Difference between revisions of "Part:BBa K2587024"

Revision as of 14:27, 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 comunication 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. luorescence is present in the case where the pLux promoter is activated by the quorum sensing system (Figure 2A), whilst no 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 untreated 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/ negative control) 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. 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 incubation. Here as well, incubation was performed at 37°C, 200rpm and 24 hours.

Results Growth is only slightly influenced and only at the end of 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.

Optimisation idea For further experiments, if more cell lysis is desired, the concentration of quorum sensing molecule can be increased. This remains to be further evaluated.

Conclusion

We have successfully developed a construct able to reduce the cell density of E.coli. This is especially important for our 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 cell´s growth. We achieved this by the assembly of an inducible self lysis construct, which as evidenced, leads to a reduced cell growth of E.coli cells. Further studies are required if cell growth has to be adapted to a specific growth.

References

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

Functional Parameters