Difference between revisions of "Part:BBa K1893019"

Latest revision as of 15:41, 12 October 2023

T7 phage gene product 2 (Gp2)

The T7 phage is a bacteriophage that infects Escherichia coli and leads to cell lysis of the host. It is also the source of the T7 promoter, which is commonly used in synthetic biology with T7 RNA polymerase for tight control of gene expression. Infection by T7 phase is facilitated by a number of viral genes encoded in its 40kb T7 genome, including gene product 2.

Usage and Biology

Gene product 2 (gp2) is a small 7 kDA protein that plays a key role in the late stages of T7 phage infection. It binds to the β’ subunit of RNA polymerase (RNAP) in the E. coli host, which inhibits host transcription by preventing formation of the active RNAP holoenzyme. This allows the phage-encoded RNAP to transcribe the phage proteins required for successful infection without interference from the host transcriptional machinery. An additional effect of inhibited host transcription is a decrease in the growth rate of the host.

We selected gp2 as a candidate for the growth-regulation module of our GEAR system for its ability to inhibit growth without causing cell death. gp2 also allows for growth inhibition without the need to manipulate growth media or generate knockout strains, unlike some of the other candidates we had considered.

Characterisation (Imperial College 2016)

In order to investigate the effect of gp2 on E. coli growth, we placed the gp2 coding sequence under the control of an arabinose-inducible promoter (BBa_K1893015) and characterised cell growth at varying levels of gp2 expression. The results of these experiments can be found here (BBa_K1893016).

Colonies of E. coli transformed with gp2 constructs appeared to be irregular and smaller in size than wildtype E. coli colonies, even when the gp2 sequence was not prefaced by an active promoter. This could suggest leaky expression of the gp2 coding sequence in E. coli.

Characterisation (Imperial College 2018)

In order to test our PixCell electrogenetic part library we constructed a biocontainment device to prototype one of our proposed applications. This was constructed using an existing part in the iGEM registry: the growth inhibitor Gp2. By placing this downstream of pSoxS (BBa_K2862006) and expressing SoxR (BBa_K2862014) from a constitutive promoter (BBa_K2862019) we could inhibit cell growth in the oxidising conditions that can be generated by our electrochemical set up. We envisioned this being used with our array in a biocontainment device in which any cells that spread to the edges of the device have their growth seized by an oxidising potential. This "microbial electric fence" could be used to prevent the accidental release of GMOs.

Figure 1: Gp2 construct diagram.

This application construct and a control replacing GP2 with GFP were grown in both the reducing (OFF) condition (2.5μM pyocyanin, 0.02% sodium sulfite, 10mM ferrocyanide) and the oxidising (ON) condition (2.5μM pyocyanin, 0.02% sodium sulfite, 10mM ferricyanide) of our electrogenetic system. The Gp2 construct shows a significant decrease in the maximum achievable growth rate in the oxidising condition.

Figure 2: Growth curves for cells containing the GP2 construct, in both reducing (OFF) and oxidising (ON) conditions, demonstrating reduced growth in the oxidising environment. Data obtained from five biological replicas.

In comparison no significant difference is seen in these conditions with the control construct.

Figure 3: Growth curves for cells containing the GFP control, in both reducing (OFF) and oxidising (ON) conditions, showing no reduction in growth rate in oxidising conditions. Data obtained from five biological replicas.

Looking at the steady state OD600 values, there is a clear, significant, ~2-fold reduction in the application construct in oxidising conditions. Furthermore there is no difference between the growth in the reducing conditions and the controls. This not only provides a proof-of-concept for out application, but also proves the system is not significantly leaky. This means it is feasible to use a higher copy number plasmid, or a more toxic gene (such as MazF) in this device to provide an even more effective biocontainment circuit.

Figure 4: Steady-state OD600 values for GP2 construct, and GFP control, in both oxidising and reducing conditions. Significant differences are only seen for the GP2 construct in oxidising conditions. Data obtained from five biological replicas.

iGEM Sheffield 2023 PARSE

Figure 5: Mean log2(OD) against time for gp2, concentrations refer to the [IPTG] used to induce the pLac promoter regulating gp2.

Figure 5 shows that there's a significant decrease in growth of E. coli when they are transformed with a plasmid containing gp2. The decreased mean log 2 OD observed without induction of pLac shows leaky expression of the growth slower gene has an impact on bacterial cell growth. With induction, the greater the [IPTG], there's a trend of decreased mean log2 OD that conveys the decrease in growth. This supports that gp2 causes a growth slowing impact in E. coli, and the level of growth slowing can be controlled using an inducible promoter.

Sequence and Features