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Part Improvement

Fim Standby Switch

BioBricks made and used: BBa_K2205005 (New), BBa_K1632013 (2015 Tokyo Tech part), BBa_K1632007(2015 Tokyo Tech part)

Rationale and Aim

Sensynova multicellular biosensor platform has been developed to overcome the limitations that hamper success in biosensor development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Reporter) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors.

This part can be used within the platform as a processor unit. Real world applications of biosensors are limited by many factors, one of which is that with most biosensors there is not a readout signal showing if the biosensor is working when not in use, i.e that the cells are still alive and have not lost their biosensor phenotypes. This can make them difficult to use, as well as market, since their viability comes into question as well as leading to false negatives/positives. Biosensors which rely on expression of a reporter signal may also suffer from unobserved activation due to weak or inconstant induction.

For this section of the project, as an improvement on a part by the Tokyo Tech 2015 team (BBa_K1632013), we aim to produce a biobrick compatible part which is able to constitutively express a reporter signal prior to activation (to show that it is functioning) and to amplify a weak or inconsistent induction signal by permanently switching from an [OFF] to [ON] state after induction.

Background Information

Expression of the E. coli type 1 fimbriae gene is tightly regulated and phase dependent, i.e expression is either completely [ON] or [OFF] (Klemm, 1986). This change in expression is controlled by the action of two proteins FimB and FimE which independently act upon a 300bp promoter region upstream of the fimbriae gene. The 300bp promoter region is inverted to either activate or suppress expression (McClain et al., 1991). Typical gene regulation mechanisms rely on up or down regulation of a promoter from a baseline expression, the fimbriae mechanism of ‘ALL’ or ‘NONE’ makes it a useful tool for synthetic biology applications. While the FimB protein inverts the promoter back and forth between [ON] and [OFF] states the FimE protein permanently inverts the promoter from [ON] to [OFF]. This inversion can be used to amplify weak or inconsistent induction signals.

Since the part we are making is designed to amplify a weak signal which can then be detected by a downstream ‘reporter’ cell the quorum sensing system from P. aeruginosa was adapted to allow for signal transfer between cells. The rhlI gene from P. aeruginosa produces the quorum sensing molecule N-butyryl-AHL (C4-AHL) (Parsek et al.,2000) (J64718), this molecule is membrane permeable and able to induce expression of a promoter upstream of sfGFP in another cell (K2205015).

Figure 1: SBOL Visual of the switching mechanism of the Fim Switch, in the native [OFF] state the eforRED reporter is expressed (shown in red) allowing direct visualisation of the cells. Following the inversion of the promoter region (K1632004), eforRED expression is halted and the rhlI gene is expressed (J64718), this is now the [ON] state.

Design Stage

To construct the Fim reporter switch 3 separate gBlocks were designed with overlapping adaptor regions homologous to the iGEM prefix and suffix to allow for Gibson assembly into the pSB1C3 backbone whilst retaining biobrick compatibility. The individual genes and other components are shown in (Table 1). The 1st gBlock sequence starts with a RBS (B0034) upstream of the fimE ORF (K137007) with no promoter region, this is to allow for other promoters to be cloned in upstream of the part. Downstream of the fimE gene is a double terminator (B0015). All RBS and terminator sequences used are B0034 and B0015 respectively. The switching mechanism consists of the Fim promoter sequence (K1632004) flanked by two RBS-ORF-Terminator sequences. While in the native [OFF] state the Fim promoter drives expression of eforRed (K592012) and when flipped to the [ON] state drives expression of rhlI (J64718). The rationale behind using the fimE gene instead offimB is that it permanently inverts the promoter region meaning weak induction signals can be amplified by the Fim switch.

Table 1: Table of parts used for constructing the Fim Switch.

Implementation

To assemble the Fim switch part the isothermal Gibson assembly cloning method was chosen as it would significantly shorten the time taken to assemble 3 separate sequences compared to traditional cloning methods. The 3 gBlock DNA fragments shown in (Table 1) were amplified by high fidelity Q5 PCR and the pSB1C3 backbone was digested with restriction enzymes EcoRI and PstI.

Figure 2: High fidelity amplification of the 3 gBlock fragments for assembly of the Fim Switch. The gBlock-1 amplification is shown in lanes 1+2 (819 bp), gBlock-2 amplification is shown in lanes 3+4 (1148 bp) and the gBlock-3 amplification is shown in lanes 5+6 (939bp).

The Gibson assembly reaction re-forms the iGEM prefix and suffix regions at the 5’ and 3’ ends of the Fim switch part making the component biobrick compatible while leaving no scarring regions. Following assembly, the plasmid was transformed into chemically competent DH5α E. coli and colonies patched onto LB Chloramphenicol agar plates. A single patch showed the correct red colour indicative of the eforRed chromoprotein (see Figure 3).

Figure 3: Patches of the Fim Switch transformants. Patch number 6 shows the correct red colour which indicates expression of the eforRed chromoprotein.

The red patch was cultured in LB chloramphenicol overnight and the plasmid DNA extracted by miniprep. The plasmid was digested with restriction enzymes XbaI and PstI. The image in Figure 4 shows the DNA bands from the digested Fim switch plasmid.

Figure 4: Restriction digestion of the Fim switch plasmid to confirm successful integration into the iGEM pSB1C3 backbone. The Fim switch plasmid (Lane 1) was digested with XbaI and PstI with expected band sizes of (2840 bp and 2044 bp). The pSB1C3 plasmid (Lane 2) containing sfGFP as a control was also digested with XbaI and PstI with expected band sizes of (811 bp and 2044 bp).

The Fim switch insert is 2882 bp in length which makes performing standard short sequencing reads challenging as multiple reactions are required to completely sequence the entire part. To overcome this we used our in-house Illumina MiSEQ to completely sequence the entire plasmid. Following quality control analysis the sequence was assembled and shown to be a match to the expected Fim switch part.

A problem we found with the Fim switch was that a subset of the colonies were prematurely switching from red to white. This is likely due to a low level of leaky expression of the fimE gene which then inverts the promoter region upstream of the eforRed gene. A single white colony was picked and cultured for use in downstream testing as a control as the switching of the promoter should express the rhlI gene and therefor produce the C4 quorum sensing molecule.

Characterisation

To test the functionality of the Fim switch, ensuring that C4 AHL is produced, the strain was cultured with a reporter strain (K2205015) which produces GFP in response to the quorum sensing molecule C4 AHL. Due to a small sub-population of the Fim switch strain being white, a single white colony was picked and cultured separately. This strain was used as a positive control as it should produce C4 AHL. Both the majority (red) Fim switch strains and minority flipped (white) Fim switch strains were tested for C4 AHL production by co-culture with the reporter strain. Initially the Fim switch strains were spotted onto a lawn of the reporter strain (Figure 5) followed by quantitative analysis of the strains by co-culture in a 96 well microplate (Figure 6). This culture is then co-cultured with the reporter cell. This reporter cell detects C4 AHL and expresses GFP in response.

Figure 5: Initial test of the red and white fim switch strains which were spotted onto a lawn of the reporter strain (BBa_K2205015).


Figure 6: Expression of GFP in the reporter (BBa_K2205015)strain in co-culture with the Fim switch strains. The assay was performed using methods described in Fim 96 Plate assay Protocol. The data shows the expression of GFP in the reporter strain over a standard growth curve. The FimW and FimR strains represent the white and red variants of the Fim switch strain respectively, these were co-cultured with the reporter strain in a 1:14 ratio. Each data point is the mean of 3 biological repeats. RFU stands for relative fluorescence units.

Conclusions

The aim of the Fim switch part was to make a processor module which can be visually inspected for functionality. The Fim switch has been shown to expresses the eforRed chromoprotein under normal (uninduced) conditions which allows the user to both determine that the strain is alive and has maintained the Fim switch plasmid. Following induction, the Fim promoter flips direction and begins expressing RhlI which synthesises the C4-AHL quorum sensing molecule. This has been shown to successfully induce expression of sfGFP in the reporter strain (BBa_K2205015).

Despite several attempts we were unable to produce a Fim switch testing construct where fimE expression could be controlled using the E. coli arabinose inducible promoter. Though transformations did yield some colonies when trying to make this part, none were red in colour. This possibly indicates that the arabinose inducible promoter (even when grown on 0.5% w/v glucose) is still too active. The design for this construct has been submitted (BBa_K2205006).

Future Work

Since there is some leaky expression of the fimE gene (even without a promoter) to fine tune the Fim switch the lac operator could be inserted upstream of the fimE RBS to repress unwanted expression. The part could then be used following the addition of IPTG. An alternative method would be to clone a transcriptional terminator upstream of the fimE RBS to prevent leaky expression from elsewhere in the plasmid.

References

P. Klemm, Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J 5, 1389-1393 (1986).

M. S. McClain, I. C. Blomfield, B. I. Eisenstein, Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli. J Bacteriol 173, 5308-5314 (1991).

M. R. Parsek, E. P. Greenberg, Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc Natl Acad Sci U S A 97, 8789-8793 (2000).