In this section, a genetic module for cell-cell communication in a cell density- dependent manner was designed and constructed on the basis of the lux quorum sensing regulatory system in V. fischeri as introduced in Section 6.1.1.
Figure 6.2 shows the design of the genetic module. The module was created on the basis of the synthetic Plux promoter that has been characterised in Chapter 4 and the luxR and luxI regulatory genes from the lux quorum sensing system. Although the natural lux regulatory system has been isolated and demonstrated to have the population density-dependent behaviour, a modular design approach was taken here in
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building the module using standardised BioBrick parts based on the intrinsic mechanisms of quorum sensing (see Section 6.1.1). The module is different from the natural one, but, essentially, employs the same positive feedback mechanism. The Plux promoter contains the lux box and the right PluxI promoter from the native lux system. The luxR gene is constitutively expressed under the Ptet promoter. The luxI and gfp are expressed as an operon under the regulation of the Plux promoter. Thus, when at low cell densities, LuxI and GFP proteins are expressed at low levels and the AHL molecules synthesised from LuxI is also at a low level and can not sufficiently bind LuxR to activate the transcription from Plux. When the cells continue to grow, the AHL accumulates in the environment and reaches a threshold at a certain cell density, at which it sufficiently binds LuxR to activate the positive feedback loop as shown in the graph and then more LuxI and GFP proteins are expressed. As a result, the output in terms of fluorescence increases with cell density, and the module has the population density-dependent behaviour.
To conform to the BioBrick standard, all the module components (promoters and genes) were obtained from the Registry of Standard Biological Parts and were assembled on the BioBrick standard vector pSB3K3 as shown on the right of Figure 6.2a. Since the luxI and gfp are expressed as an operon in the module, the gfp expression level is likely different from the one that is first expressed downstream from the Plux promoter even with the same RBS used. Polarity is known to occur in bacterial systems, and later transcribed genes in an operon are often not as highly expressed as those initially transcribed. Such effects can be due to changes in the amounts of message where the message is degraded from its 3' end, competing RNA structures that disfavour translation, and so on. This view is borne out by the initial experimental results which showed that, for the constructs with rbs33-gfp or rbsH-gfp downstream the luxI gene, there were nearly no fluorescent outputs of the cells harbouring the module for all 6 RBSs (rbs30 - rbsH) used for luxI (data not shown). Thus, the strong RBS – rbs30 was finally chosen for obtaining measurable levels of
gfp expression, which gave a clear fluorescent output for cells harbouring the module.
The weak RBS – rbs33 for luxI in the final construct was chosen from the screenings which showed that, for all 6 RBSs (rbs30 – rbsH) used for luxI gene (luxI-rbs30-gfp),
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P
lux luxR luxI GFPP
tetrbs34 rbs33 rbs30AHL
luxR luxR luxI b 10-2 10-1 100 0 10 20 30 40 50 60 70 80 90 100 OD600 Mean f luor es ce nc e ( a u)sample culture of the module
Figure 6.2 The engineering of a cell density-dependent module. a, The regulatory module is designed on the basis of the synthetic AHL-responsive Plux promoter and the luxI gene, which forms a positive feedback loop in response to the density of the cell population. On the left is the plasmid construct of the module. The luxI gene is modified with a LVA degradation tag. b, The characterised results of the module in E.
coli MC1061 after 5 hours growth in M9-glycerol at 37 °C. The sample cultures are
initially inoculated with various numbers of cells of OD600 at (bottom to top) 1 × 10-4, 3 × 10-4, 5 × 10-4, 1 × 10-3, 2 × 10-3, 5 × 10-3, 1 × 10-2, 2 × 10-2 and 5 × 10-2. The negative control is the culture of cells carrying the gfp-free construct, and the positive control is the culture of cells carrying the functional construct induced with 100 nM AHL. The inoculation density for both the negative and positive controls is of OD600 at 0.02. On the right is the plotted curve for the module output (mean fluorescence from FL1 filter) as a function of cell density for the FACS assays.
where there was only significant output difference between the non-induced culture and fully induced culture (by 100 nM AHL) for cells harbouring the module with
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gfp respectively in the module to achieve the cell density-dependent output property.
The constructed module (Figure 6.2a) was next characterised in E. coli MC1061 by FACS assay and the results are shown in Figure 6.2b. To prepare the day cultures, the overnight cultures were pelleted by centrifugation, and re-suspended in pre- warmed M9 media and left for 10 mins before being diluted into the day cultures at various cell densities. This washing step is important for removing the AHL molecules expected to have built up in the media because the overnight culture was likely saturated with high level of AHL molecules after a long time of growth. The diluted day cultures were next grown at 37 °C for 5 hours to various cell densities (measured by spectrophotometer at 600 nm) before being harvested for analysis by flow cytometry. Referring to Figure 6.2b, it shows that the cells containing the engineered module displayed the desired cell density-dependent property. The fluorescent outputs of the sample cultures with various initial cell densities varied from each other after the same length of time of growth, and the output increases with the density of the cell culture. Extrapolating back towards a fully “off” low density cell culture, it seems that the positive feedback of the module was already partly switched on at a lower cell density than the lowest density shown in the graph. However, due to the detection limit of the spectrophotometer used, the lowest OD600 that can be accurately measured for the cell culture is 0.01. Although the completely “off” culture was not directly observed during the experiment, the cell cultures analysed indeed exhibited the density-dependent behaviour. It is known that AHL (Acyl-homoserine lactones) molecules are quite stable in mildly acidic or neutral pH environments (Leadbetter and Greenberg, 2000). The quorum-sensing signalling molecules AHL are found to degrade significantly only in some specific bacterial species like Bacillus bacteria which can express the AiiA enzyme to degrade the signalling molecules (Dong et al., 2000) or in Variovorax paradoxus which can metabolise the signalling molecules (Leadbetter and Greenberg, 2000). Thus, the effect of AHL degradation can be negligible in the E. coli chassis used here. However, if used in an E. coli chassis expressing the AiiA degrading enzyme, it might slow down the accumulation of AHL in the media and lead to a delayed density switching response, i.e. switching at a higher cell density.
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