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Team:Exeter/Parts
From 2014.igem.org
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Contents
The XenB Construct: (BBa_K1398001)
XenB is an NADH-dependent flavoprotein (Xenobiotic Reductase B) from the soil bacterium Pseudomonas fluorescens((BBa_K1398001)). We chose XenB because:
- XenB may serve a dual purpose as a Nitroglycerin and TNT degrading enzyme (see ( The Enzymes: XenB) .
- The phylogeny of Old Yellow Enzyme memberssuggests that XenB is similar in sequence to the previously submitted PETN reductase and is comparatively well understood.
- Heterologous expression of XenB from P. fluorescens has been previously demonstrated in E. coli DH5α enhancing our chances of successful expression.
The coding sequence for P. fluorescens XenB has been deposited within GenBank (accession no. AF154062)and this forms the functional unit of our construct (BBa_K1398001) (Figure 1):
Figure 1: The XenB construct depicting the constituent parts of the construct.
The construct contains the coding sequence for XenB (BBa_K1398000). Expression of XenB is driven by a Lactose-inducible promoter (BBa_R0010) coupled with a strong RBS (BBa_B0034). The construct is terminated using a double terminator made up of BBa_B0010 and BBa_B0012.
The NemA Construct: (BBa_K1398003)
With the nemA DNA sequence identified28 we were able to design our construct to evaluate NemA as follows (Figure 2):
Figure 2: The NemA construct depicting the constituent parts of the construct.
The construct contains the coding sequence for NemA (BBa_K1398002), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism. The construct also contains a Lactose-inducible promoter (BBa_R0010), a strong RBS (BBa_B0034) and a double terminator made up of BBa_B0010 and BBa_B0012). The protein has been codon-optimised for expression in E. coli.
The NemR promoter constructs
The development of the NemR promoter was originally as part of the design of a kill switch we intended to implement. However, it was clear that impressive kill switches have already been widely developed and used. We decided instead to focus on a NemR switch that could be used to regulate them such that the bacterium would die in the absence of TNT. When we designed the NemR promoter, we debated where the ‘NemR box’ would be inserted into the construct sequence to enable successful gene repression and expression. We eventually opted for two constructs, one with the entire NemR region was placed upstream of the promoter (The intergenic reporter BBa_K1398004) and one where the NemR box was placed in-between the -12 and -33 region of the promoter (BBa_K1398007 as this region appeared reasonably conserved across the Anderson promoter group available to us on the registry: http://parts.igem.org/Promoters/Catalog/Anderson.
The NemR intergenic reporter construct(BBa_K1398004)
We designed the NemR upstream intergenic construct, BBa_K1398004, as follows:
This construct was created to test the effectiveness of the NemR upstream intergenic region (BBa_1398005). The construct contains the entire NemR upstream region (BBa_K1398005), which contains a promoter and RBS. It is followed by the fluorescent reporter iLOV (BBa_K660004), a double STOP codon and a double terminator made up of BBa_B0010 and BBa_B0012.The NemR box promoter construct(BBa_K1398007)
We designed the NemR box insert construct, BBa_K1398007, as follows:
Figure 4: The NemR construct depicting the constituent parts of the construct.
The construct begins with the synthetic promoter NemR, which combines a high-expression promoter with the NemR recognition box (BBa_K1398008). It is followed by a strong RBS (BBa_B0034), the fluorescent reporter iLOV (BBa_K660004), a double STOP codon and a double terminator made up of BBa_B0010 and BBa_B0012.
The addition of the iLOV reporter gene was also to help further characterise part BBa_K660004 submitted into the iGEM database in 2011 by the team from Glasgow: https://2011.igem.org/Team:Glasgow/LOV2. Given that this promoter would respond to TNT, this means we also aim to generate a biosensor for TNT.
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