Team:Glasgow/Project/Overview/Bistable

Summary

Aim: To test the function of the SrpR and PhlF repressor proteins using GFP and RFP reporter genes. To demonstrate the utility of these two repressors by building a classic Synthetic Biology genetic circuit; a bistable switch in which each repressor represses the expression of the other.

Results Overview: Our results indicate that SrpR (K1725060) is expressed, rather than PhlF (K1725040), in both versions of the bistable switch we built (K1725100 and K1725101). We have not yet added a way to flip the switch from one state to the other.

Parts Submitted:
• BBa_K1725043 - PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and B0010+ terminator
• BBa_K1725063 - SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and B0010+ terminator
• BBa_K1725100 - SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and B0010+ terminator and PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and B0010+ terminator
• BBa_K1725101 - PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and B0010+ terminator and SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and B0010+ terminator
• BBa_K1725102 - SrpR repressible promoter driving the expression of RFP with B0030 RBS and B0015 terminator
• BBa_K1725103 – SrpR repressible promoter driving the expression of RFP with B0030 RBS and B0015 terminator and PhlF repressible promoter RBS driving GFP expression with B0032 RBS

Introduction

A bistable switch is composed of two repressor proteins and the promoters they repress. (for a more detailed explanation of how these transcriptional repressors function, see our page ). The system is set up so that each repressor inhibits the promoter that is used to transcribe the other repressor, as shown in figure 1. (Gardner et al, 2000). The system can therefore fall into two different stable states. If repressor 1 is on, it turns off repressor 2 so that repressor 1 continues to be expressed. If repressor 2 is on, the opposite happens. In addition it is useful to have inducer chemicals that interact with the repressor proteins, preventing their association with their operator sequence and preventing repression. Adding the appropriate inducer can switch which repressor is expressed – changing the state of the switch. It has been shown that bistable switches can maintain their state for a long time, without changing spontaneously unless one of the inducers is added. This type of switch has been utilised to develop bacteria of the gut microbiome with memory that can be used in diagnostics by recording and reporting changes in the environment of an organism. (Kotula et al, 2013)

Fig 1: The mechanism of a bistable switch. In the absence of any inducers, expression of repressor 1 leads to inactivation of promoter 1 which stops transcription of repressor 2, hence maintaining expression of repressor 1 from promoter 2. The alternate state with promoter 1 active and promoter 2 inactive is also stable. The state can be switched by adding an inducer which blocks the action of the currently acting repressor. When promoter 2 is on, a reporter is also transcribed along with repressor 1. (Gardner et al, 2000)

Our goal was to design a bistable switch that would allow further characterisation of our repressors, and to show that they could be used to build a classic Synthetic Biology genetic circuit. To achieve this, we used the two repressors, PhlF and SrpR, and their repressible promoters Psrpr and Pphlf, as well as reporter plasmid with the same promoters driving RFP and GFP expression independently. First, we created a plasmid with each promoter driving the expression of the other repressor protein, to emulate the bistable switch set up by Gardner et al. (2000) as shown in Figure 1, and also our reporter plasmid as shown in Figure 2. The cells transformed with both of these plasmids should fluoresce either red or green, depending on which repressor is expressed. Therefore, if SrpR is activate, Pphlf that is driving its expression should not be repressed. Subsequently, GFP should be expressed as it is also controlled by Pphlf. However, if PhlF is the being expressed, RFP should also be expressed as they both have Psrpr as their promoter.

Fig 2: Our system's bistable switch mechanism. SrpR represses Psrpr thus turning off transcription of both PhlF and RFP. In this case Pphlf would be active and GFP would be expressed. PhlF represses Pphlf which controls transcription of both SrpR and GFP. Therefore if PhlF is active, SrpR would be off and we would get red colonies. Pictured as black dots are the terminators.

Methods

E. colistrain used: DH5α. Biobricks that used and assembled in plasmids for the bistable switch are shown below.

• BBa_K1725000 – PhlF repressible promoter (Pphlf)
• BBa_K1725002 – PhlF repressible promoter driving GFP expression with B0032 RBS
• BBa_K1725020 – SrpR repressible promoter (Psrpr)
• BBa_K1725041 – Phlf repressible promoter with B0032 RBS and B0010+ terminator
• BBa_K1725043 – PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and B0010+ terminator
• BBa_K1725061 – srpr repressor with B0032 RBS and B0010+ terminator
• BBa_K1725063 – SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and B0010+ terminator
• BBa_K1725100 – SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and B0010+ terminator and PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and B0010+ terminator
• BBa_K1725101 – PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and B0010+ terminator and SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and B0010+ terminator
• BBa_K1725102 – SrpR repressible promoter driving the expression of RFP with B0030 RBS and B0015 terminator
• BBa_K1725103 – SrpR repressible promoter driving the expression of RFP with B0030 RBS and B0015 terminator and PhlF repressible promoter RBS driving GFP expression with B0032 RBS

The above biobricks were tested by restriction digests to confirm their sequence.
First we had to show that the GFP/RFP reporter plasmid (K1725103) in pSB3K3 with the two promoters expresses both GFP and RFP when there are no repressors present. The plasmid was transformed into DH5α and scanned to detect fluorescence.
Then we had to test that each of the repressors in K1725043 and K1725063 work normally, and that one turns off the GFP and the other turns off the RFP of the reporter plasmid. We therefore transformed each plasmid in pSB1C3 containing one of the repressors into DH5α cells with the reporter plasmid. The colonies were scanned for GFP and RFP detection.
After testing PhlF and SrpR repressors individually, we had to test both repressors with the GFP/RFP reporter plasmid. We transformed both versions of the bistable switch (K1725100 and K1725101) in pSB1C3 into DH5α cells already containing the reporter plasmid. Fluorescence scans followed to reveal which of the promoters is expressed.
For the fluorescence scanning, the Typhoon scanner was used. Green fluorescence was detected using channel 1 (lager 473nm; filter BPB1; PMT 450V) and red fluorescence from channel 2 (lager 532nm; filter LPG; PMT 450V). BPB1 was at 530nm (+/- 20) and CPG at >575nm. The protocols followed for the experiments, including the production of CaCl2 competent cells, transformation, plasmid preparation, restriction digest, gel electrophoresis, ethidium bromide and Azure A staining, gel extraction, and ligation, are available on our Protocols page.

Results

Our reporter plasmid, K1725103 in pSB3K3, carries the gfp gene expressed from the PphlF promoter and rfp expressed from the PsrpR promoter. This plasmid was introduced into DH5α cells and colonies were scanned for expression of GFP and RFP (fig 3). In the absence of any repressor all the colonies expressed both GFP and RFP and are yellow when the red and green images were overlaid (Figure 3d) Controls were done with plasmids expressing no GFP or RFP (K1725083) (Figure 3a) , just GFP from the PphlF promoter (K1725002) (Figure 3b) or just RFP from the PsrpR promoter (K1725102) (Figure 3c), showing that the scanner was correctly detecting GFP and RFP, and that our reporter plasmid expresses both fluorescent proteins in the absence of any repressors.

Fig 3: Detection of both RFP and GFP from our reporter plasmid using the typhoon scanner. a) The first plate is the negative control for which K1725083 was used that should not express any fluorescent protein. b) The second plate contains K1725002 that only expresses GFP from PphlF. c)

The third plate is K1725102 that expresses RFP only from PsrpR. d) In the last column, cells with K1725103 express both GFP and RFP.
The repressors SrpR and PhlF expressed in K1725043 and K1725063 respectively were tested in DH5α cells containing the reporter plasmid. The purpose was to see if the repressors are successful in repressing the correct promoter in the reporter plasmid. SrpR repressed PsrpR, and therefore RFP was not expressed. (figure 4a) Similarly, PhlF repressed PphlF while PsrpR could drive expression of RFP. (figure 4b)

Fig 4: Detection of RFP and GFP showed that cells expressing SrpR only produced green colonies (a) and cells expressing PhlF only produced red colonies (b).

The genetic circuit was built by introducing K1725100 and K1725101 into DH5α cells with the reporter plasmid. K1725043 and K1725063 with individual repressors were used as controls for GFP and RFP respectively. The yellow colonies in the last plate are a negative control where both GFP and RFP are expressed as a response to the lacI promoter. For both variations of the bistable switch, SrpR was the active repressor, thus PphlF was driving expression of GFP. RFP was absent, indicating that PsrpR was repressed.

Figure 6 Characterising Repressors. Repressor constructs in pSB1C3 backbone; promoter driving GFP constructs in pSB3K3 backbone; in DS941 cells. The DS941 genotype can be found on our Protocols page. Cells were grown overnight in 100μM IPTG, to induce expression of the repressor proteins. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.

In addition to showing that K1725042 and K1725083 were capable of repressing K1725001 and K1725082, respectively, quantification of repression of GFP expression was calculated. Figure 7 shows that K1725082 represses K1725083 GFP expression by 33-fold, whereas K1725042 represses K1725001 GFP expression by 83-fold.

Figure 7 Fold Repression. Repressor protein expression induced with 100μM IPTG. Values and error bars from experiments described above.

To further characterise K1725042 and K1725083, the concentration of IPTG used to induce repressor expression was reduced to investigate the range of regulation of GFP expression. Figure 8 shows that K1725083 has a wider range of regulation, whereas K1725042 shows no significant difference between 100μM and 10μM IPTG.

Figure 8. Repressor constructs with pSB1C3 backbone; promoter driving GFP constructs with pSB3K3 backbone; in DS941 cells. Cells were grown overnight in 100μM, 30 μM, 10 μM, 3 μM, and 0 μM IPTG, to induce expression of the repressor proteins. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.

Conclusion

New Basic Parts, BBa_K1725000 (PhlF repressible promoter), BBa_K1725020 (SrpR repressible promoter), and BBa_K1725040 (phlF encoding PhlF repressor) were successfully characterised, and submitted to the iGEM registry. BBa_K1725060 – (srpR encoding SrpR repressor) was characterised on our Bistable Switch page.

K1725000 drives expression of GFP in the BioBricks K1725001 and K1725002, and is significantly stronger than our control promoter, R0040. K1725020 drives expression of GFP in the BioBricks K1725021 and K1725022, and is of equivalent strength to our control promoter, R0040, when both were coupled with the strong Ribosome Binding Site, B0034. K1725040 successfully represses K1725000 driven GFP expression 83-fold, and does not repress promoters K1725020 or R0040.

The next steps in characterisation of our repressor BioBricks would be to construct the BioBrick K1725062 (by insertion of the lacI regulated promoter, K1725080, upstream of K1725061, in order to express the repressor protein) then to repeat our fluorescence experiments to characterise K1725060 further. Additionally, GFP purifed from E. coli would be used in place of iLOV fluorescent protein to calibrate the Typhoon FLA 9000, so the molecules of GFP per cell calculations would give a value closer to an absolute value for fluorescence.

References

Abbas, A., Morrissey, J.P., Marquez, P.C., Sheehan, M.M., Delany, I.R., and O’Gara, F. (2002). Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113. J. Bacteriol. 184, 3008–3016.

Alberts et al (2008). Molecular Biology of the Cell (Garland Science, Taylor and Francis Group). Chapter 7, p337-434

Buckley, A. Petersen, J. Roe, A. Douce, G. Christie, J. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology. 27 (1), p39–45.

Sheehan, M.M., Delany, I., Fenton, A., Bardin, S., O’Gara, F., and Aarons, S. (2000). Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor. Microbiology 146, 537–546.

Stanton, B.C., Nielsen, A.A.K., Tamsir, A., Clancy, K., Peterson, T., and Voigt, C.A. (2014). Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat. Chem. Biol. 10, 99–105.

Sun, X., Zahir, Z., Lynch, K.H., and Dennis, J.J. (2011). An Antirepressor, SrpR, Is Involved in Transcriptional Regulation of the SrpABC Solvent Tolerance Efflux Pump of Pseudomonas putida S12. J. Bacteriol. 193, 2717–2725.

Wery, J., Hidayat, B., Kieboom, J., and Bont, J.A.M. de (2001). An Insertion Sequence Prepares Pseudomonas putida S12 for Severe Solvent Stress. J. Biol. Chem. 276, 5700–5706.

Team:Glasgow/Project/Overview/Bistable

Summary

Introduction

Methods

Results

Conclusion

References

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Protocols

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Terminator

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