Difference between revisions of "Part:BBa K1893019"

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T7 phage gene product 2 (Gp2)

Usage and Biology

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.

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

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.

Sequence and Features