Difference between revisions of "Part:BBa K2862018"

Latest revision as of 02:27, 18 October 2018

SoxR from Phytobacter sp. SCO41

SoxR is a redox-sensitive transcription factor providing transcriptional activation or repression downstream of the pSoxS promoter when oxidised, 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 SoxR to build electrogenetic devices where electrical inputs modulated gene expression.

This SoxR CDS was taken from Phytobacter sp. SCO41, and is codon optimised for E. coli. It forms part of the PixCell library of electrogenetic and redox-sensing parts.

This part has been designed to be fully modular, being compatible for BioBrick, BASIC and Golden Gate assembly.

Biology

SoxR is a homodimeric transcription factor conserved amongst various species of both gram-positive and gram-negative bacteria. The protein is constitutively expressed from the pSoxR portion of the pSoxR/pSoxS bidirectional promoter, with a steady state concentration of about 75 SoxR proteins per cell in E. coli under normal conditions. Each monomer of the protein contains a single 2Fe-2S cluster. Direct interactions with redox-cycling drugs or oxidative stress caused by superoxide ions (either produced directly or via other chemical species) cause oxidation of these clusters. The resulting structural change allows SoxR to bind and provide transcriptional activation downstream of the pSoxS portion of the bidirectional promoter. This allows for activation of the soxRS regulon which in turns provides adaptation to conditions of oxidative stress.

Usage

SoxR combined with pSoxS acts as 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, SoxR can be coupled with various different parts to tune the dose response of the system to suit the device being constructed. This library includes a mutant promoter where SoxR acts as a transcriptional repressor.

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 1.4 suggesting the part is not cytotoxic.

Figure 1: Growth curve of part BBa_K2862018.

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.

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 49
Illegal BsaI.rc site found at 526
Illegal SapI.rc site found at 357

@@ Line 25: / Line 25: @@
 ===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 X suggesting the part is not cytotoxic.
+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 1.4 suggesting the part is not cytotoxic.
-Fig X23 Growth curve of part
+[[File:T--Imperial College--growthcurveBBa K2862018.png|thumb|center|Figure 1: Growth curve of part BBa_K2862018.]]
 Sanger sequencing was used to confirm the sequence of the part within the submission vector.
-Fig X24 Sequencing quality scores? Phred scores?
-Characterisation was performed using a simple reporter device assembled by BASIC assembly. This SoxR was expressed from an improved version of the J23101 promoter (BBa_K2862019) and a medium strength RBS. Activation of GFP expression under the control of every pSoxS promoter in the library was determined.
+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.
-[[File:T--Imperial College--diagramofreportercircuit.png|thumb|center|Figure 2: Diagram of reporter circuit]]
+[[File:T--Imperial College--newlidconst.png|thumb|center|Figure 2: Diagram of reporter circuit]]
-The following heat maps show the fold induction and RFU values of all members of the library, growing cells for 15 hours with either 0μM or 2.5μM of pyocyanin.
+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.
-[[File:T--Imperial College--heatmapswht4.png|thumb|center|Figure 3: Relative promoter units (RPU) of PixCell library, growing cells for 15 hours with either 0μM or 2.5μM of pyocyanin.]]
-Some members of the library form orthogonal pairs that can be used together with minimal cross-talk.
-Fig X26: List of Orthogonal Pairs
+[[File:T--Imperial College--heatmapswht4.png|thumb|center|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.]]