Parts

Gp79

[BBa_K4451009]

A transcriptional inhibitor from phiEco32 phage, which is able to cause growth rate suppression in E. coli.
The Gp79 gene encodes a ~10kDa protein that has been shown to bind to the core of the RNA polymerase σ70 holoenzyme, which prevents transcription bubble formation in many the housekeeping genes in E. coli and thus enacts global transcriptional downregulation. Whilst the exact molecular mechanism has yet to be elucidated, it is likely that Gp79 is utilised to switch the specificity of host RNA polymerase to that of phiEco32 late genes in the infection cycle. [3]

Gp104

[BBa_K4451011]

An inhibitor of DNA replication from the 77 phage, which causes growth rate suppression in Staphylococcus aureus.
The gp104 gene encodes a small inhibitory protein that targets and inhibits the DnaI protein in S. aureus, itself responsible for helicase loading onto replicating DNA and thus a crucial part of the replisome. Its inhibition shows potent growth rate suppression due to the stalling of DNA replication initiation. To date, no evidence has been shown to suggest its function in E. coli, and it is worth noting that E. coli itself does not have a closely related DnaI homolog. [4]

AsiA

[BBa_K4451010]

A transcriptional inhibitor from T4 phage, which causes growth rate suppression in E. coli.
The AsiA gene encodes an anti-σ factor, which is able to bind to σ70 and prevent the RNA polymerase σ70 holoenzyme from recognising σ70 specific promoters, thus enacting a transcriptional downregulation of housekeeping genes and subsequently causing growth rate suppression. AsiA further enables the transcription of phage middle genes as part of the T4 infection cycle. [5]

Alc

[BBa_K4451008]

A transcriptional terminator from T4 bacteriophage that has been shown to suppress growth rate in E. coli.
The Alc gene encodes a site-specific termination factor, which binds to commonly found sites in the E. coli genome and causes early transcriptional termination, thus reducing transcriptional output and reducing growth rate. Termination is only possible on actively processing RNA polymerases, any stall within 15bp of the Alc site abolishes early termination. This, alongside AsiA, is utilised to trigger transcriptional redirection toward phage genes in the T4 infection cycle. [6]

Other Parts

vsfGFP-0:

[BBa_K4939000]

An engineered variant of sfGFP, which has shown enhanced stability and fluorescence in in vitro and in vivo models.
It has been widely reported that the binding of certain single domain antibodies (nanobodies) allows for the stabilisation of GFPs (which can be inherently unstable), and thus enhances their fluorescence. One particular nanobody - the enhancer nanobody - was shown to significantly increase fluorescence when bound to GFP. Eshagi et. al [1], reasoned that directly fusing this nanobody to sfGFP could create a novel GFP with enhanced fluorescence, since the stabilisation was now intramolecular and thus built into the fold of the protein. Indeed, they were correct and the newly designed vsfGFP-0 showed ~3 times more fluorescence than sfGFP. Notably, the group developed two versions: vsfGFP-0, in which the fusion of the enhancer nanobody gave rise to a dimerisation prone GFP; and vsfGFP-9 in which a 9aa linker was utilised between the domains which promoted monomeric folding - the former (i.e., the dimerised variant) is the one discussed here. [7]

pBmo

[BBa_K4939002]

A butanol sensitive σ54 dependant promoter from Thauera butanivorans, with an included upstream activation site 193 bp upstream of the -24 and -12 RNAP binding sites.
Originally isolated from sludge at a Japanese oil refinery, T. butanivorans is a gram negative alkanotroph that can metabolise C2-C9 alkanes. The metabolic operon with which the cells can carry out this metabolism is under the control of pBmo - a butanol sensitive, σ54 dependent promoter that itself is regulated by the BmoR repressor (an alcohol-responsive bacterial enhancer protein (bEBP)). In typical σ54 fashion, the activation of the associated operon involves an upstream activation sequence (UAS) some 193 bp upstream of the RNAP binding site. Therefore, BmoR binding triggers significant DNA bending to achieve gene activation. Thus, for the use of a functional butanol biosensor in vivo, the strain in question must also contain the UAS and the transcriptional unit for BmoR. Butanol biosensing has been achieved in E. coli using this system.

bmoR

[BBa_K4939001]

Encodes the BmoR transcription factor, an alcohol responsive bacterial enhancer protein (bEBP) that activates the butanol sensitive pBmo promoter and thus allows for butanol biosensing.
Originally isolated from sludge at a Japanese oil refinery, Thauera butanivorans is a gram negative alkanotroph that can metabolise C2-C9 alkanes. The metabolic operon with which the cells can carry out this metabolism is under the control of pBmo - a butanol sensitive, σ54 dependent promoter that itself is regulated by the BmoR repressor (an alcohol-responsive bacterial enhancer protein (bEBP)). In typical σ54 fashion, the activation of the associated operon involves an upstream activation sequence (UAS) some 193 bp upstream of the RNAP binding site - to which a butanol dependant oligomerized BmoR protein binds and enacts transcriptional activation. Therefore, BmoR binding triggers significant DNA bending to achieve gene activation. Thus, for the use of a functional butanol biosensor in vivo, the strain in question must also contain the pBmo and associated UAS. Butanol biosensing has been achieved in E. coli using this system.