. K., Zhang, H., Gunawan, E., & Poh, C. L. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic acids research, 2016, 44(14):6994–7005.
EL222 Driver Circuit
Blue light Driver Circuit
Design: As discovered when testing pGL3CM-H (BY474B), expression has to be driven up further. Before considering additional genetic measures, it was hypothesized that the colour of the media could in fact affect optogenetic expression, as currently pGL3CM-H (BY474B) is cultured in YPD-HygB, which has an orange tint to it. As orange is a contrary colour to blue, blue light may not penetrate the culture as well. As such, the 3xC120-CYC-LacO-mKO cassette was first shifted to a plasmid that relied on uracil auxotrophy to test the possible effect of medium on blue light induction.
Build: Using Gibson assembly, the 3xC120-CYC-LacO-mKO cassette was amplified and premptively inserted into a plasmid containing the eventual red light sensitive promoter, producing the plasmid pRL3CM-U. pRL3CM-U was transformed into BY474B to form the strain pRL3CM-U(BY474B)
Test: Blue light induction protocol was carried out on pRL3CM-U(BY474B) cultured in YNB-URA, compared against pGL3CM-H(BY474B) cultured in YPD-HygB for 6 hours. As a precaution for future red light integration, the possible cross talk with red light was also tested by following an identical protocol for blue light induction but replacing blue light with red light.
Figure 32: Induction fold of pGL3CM(BY474B) when cultured in YPD-HygB compared to induction fold of pRL3CM(BY474B) in YNB-URA for 6 hours. Results shown are mean±standard deviation(n=3).
Figure 33: pRL3CM-U(BY474B) illuminated in blue light, red light and darkness. Results shown are mean±standard deviation(n=3).
Learn: Culturing pRL3CM-U (BY474B) in YNB-URA demonstrated around 3 times higher overall expression in both darkness and light compared to pGL3CM(BY474B) culture in YPD-HygB (Figure 33). Since the promoter and expression cassette in these two plasmids are identical, it can be reasoned that the likely contributing factor for this is the switch in medium. As such, going forward, auxotrophy plasmids will be used for light induced constructs as YNB media may allow for more comprehensive penetrance. It was also ascertained that there was no cross-talk from red light signals, as pRL3CM-U cultured in red light showed minimal difference from culture kept in the dark.
Design: To further enhance the activity of the C120-CYC-LacO promoter, the expression of EL222 was examined. Although in our models, it was assumed that the concentration of EL222 is constant, our modelling team relayed that this need not be the case. As such, a feedforward driver circuit was proposed, such that blue light induction increases the expression of EL222. Due to the positive feedback, this increment is greater than a linear increase, and results in increasing expression of EL222 and promoter activity.
In this circuit, in addition to constitutively expressed EL222 construct powered by native yeast transcriptional machinery, a secondary construct an identical EL222 ORF under the control of the 3C120-CYC-LacO promoter would be integrated into the genome. Thus, in the presence of blue light, increasing amounts of EL222 would be produced in a positive feedback loop, until blue light is removed and produced EL222 undimerizes. This was hypothesized to aid expression by 1. Increasing the overall concentration of EL222 to activate the 3C120-CYC-LacO promoter only in the presence of blue light and 2. Insulating the production of EL222 from fluctuations in the native metabolism of the cell. In the initial circuit, EL222 was produced by constitutive promoter ACTp, however, under certain conditions, the cell may choose to divert it’s metabolic flux away from the Act gene, which will inadvertently affect EL222 expression. By placing EL222 in an auto-regulated loop, it ensures that expression will not be directly hindered by cell metabolism patterns.
Build: Using Gibson assembly, EL222 was duplicated in the pEL222_integrate plasmid, and inserted under the control of the 3C120-CYC-LacO promoter to form the plasmid pDriver_integrate. pDriver_integrate was then digested with EcoRI, and integrated into the genome of BY4741 to form the strain BY474D. pRL3CM-U was then transformed into BY474D to test the effects of the driver circuit, forming the strain pRL3CM-U (BY474D)
Test: Blue light induction protocol was carried out on pRL3CM-U (BY474D) and pRL3CM-U (BY474B) to ascertain the effect that the driver circuit had in comparison to the standard constitutive expression of EL222. As a negative control, pRL3CM-U was transformed into BY4741, thus had no constitutive expression of EL222.
Figure 34: Effect of a backend positive feedback loop for EL222 on expression of pRL3CM-H. Cells with either no EL222 expression (BY4741), standard EL222 expression (BY474B) or the feed forward driver loop(BY474D) were transformed with pRL3CM-H and their fluorescence over 24 hours measured. Results shown are mean±standard deviation(n=3).
Learn: Figure 34 demonstrates that pRL3CM-H showed much higher expression when expressed alongside the blue light induced driver circuit compared to a standard EL222 expression cassette or no EL222 expression within the first 6 hours, being able to express more than double the amount, without a corresponding fold increase in leakiness. In addition, greater expression was maintained over 24 hours compared standard EL222 circuit, while maintaining similar leakiness to pRL3CM-H transformed with the wildtype genome. It should be noted that pRL3CM-H (BY4741) still demonstrated some level of leakage over 24 hours, confirming that the synthetic promoter itself was leaky independent of unspecific induction from EL222.
Design: To test out the functionality of the driver circuit, a construct with 3C120-CYC-LacO controlling the FLO1 gene was designed. Anticipating the need to test integration, this plasmid was also designed to include the HBD2 gene under the control of the GAL1 promoter.
Build: Using Gibson Assembly, FLO1 from pGFT-H and GAL1-mfa-HBD2 from pGmFaHBD-H were integrated into the pRL3CM-H plasmid, forming the plasmid pGH3CF-U. pGH3CF-U was then transformed into BY474D to form the strain pGH3CF-U (BY474D).
Test: Blue light induced flocculation protocol was replicated for pRL3CM(BY474D), and the reduction in OD600 after 24 hours of flocculation as well as the unflocculation index plotted.
Figure 35: Change in OD600 of supernatant of static pGH3CF-U(BY474D) culture after 24 hour in either dark or blue light, compared to pC120-Flo-EL222-U(BY4741). Results shown are mean±standard deviation(n=3).
Figure 36: Unflocculation index of pGH3CF-U(BY474D) compared to pC120-Flo-EL222 (BY4741) and pGFT-H (BY4741). Results shown are mean±standard deviation(n=3).
Learn: From Figure 35 it can be seen that utilizing the driver circuit and the new 3C120-CYC-LacO promoter, the OD600 of the supernatant drops much more rapidly after static incubation, and the overall unflocculated cell mass has been reduced according to the Unflocculation index (Figure 36).
Figure 37: Images taken of pRL3CF-U (BY474D) after 24 hours of either culturing in the dark or light. Cells that were cultured in the light form a visible pellet at the bottom of the tube.
Design: In order to implement a biosafety layer into the construct, a kill switch needed to be developed. Part BBa_K1159105 was chosen as a desirable kill switch as it functioned as an endonuclease, and thus not only prevented live organisms from escaping the bioreactor, but also would destroy modified DNA, reducing the chance of horizontal gene transfer.
Build: Sequence for BBa_K1159105 was ordered from IDT, and using Gibson Assembly it was inserted into the pGmFaHBD-H plasmid to form pGNucA-H. pGNucA-H was transformed into BY4741 forming the strain pGNucA-H (BY4741).
Test: pGNucA-H (BY4741) was cultured in YPD-HygB for 24 hours, and a CFU assay was carried out 5 serial dilutions to a cell count of 10-5 and then plated on either YPD-HygB agar as a negative control, as well as YPGR-H to induce the production of NucA.
Learn: Culture that was induced showed only a 20% decrease in cell mortality, and it was decided that the part needed to be improved upon.
Design: In order to improve the efficacy of the nuclease, a NLS sequence was added to the N-terminus of the part, as this was expected to increase the amount of NucA that is translocated to the nucleus to digest the genome and DNA, which in turn will increase the mortality rate of the cell culture.
Build: Gibson Assemble was used to insert a 7 amino acid NLS sequence at the N-terminus of pGNucA-H forming the plasmid pGNLSNucA-H. pGNLSNucA-H was transfomed into BY4741 forming the strain pGNLSNucA-H (BY4741).
Test: pGNLSNucA-H (BY4741) was in YPD-HygB for 24 hours, and a CFU assay was carried out 5 serial dilutions to a cell count of 10-5 and then plated on either YPD-HygB agar as a negative control, as well as YPGR-H to induce the production of NucA.
Learn: By adding the NLS, the overall mortality rate of the cells with nuclease induced increased as assessed from the CFU assay, indicated by a greater decrease in the colonies observed growing from the induced to uninduced cultures.
Figure 38: CFU assay results from pGNucA-H(BY4741) and pGNLSNucA-H(BY4741), ratio of colonies counted from plates with the nuclease induced to the colonies counted from the plates with nuclease uninduced. Nuclease with the NLS attached reduces the total number of live colonies on the plate. Results shown are mean±standard deviation(n=2).
Red Light Induction
Design: For the red light induction, the PhiReX system reported in literature was chosen as a red light induction system that was demonstrated in S.cerevisiae. Plasmids for the PhiReX system were ordered from addgene, however, an issue encountered was that two integrative plasmids were required to fully implement the PhiReX system (pRL_GI_PCB28, Figure 39, pRL_CT_del1, Figure 40). However, both these plasmids were designed to integrate into the auxotrophic markers of the YPH500 strain of S.cerevisiae. Our team only had access to the BY4741 strain, which lacked the required auxotrophic markers for integration.
Figure 39: pRL_GI_PCB28, designed to integrate into the truncated HIS gene in YPH500
Figure 40: pRL_CT_GI_del1, designed to integrate into the truncated URA gene in YPH500
Build: To remedy this issue, our first solution was to use Gibson Assembly to move the relevant pathways to a different integrative plasmid that was compatible with BY4741, which was successfully achieved with pRL_GI_PCB28, yielding the plasmid pH_integrate (Figure 41)
Figure 41: pPH_Integrate, for porting expression of HY1 and PcyA to a BY4741 compatible plasmid
pPH_integrate was successfully integrated into the BY4741 genome and selected for with URA auxotrophy. However, genes in pRL_CT_GI_del1 could not be amplified with PCR. Upon discussing with the author of the system, Dr. Lena Hocherein, we determined that the SynTALE construct interfered with PCR as it contained many tandem repeats. Thus, a different strategy had to be adopted to integrate the system on the pRL_CT_GI_del1, that did not rely on editing the plasmid itself.
Recognizing that URA markers could be counter selected for with FOA medium, and that pPH_integrate was successfully integrated, a method was devised to artificially insert the integration site for pRL_CT_GI_del1 into the genome.
Figure 42: Artificially inserting integration site for pRL_CT_GI_del1 to integrate into the genome, using counter-selection for the URA marker in pPH_integrate
A gene fragment was ordered from IDT containing sequences flanking the URA marker used to select for successful integration of pPH_integrate. This fragment internally contained the left and right flanking sequences of pRL_CT_GI_del1 linearised with the restriction enzyme PmeI. BY4741 with pPH_integrate integrated were transformed with the new fragment, which integrated into the URA marker present in pPH_integrate. This would introduce the correct integration site for a double crossover with a linearized pRL_CT_GI_del1, and was selected for with FOA medium to select for the loss of the URA marker (Figure 42).
Figure 43: Linearized pRL_CT_GI_del1 integrating into edited pPH_integrate site in the genome
pRL_CT_GI_del1 was linearized, and transformed into BY4741 with the edited pPH_integrate site, and successfully selected for using the original LEU3 marker on the plasmid (Figure 43), forming the strain BY474R. Genome integration was confirmed by genome isolation and PCR verification.
Finally, the plasmid pRL_yeGFP, also provided by the PhiReX paper, containing a GFP gene under the control of a promoter activated by their red light systems, was transformed into the strain BY474R.
Test: Red light induction was carried out according to specifications in the PhiRex paper, cells were cultured in selective YNB-URA medium in darkness for 24 hours, and then exposed to far red light and cultured for another 6 hours, before cells were either exposed to pulses of red light for another 16 hours or kept in darkness to suppress induction. As a negative control, BY4741 without a plasmid and BY4741 transformed with pRL_yeGFP were also cultured and underwent an identical protocol.
Figure 44: Induction of RL_yeGFP(BY474R), wildtype BY4741 and BY4741-RL_yeGFP in response to red light and darkness. Results shown are mean±standard deviation(n=3).
Learn: Red light did not seem to have any significant induction fold on the system RL_yeGFP (BY474R). As the wildtype transformed with RL_yeGFP showed minimal expression, it was deduced that it was not a result of the plasmid leakiness itself. Rather, the transcription factors integrated into the genome were constitutively activating the plasmid.
Unfortunately, there was insufficient time to complete the engineering of the red light transcription factors.
- Hochrein, Lena et al. “PhiReX: a programmable and red light-regulated protein expression switch for yeast.” Nucleic acids research, 2017, 45(15): 9193-9205.