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 with B0032 RBS and K1725081 terminator
• BBa_K1725063 - SrpR repressible promoter drives the expression of phlF with B0032 RBS and K1725081 terminator
• BBa_K1725100 - SrpR repressible promoter drives the expression of phlF with B0032 RBS and K1725081 terminator and PhlF repressible promoter drives the expression of srpR with B0032 RBS and K1725081 terminator
• BBa_K1725101 - PhlF repressible promoter drives the expression of srpR with B0032 RBS and K1725081 terminator and SrpR repressible promoter drives the expression of phlF with B0032 RBS and K1725081 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 P_srpR and P_phlF, 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, P_phlF that is driving its expression should not be repressed. Subsequently, GFP should be expressed as it is also controlled by P_phlF. However, if PhlF is the being expressed, RFP should also be expressed as they both have P_srpR as their promoter.

Fig 2: Our system's bistable switch mechanism. SrpR represses P_srpR thus turning off transcription of both PhlF and RFP. In this case P_phlF would be active and GFP would be expressed. PhlF represses P_phlF 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 K1725081 terminator
• BBa_K1725061 – srpr repressor with B0032 RBS and K1725081 terminator
• BBa_K1725063 – SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and K1725081 terminator
• BBa_K1725100 – SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and K1725081 terminator and PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and K1725081 terminator
• BBa_K1725101 – PhlF repressible promoter drives the expression of srpr repressor with B0032 RBS and K1725081 terminator and SrpR repressible promoter drives the expression of phlf repressor with B0032 RBS and K1725081 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.

Fig 5: Detection of both RFP and GFP from our reporter plasmid using the typhoon scanner. a) The first plate is a control for which K1725043 was used that only expresses GFP from PsrpR. b) The second plate contains K1725063 that only expresses RFP from PsrpR. c) The third plate is K1725100 with the reporter plasmid that expresses GFP only from PphlF. d) The third plate is K1725100 with the reporter plasmid that also expresses GFP from PphlF. e) the last plane contains K1725080 cells with K1725103 that is a LacI promoter and express both GFP and RFP.

Discussion

The results showed that K1725103 is expressing both reporters in the absence of repressors. The repressors allowed expression of one fluorescent protein when found individually in a cell with the reporter plasmid. As expected, PhlF was repressing PphlF and therefore only RFP was expressed, whereas SrpR was allowing GFP expression only as PsrpR was repressed.
A bistable switch was successfully constructed using K1725103 with K1725100 or K1725101. It was observed that only GFP was expressed consistently regardless the order of the K1725100 or K1725101. That means that RFP is not expressed because Psrpr is repressed. However, GFP is normally expressed because Pphlf remains active. From the repressors part of our project ( the page can be found in this link ), promoter strength measurements suggest that Pphlf is stronger than Psrpr, which could explain these results.
To further characterise the bistable switch, we would insert the lacI regulated promoter in front of with K1725100 and K1725101. We would expect that the stable state that the switch is currently observed at could switch by insertion of this promoter. This is because in each case, a gene is forced to be expressed by the additional promoter.

References

Kotula, J., Kerns, S., Shaket, L., Siraj, L., Collins, J., Way, J. and Silver, P. (2014). Programmable bacteria detect and record an environmental signal in the mammalian gut. Proceedings of the National Academy of Sciences, 111(13), pp.4838-4843.
Collins, J., Gardner, T. and Cantor, C. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature, 403(6767), pp.339-342.