Difference between revisions of "Part:BBa K2862010"

Latest revision as of 02:03, 18 October 2018

pSoxS Mutant 3

pSoxS is one half of the pSoxR/pSoxS bidirectional promoter. Transcription downstream of pSoxS is activated in response to oxidation of the SoxR transcription factor, either directly by redox-cycling drugs or by oxidative stress. It is therefore inducible by various redox-cycling drugs, toxins, antibiotics, heavy metals, hydrogen peroxide and nitric oxide, providing various applications in the development of environmental and therapeutic devices. By coupling oxidation of redox-cycling species to an electrode, the 2018 Imperial College London iGEM team (PixCell) used pSoxS to build electrogenetic devices in which electrical inputs modulated gene expression.

This pSoxS promoter represents an improvement on Part:BBa_K387001. Our primary contribution is enhanced modularity, allowing its incorporation into a library of promoters and corresponding transcription factors; we also have evidence of new functionality (transcriptional repression) in combination with one of the transcription factors in this library. The promoter was taken from E. coli, with a 2bp deletion introduced between the -35 and -10 site in order to convert the induction of the promoter from transcriptional activation to transcriptional repression. The promoter was also engineered to be unidirectional by knocking out activity of the pSoxR portion. It was designed with an upstream terminator to remove the need for these to be added as parts in large assemblies. It also contains a downstream ribozyme to reduce context-dependency. It forms part of the PixCell library of electrogenetic and redox-sensing parts.

This part is compatible for BioBrick, BASIC and Golden Gate assembly.

Biology

pSoxS is the cognate promoter to the SoxR transcription factor and one half of the pSoxR/pSoxS bidirectional promoter. When SoxR is reduced, the promoter pSoxS is inactive, but upon oxidation of SoxR by redox-cycling drugs or oxidative stress transcriptional activation is triggered downsream of pSoxS. This in turn allows for activation of the soxRS regulon and a response to oxidative stress. Although SoxR is able to bind pSoxS in both its oxidised and reduced forms, only the oxidised form provides transcriptional activation.

The promoter is conserved amongst various species of both gram-positive and gram-negative bacteria. A long 19bp spacer region between the -10 and -35 consensus sequences (16-18bp is standard) allows for the promoter to be constitutively inactive. Crystallographic data shows that interactions between oxidised SoxR and the spacer reduced the spacer length by ~3.4Å, the equivalent of ~1bp, thereby returning the spacer region to a more standard length. This allows for transcription to occur from the promoter, demonstrating the promoter's mechanism of transcriptional activation. Previous papers proved how deletions of 2bp from the spacer region can manipulate this mechanism to create a constitutively active promoter with induction causing transcriptional repression due to shortening of the spacer region past the threshold for activity.

Usage

pSoxS combined with SoxR acts as a functional sensor of redox-cycling drugs and oxidative stress, making it a useful part for the creation of biosensors or devices activated by redox-cycling drugs, toxins, antibiotics, certain organic molecules, heavy metals, nitric oxide and hydrogen peroxide: all of which can exert oxidative stress on cells.

The 2018 Imperial College London iGEM project (PixCell) utilised SoxR in electrogenetic devices capable of activating gene expression in response to an electrical stimulus. This was achieved via oxidation and reduction of redox-mediators at an electrode. These systems provide programmable spatiotemporal control of gene expression with an inexpensive experimental set up.

The induction of this system by redox-cycling drugs makes it a particularly cheap system to use for chemical induction of gene expression, with the molecule PMS (phenazine methosulfate) being cheaper per reaction than several other common chemical inducers.

As part of the PixCell library, pSoxS can be coupled with various different parts to tune the dose response of the system to suit the device being constructed. This particular promoter is a mutant providing transcriptional repression rather than the transcriptional activation of wild-type pSoxS.

Characterisation

Prior to construction of this part into reporters, growth curve data of the part stored in an iGEM submission vector were taken to determine the cytotoxicity of the cells. DH5-α cells containing this plasmid grew to a final OD600 of 1.43 suggesting the part is not cytotoxic.

Figure 1: Growth curve of part BBa_K2862010.

Sanger sequencing was used to confirm the sequence of the part within the submission vector.

Characterisation was performed as part of a library of mutated SoxR transcription factors and mutated pSoxS promoters, using a simple reporter device assembled by BASIC assembly (Figure 2). Different SoxR transcription factors were expressed from a J23101 promoter and a medium strength RBS. Activation of GFP expression from combinations of mutated promoters and transcription factors was then determined.

Figure 2: Diagram of reporter circuit

The following heat maps (Figure 3) show the fluorescence in 2.5μM pyocyanin relative to the library promoter seed Part:BBa_K2862006, and the change in absolute values of fluorescence between 0μM and 2.5μM pyocyanin for a given promoter/transcription factor pair, have grown cells for 15 hours. We have evidence that the particular mutant promoter BBa_K2862010 presented on this page, in combination with the transcription factor BBa_K2862018, shows repression of GFP transcription in response to increased concentrations of pyocyanin.

Figure 3: Performance of library. Left: GFP fluorescence/OD600 for promoter/transcription factor pairs for cells grown for 15 hours. "0" indicates a failed experiment in which cells with a functioning construct were not observed. Right: absolute changes in GFP flourescence/OD600 for functioning constructs; the BBa_K2862010 / BBa_K2862018 pair shows evidence of repression in response to increased pyocyanin concentrations.

Sequence and Features