Difference between revisions of "Part:BBa K2587024"

Revision as of 15:11, 9 October 2018

luxI_luxR_P_lux_E

This part is an assembly of different genes that yield a final plasmid, able to self lyse 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 communication tool amongst those organisms. In this case with the addition of a lysis gene it will induce 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 promoter pLux, which will activate the synthesis of the lysis protein E from the bacteriophage phiX174E.

• Acyl homoserine lactone synthase: LuxI
• Regulator: LuxR
• Promoter: pLux, inducible by the quorum sensing molecule acyl homoserine lactone bound to LuxR
• Lysis gene: Lysis gene E from bacteriophage phiX174E
• 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 slowing it down
• This part contains Type II S 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 the promoter, pLux, is really induced by the binding of the acyl homoserine lactone to the LuxR, a further construct (BBa_K2587027) is created which has the lysis gene E exchanged by a GFP gene. Here the relative fluorescence units are measured in comparison to the wild type E.coli cells. In order to exclude that the lack of fluorescence of the wild type cells is due to their scarce growth, a measurement of the optical density was done as well (Figure 1). The performance of the pLux promoter is further confirmed by fluorescence microscopy (Figure 2).

Results It can clearly be observed, that fluorescence increases upon growth of the cells. Wild type cells, 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 the pLux promoter is activated by the quorum sensing system (Figure 2A), whilst no fluorescence is present in the corresponding untransformed cells (Figure 2B). As a control, GFP expression under the control of a constitutive promoter is checked (Figure 2C). When comparing the expression of GFP under the control of the different promoter (pLux and constitutive promoter J23102), as expected, a higher expression is visualized in the latter case.

Figure 1: Fluorescence intensity and optical density at 600 nm of E. coli cells harbouring a fluorescent protein under the control of the Quorum sensing system (LuxI-LuxR-pLux-GFP) and wild type E. coli cells (E. coli BL21 (DE3) C43) over time. GFP extinction was measured at 485 nm and emission at 520 nm. Mean values of triplicates subtracted from the respective blank are plotted.

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

Characterization using growth measurement by OD 600

We analysed this construct by measuring the optical density of wild type E.coli BL21 (DE3) C43 cells, and the corresponding transformants harbouring the lysis construct. After one day incubation, cells were diluted to an OD of 0.1 in LB medium with 100µg/mL final ampicillin concentration. 200µl were then added to a 96 well plate and OD600 has been measured 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 wild type E.coli cells (Figure 3). Cells harbouring this plasmid grow slower. Observation of their growth shows a remissive exponential phase, reaching the stationary phase with a lower cell density than wild type. As a control, also the OD600 of cells harbouring a plasmid (LuxR/pLux/GFP) with an antibiotic resistance is shown, demonstrating that a decrease in growth is not due to the absence of antibiotics when growing wild type cells.

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

Characterisation 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 further quorum sensing molecule, a further test was performed. Here cells were induced with 0.05mM 3-oxo-C6-HSL prior to incubation. Here as well, incubation was performed at 37°C, 200rpm and over 24 hours.

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

Figure 4: Optical density of E.coli wild type 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 to the others, it was important for us to be able to regulate the cells growth. We therefore applied the Quorum sensing control system, which is a reliable and well characterized tool of synthetic biology (2) and thought to accomplish our aim by choosing a lysis gene able to modulate cell wall synthesis. First the promoter pLux is characterized, showing efficient function 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 proteolysis (1). In this way protein E is able to interact with MraY at the host membrane, which blocks MraY transclocase, necessary for lipid I catalysis (1). The latter is an essential component for host cell wall synthesis (3). We demonstrating that growth, based on OD600 measurements, is decreased in cells harbouring the lysis plasmid. Based on literature, lysis is occurring because the lysis protein E impairs peptidoglycan synthesis (3). This fact is indeed consistent with our finding, since cell lysis results in less cells, shown by a lower OD600 measurement. Moreover, as according to literature, lysis occurs only when cells reach a certain threshold of quorum sensing molecule AHL, probably during the stationary phase and induce a lysis event (2). Also here, similar to what Scott and colleagues discovered, population reduction is observable at around ten hours of growth (2). In Figure 4 a much higher degree of lysis due to the addition of 0,5mM AHL was expected. Here further improvements could be performed to achieve better results. All in all it was evidenced, that cell growth of E.coli cells measured by optical density variations is influenced with lysis gene expression under the control of the Lux- Quorum sensing system. With this discovery we have opened up new ways of how to control the growth of a population.

Optimisation idea Further studies are required if 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 can 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

Functional Parameters