Team:Duesseldorf/Demonstrate

Demonstrate

logo Growthyeast title= Selfregulation title= Lysine title= Sucrose title=

Self regulating Escherichia coli

In this part of the project, the aim was to regulate cell growth of E. coli BL21(DE3)C43 cells. We wanted to accomplish this by expressing a lysis gene under the control of the quorum sensing promoter Plux. To test whether the cell growth was impaired by the presence of a lysis protein, different experiments were conducted.
E. coli cells harboring different plasmids, the lysis plasmid and the different controls, were incubated overnight at 37 °C, 220 rpm. On the following day, the cells were diluted to a final OD600 of 0.1. 200 µl of these dilutions were distributed on a 96-well plate for plate reader measurements. Each sample was measured in technical triplicates during all experiments. In a final approach, cells harboring the luxI-luxR-Plux -phiX174E lysis construct were grown in LB-medium until an OD600 of two. Culture supernatant was harvested, sterile filtered and then mixed 1:1 with pure LB medium.

Results

To check the functionality of the Plux promoter upon induction by the quorum sensing molecule acyl homoserine lactone (AHL) and the respective transcriptional activator protein LuxR, an experiment based on the expression of gfp under the control of the respective quorum sensing promoter was performed (Figure 1). It can be seen that GFP fluorescence increases over time, while the untreated cells show low or no fluorescence at all. This observation led us to the conclusion that the promoter Plux is induced upon synthesis of AHL by the LuxI synthase and subsequent binding to the LuxR regulator. Therefore, this promoter is characterized as functional for further use within our project, as well as within the iGEM registry.
In order to exclude that the lack of fluorescence of the progenitor cells is due to poor growth, a measurement of the optical density was included (Figure 1). As expected, the cells grow similar, therefore it can be concluded that the lack of fluorescence of the progenitor cells is not due to a smaller population size.

The performance of the Plux promoter is further confirmed by fluorescence microscopy (Figure 1). Here it can be seen that fluorescence is present in the case of activation of the Plux promoter by the quorum sensing system (Figure 1A), while no fluorescence is present in the corresponding untreated cells (Figure 1C). As a positive control, GFP production under the control of a constitutive promoter was checked (Figure 1B). When comparing the expression of gfp under the control of different promoters (Plux and constitutive promoter J23102), higher fluorescence could be visualized in the latter case.

Figure 1: Confocal fluorescence microscopy of A: E. coli cells with luxI-luxR-Plux-gfp construct; B: E. coli cells with constitutive expression of gfp and C: E. coli BL21(DE3)C43 cells.

Next, the efficiency of the lysis plasmid containing the lysis gene E from bacteriophage phiX174 was tested (Figure 2). This was done by measuring the optical density of the cells containing the lysis plasmid. Progenitor E. coli cells and cells harboring an empty vector control were included as negative controls. The latter was done to rule out a higher cell density of the progenitor strain than the other cells due to the lack of antibiotics in the culture medium. It is noticeable that growth of the cells is influenced by the presence of the lysis plasmid. This is especially observable during the stationary phase, starting after 10 hours, as the cells do not reach the same maximum density as the wild type cells or the cells harboring the uninduced plasmid (luxR-Plux-gfp).

Figure 2: Effect of lysis plasmid in E. coli cells compared to progenitor E. coli cells. As a control, the progenitor strain (E. coli BL21(DE3)C43) as well as another strain carrying the luxR-Plux-gfp construct was used. The different colors represent different E. coli strains. Measurements were performed over a time period of 24 h. Mean values of triplicates subtracted from the respective blank are plotted.

Conclusion

The aim of this subproject was to construct a strain that grows slower than its progenitor counterpart. We consider this step to be essential in establishing the first part of our co-culture. Our priority here lies on us being able to make the co-culture populations achieve balance. We therefore applied the quorum sensing system, which is a reliable and well characterized tool of synthetic biology2 and wanted to accomplish this by choosing a lysis gene able to inhibit cell wall synthesis. As compared to the constitutive expression of gfp, a lower fluorescence intensity is visible (Figure 1A/B). This might be due to the smaller amounts of AHL synthetized by LuxI, which might lead to more limited activation of the Plux or, more likely, different promoter strengths. In this case, lysis protein E is used which is able to interact with the host's SlyD and therefore enables protein E to be protected from proteolysis. This way, protein E is able to interact with MraY (an essential membrane enzyme for bacterial cell wall synthesis) at the host membrane, which blocks MraY translocase, necessary for lipid I catalysis. The latter is an essential component for host cell wall synthesis3.
As suggested by Dr. Spencer Scott, an expert for quorum sensing regulation, whom we asked for advice as Integrated Human Practices, we let the cells grow for only 24 hours. We showed efficacy of our system by demonstrating that growth, based on OD600 measurements, is decreased in cells harbouring the lysis plasmid. Based on literature, lysis occurs because the lysis protein E impairs peptidoglycan synthesis4. This is consistent with our finding, since cell lysis results in less cells, shown by a lower OD600 measurement. Longer measurements resulted in lower OD in the control strains as well, likely due to cell death from starvation, which often happens in older cultures. Moreover, as was described in literature, lysis occurs only when the medium reaches a certain concentration threshold of the quorum sensing molecule AHL, probably during the stationary phase and induces a lysis event1. Also, similar to what Scott and colleagues discovered, population reduction is observable at around ten hours of growth1.

Lysine Supply by E. coli

In our auxotrophy system the aim was to establish an auxotrophic dependency of S. cerevisiae BY4742 (S. cerevisiae in the following) and E. coli BL21(DE3)C43 harboring lysC for lysine overproduction (E. coli_lysC in the following). In order to analyze the growth of E. coli and S. cerevisiae in mono- and co-culture, we performed a growth experiment over 36 hours. For all experiments, M2 medium, supplemented with 1.5% glucose, was used. To show the functionality of our approach, E. coli expressing a codon optimized and feedback resistant version of lysC under the control of a constitutively active promoter Bba_J23100, was cultivated together with S. cerevisiae which is auxotrophic for uracil, histidine, leucine and lysine.
Both strains were cultivated in monocultures to observe their growth with the four additional amino acids as a control. In our second approach, both were cultivated in a co-culture without lysine. Furthermore, the medium of the E. coli_lysC strain was sterilized and reused to cultivate S. cerevisiae to observe growth behavior in medium containing lysine produced by E. coli . In this approach, three amino acids, but no lysine, were added.

Figure 1: Growth of S. cerevisiae in reused M2 cultivation medium of E. coli_lysC. Comparison of S. cerevisiae growth rates in the positive control with four additional amino acids (Standard Medium) and reused M2 cultivation medium of E. coli_lysC over 36 h. In the other culture the supernatant of E. coli_lysC cultures was used and no lysine was added (Supernatant of E. coli). The y-axis shows the CFU in 100 µl.

Figure 1 shows the growth of S. cerevisiae monocultures. Here, the growth of the positive control and the experiment with the reused medium, described in the experimental design, are compared. The results show that it was possible to observe growth of S. cerevisiae in the medium which was lysine-enriched by E. coli_lysC. After around 4 hours, the cells in both cultures began to grow. After 12 hours, cell density reached its maximum in both cultures while the total density was higher in the experiment with the reused medium. After the highest amount of cells was reached, the cell density decreased faster in the medium experiment compared to the control. After 36 hours the cells reached a similar cell density. These results show that the lysine produced by E. coli_lysC, is able to allow S. cerevisiae to grow as well as it would in lysine containing medium. This can also be seen as a proof of principle for synthetic dependencies based on amino acid auxotrophies in general.

Conclusion

With the experiment using the reused medium enriched with lysine by E. coli_lysC, we could show that our approach with the auxotrophy system is very promising. Auxotrophy based dependencies between organisms are also used in recent research5. It would be interesting to improve our system by trying to cultivate the cultures under different conditions like inoculating the different organisms at lower cell density. One other approach and the next step for our desired, working co-culture would be the growth inhibition of E. coli using our self regulating system. This could prevent the E. coli cells from overgrowing the slower growing organisms.
We were able to show that the lysine produced by E. coli_lysC was sufficient to let S. cerevisiae grow in the lysine enriched medium. At the end of the measurement the cell density was at the same low level in both cultures. It would be interesting to observe whether M2 medium pre-incubated with E. coli_lysC for a longer period of time would lead to a higher growth rate or to a slower decrease of the cell density of S. cerevisiae.

Sucrose Production of S. elongatus

To detect the amount of sucrose production and secretion of our carbon source provider S. elongatus in the co-culture, we performed a biochemical assay using the YSI 2950 Biochemistry analyzer6. This method relies on the detection of sucrose mediated by a voltage change by immobilized enzymes on a specific membrane.
For this purpose, we inoculated the S. elongatus PCC 7942 cscB:::NS3 strain at an OD750 of 0.3 and incubated it under standard conditions for two days. After two days, the culture was induced with 1 mM IPTG and 150 mM NaCl. The first samples were taken before induction, then after two days, four days and six days. As a control, wild type S. elongatus was treated in the same manner and samples were taken at the same time points, respectively. To measure sucrose content, each sample was centrifuged at 4000 rpm for three minutes and the supernatant was sterile filtered for analysis.
A sucrose standard curve ranging from 1 mg/mL to 0.0625 mg/mL (1:2 dilutions each) was prepared in order to determine the final sucrose concentration in the tested samples. It can be seen that the amount of sucrose increases after induction. The media of wild type S. elongatus before induction does not show any presence of sucrose, while small amounts of sucrose are detectable before the induction of S. elongatus PCC 7942 cscB:::NS3.

Figure 1: Sucrose content in medium supernatant of S. elongatus and PCC 7942 cscB:::NS3 strain in days (d) post induction as well as pre induction with 150 mM NaCl and 1 mM IPTG. Values are calculated from triplicates and measured based on a sucrose standard curve.

Conclusion

We could clearly demonstrate that upon induction with NaCl and IPTG, the sucrose content is higher than in the uninduced variant of the S. elongatus PCC 7942 cscB:::NS3 strain. Moreover, it could be shown that the wild type, as expected, does not show any presence of sucrose in the corresponding medium. This phenomenon can be explained by the heterologous sucrose transporter CscB in the S. elongatus PCC 7942 cscB:::NS3 strain7. Upon induction, sucrose export is stimulated in the cells harboring this transporter. Wild type cells, which lack the transporter, cannot secrete sucrose in this manner. As a consequence, no sucrose is detectable in the medium. The amounts of sucrose secreted are comparable to literature values and even though the CscB transporter is inducible, low amounts of sucrose are exported when not induced by NaCl and IPTG due to promoter leakiness, which is consistent with our data7.

Growth of S. cerevisiae on Phosphite Media

In our three-way co-culture, we want to use phosphite as a non-metabolizable phosphorus source where only our engineered S. cerevisiae strain is able to convert it to phosphate for itself, as well as the other organisms.
To test if our construct with the codon optimized ptxD gene (ptxD_opt)8 works, we performed a plate reader experiment over 52 hours with different M2 media characteristics. S. cerevisiae and E. coli , both used as negative control, were cultivated in standard M2 medium10 with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l). For some experiments, M2 medium was modified to contain phosphite (also known as phosphonic acid) instead of the originally used phosphoric acid.
The same supplements were used to ensure that the media only differed in the phosphorus source. Medium lacking uracil was used in the samples containing our construct in order to maintain selection pressure. Five different constitutive promoters were tested. All samples were measured every 30 minutes in replicates of five. The sample size was 200 µl. At each measurement point, the OD600 and temperature were measured. The experiment was performed at room temperature, while the plates were shaken vigorously.

Figure 1: Growth of S. cerevisiae BY4742 (blue) and S. cerevisiae BY4742 with the ptxD_opt and TDH3 (yellow) on M2 with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) and phosphite instead of phosphate phosphite over 52 h, measured with OD600. For the not modified S. cerevisiae BY4742 uracil (18 mg/l) were added. As expected and the proof of the function of the ptxD_opt gene the modified strain of S. cerevisiae BY4742 grow on the media, while S. cerevisiae BY4742 as progenitor first grow but than the population decrease.

In Figure 1 the graph demonstrates an initial growth of the progenitor strain S. cerevisiae, but the growth stops at an OD600 of less than 0.03 after 10 hours. After 10 hours S. cerevisiae starts to decrease, in the beginning slowly, but in the end after over 50 hours a faster decrease is recognisable. At the end of the experiment the progenitor strain has an OD600 of less than 0.02. The modified S. cerevisiae strain with ptxD_opt and TDH3 starts nearly at the same ODy600 as the progenitor strain but after a short lag phase of around 5 hours, the strain grows, slowly in the beginning and a bit faster in the end, where it reaches an OD600 of over 0.02. Both strains start at the same OD600 level. The not modified strain grows initially faster than the modified strain, but then decreases more and more, while the modified strain needs some time but then shows a slowly rise of growth.

Conclusions

Stable dependencies between organisms are often based on nutrient exchange. Phosphorus is a macro element essential for microorganisms. Creating a dependency based on the ability of S. cerevisiae to utilize an otherwise unusable phosphorus source like phosphite by expressing ptxD, thereby making it metabolically available for E. coli is therefore a very promising approach.
In general the growth was minimal, so for further experiments, higher concentrations of nutrients and phosphite might lead to better results. The slow growth rate of the modified strain is as expected, because it is comparable to what the literature shows. There, a ptxD construct with the TEF1 promoter in S. cerevisiae was used and growth was monitored over 40 hours11. Moreover, a longer measurement time would also show the behavior of the culture over a longer time. This would be interesting because we would like to create a stable culture which can be maintained as long as possible. In addition, co-culture experiments could lead to other results than monocultures. In this case it would be interesting to perform them as well with the same experimental design. For a future application in the co-culture, we suggest to use the phosphate exporter XPR1 from Homo sapiens10,12. It may help to supply the other organisms, due to the secretion of the produced phosphate.

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  2. Scott, Spencer R., and Jeff Hasty. "Quorum sensing communication modules for microbial consortia." ACS synthetic biology 5.9 (2016): 969-977
  3. Bernhardt, Thomas G., Douglas K. Struck, and R. Y. Young. "The lysis protein E of φX174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis." Journal of Biological Chemistry 276.9 (2001): 6093-6097.
  4. UniProt “ Lysis Protein E”. UniProt Consortium (2018).
  5. Johns, Nathan I., et al. "Principles for designing synthetic microbial communities." Current opinion in microbiology 31 (2016): 146-153.
  6. YSI Inc.”2950D Biochemistry Analyzer” (2018).
  7. Abramson, Bradley W., et al. "Increased photochemical efficiency in cyanobacteria via an engineered sucrose sink." Plant and Cell Physiology 57.12 (2016): 2451-2460.
  8. Kanda, Keisuke, et al. "Application of a phosphite dehydrogenase gene as a novel dominant selection marker for yeasts." Journal of biotechnology 182 (2014): 68-73.
  9. Weiss, Taylor L., Eric J. Young, and Daniel C. Ducat. "A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production." Metabolic engineering 44 (2017): 236-245.
  10. Shaw, A. Joe, et al. "Metabolic engineering of microbial competitive advantage for industrial fermentation processes." Science 353.6299 (2016): 583-586.
  11. Giovannini, Donatella, et al. "Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans." Cell reports 3.6 (2013): 1866-1873.
  12. Legati, Andrea, et al. "Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export." Nature genetics 47.6 (2015): 579.