Difference between revisions of "Part:BBa K2587024"

Revision as of 19:05, 17 October 2018

luxI_luxR_P_lux_E

This part is an assembly of different genes that yield a final plasmid, which enables inducible self-lysis in Escherichia coli cells. It is a construct based on the quorum sensing system of Vibrio fischeri. In V. fischeri, this system is used as a cell-cell communication tool. In the case of this part, the addition of a lysis gene will lead to the induction of cell lysis upon synthesis of the quorum sensing molecule acyl homoserine lactone (AHL). Upon synthesis of AHL by the LuxI gene, it will bind to the regulator LuxR and then to the P_Lux promoter, which will activate the synthesis of the lysis protein E from the bacteriophage phiX174E.

• Acyl homoserine lactone synthase: LuxI
• Regulator: LuxR
• Promoter: P_Lux, inducible by the quorum sensing molecule acyl homoserine lactone bound to LuxR
• Lysis gene: Lysis gene E from bacteriophage phiX174
• Lysis protein induces host cell lysis by inhibiting host translocase MraY activity, which is necessary for catalysis of lipid I, a factor for cell wall synthesis (Cytolysis) (1)
• This construct is able to modify E.coli´s growth, by periodically reducing population
• This part contains Type IIS restriction sites and can be further used for scarless assembly with other parts

Characterization

Characterisation of promoter by fluorescence measurement of GFP
To check whether P_lux is really induced by the binding of acyl homoserine lactone to LuxR, a further construct (BBa_K2587027) was created which encodes the lysis gene E exchanged for gfp. Here, the relative fluorescence units are measured in comparison to progenitor E.coli cells. 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). The performance of the P_lux promoter is further confirmed by fluorescence microscopy (Figure 2).

Results

It can clearly be observed that fluorescence increases upon cell growth. Progenitor cells, which served as a negative control, show little or no fluorescence at all. As expected, the cells grow similarly. It can be concluded that the lack of fluorescence of the wild type cells is not due to the absence of cells. Microscopy revealed that fluorescence is present in the case where P_lux is activated by the quorum sensing system (Figure 2A), while no fluorescence is present in the corresponding untransformed cells (Figure 2B). As a control, gfp expression under the control of a constitutive promoter was checked (Figure 2C). When comparing the expression of gfp under the control of the different promoters (P_lux and constitutive promoter J23102), a higher expression can be seen in the latter case, as expected.

Figure 1: Fluorescence intensity and optical density at 600 nm of E. coli cells expressing gfp under the control of the quorum sensing system (LuxI-LuxR-P_lux-GFP) and progenitor E. coli cells (E. coli BL21 (DE3) C43) over time. GFP was excited at 485 nm and emission was measured at 520 nm. Mean values of triplicates (respective blank subtracted) are plotted.

Figure 2: Confocal fluorescence microscopy of A: E.coli cells with LuxI-LuxR-P_lux-GFP construct; B: E.coli BL21 (DE3) C43 cells and C: E.coli cells with constitutive expression of gfp.

Characterization of growth by measurement of OD ₆₀₀

We analyzed this construct by measuring the optical density of progenitor E.coli BL21 (DE3) C43 cells and the corresponding transformants harboring the lysis construct. After an incubation period of one day, cells were diluted to an OD₆₀₀ of 0.1 in LB medium with 100 µg/mL ampicillin. 200 µl per well were then distributed on a 96 well plate and OD₆₀₀ was measured at a regular interval at 37°C at 200 rpm shaking for a total of 24 hours.

Results

Growth is clearly influenced by the presence of the lysis plasmid as compared to the progenitor E.coli cells (Figure 3). Cells harboring this plasmid grow slower. Observation of their growth shows a remissive exponential phase, reaching the stationary phase with a lower cell density than the control. As a control, the OD₆₀₀ of cells harbouring a plasmid (LuxR/pLux/GFP) with an antibiotic resistance is shown, demonstrating that the decrease in growth is not due to the absence of antibiotics when growing control cells.

Figure 3: Growth of E.coli progenitor, E.coli with the lysis plasmid and control plasmid E.coli. Cell lysis was not directly measured, but derived from the OD₆₀₀, which is a measurement of intact cells in the suspension.

Characterization using further induction with synthetized N-(3-Oxohexanoyl)-L-homoserine lactone
To check whether more cell lysis and as a consequence less cells were present upon induction with more quorum sensing molecules, another test was performed. Here, cells were induced with 0.05 mM 3-oxo-C6-HSL prior to incubation. Cells were incubated at 37°C, 200 rpm over 24 hours.

Results

Growth is reduced especially during the stationary phase as measured by the optical density at 600nm.

Figure 4: Optical density of E.coli progenitor cells, E.coli with lysis construct and E.coli with lysis construct and AHL.

Conclusion

We have successfully developed a construct able to reduce the cell density of E.coli cells. This is especially important for a system which aims to grow organisms with different characteristics in a mixed co-culture. Growth properties in our co-culture vary a lot and in order to prevent overgrowth of one species, it was important to us to be able to regulate the cell growth. We therefore applied the quorum sensing control system, which is a reliable and well characterized tool of synthetic biology² and decided to accomplish our aim by choosing a lysis gene able to modulate cell wall synthesis. First, the promoter P_lux is characterized, demonstrating efficient functionality by expression of gfp (Figure 1/2A). As compared to the constitutive expression of GFP, a lower fluorescence intensity is visible (Figure 2A/C). This might be due to the small amounts of acyl homoserine lactone synthetized by LuxI, which might lead to the limited activation of the pLux promoter. For cell wall synthesis modulation, lysis protein E is used, which is able to interact with the host SlyD and therefore enables protein E to be protected from proteolysis¹. In this way protein E is able to interact with MraY at the host membrane, which blocks MraY transclocase, necessary for lipid I catalysis¹. The latter is an essential component for host cell wall synthesis³. We demonstrate 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 synthesis³. This is indeed consistent with our finding, since cell lysis results in less cells, shown by a lower OD₆₀₀. Moreover, as according to literature, lysis occurs only when cells reach a certain threshold of the quorum sensing molecule AHL, probably during the stationary phase, in order to induce a lysis event². Also here, similar to what Scott and colleagues discovered, population reduction is observable at around ten hours of growth². In Figure 4, a much higher degree of lysis due to the addition of 0,5 mM AHL was expected. Here, further improvements could be performed to achieve better results.
All in all it was shown that cell growth of E.coli cells measured by optical density variations is influenced by lysis gene expression under the control of the Lux - quorum sensing system.
With this discovery we have opened up new ways to control the growth of a population.

Optimization idea Further studies are required, for example whether cell growth has to be adapted to a specific growth as for instance the exchange of the promoters controlling LuxI and LuxR expression. For further experiments, if more cell lysis is desired, the concentration of quorum sensing molecule could be increased.

References

(1) https://www.uniprot.org/uniprot/P03639

(2) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603278/

(3) https://www.ncbi.nlm.nih.gov/pubmed/11078734