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Contents 1. Our Parts 2. Basic Parts 3. Composite Parts

Our Parts

The 2014 Exeter iGEM Team has submitted eight parts to the iGEM Registry this year. Four of them are basic parts while four of them are composite parts. Each of the four basic parts is a simple protein coding or regulatory sequence; they each have a specific composite part in which they have the complementary sequences surrounding them; the enzymes have regulatory features such as an inducible promoter and terminator sequence, and the regulatory sequences have a reporter gene following them, so we can examine their expression.

When one of our parts is said to be codon optimised for E. coli we mean that the genes encoding the protein from the original organism were reverse-translated and codon-optimized for expression in E. coli using DNA2.0 GeneGPS Technology, and synthesized as BioBrick RFC10-compatible parts and cloned into an expression optimized Vector. The constructs were transformed into the iGEM vector pSB1C3 for submission to iGEM HQ. This service was provided by DNA2.0 Inc.

Basic Parts

BBa_K1398000 : XenB (Xenobiotic Reductase B)

Xenobiotic Reductase B, created for use as a Trinitrotoluene and Nitroglycerine degrading protein.

A monomeric flavin that can reduce certain nitro- groups to nitrate-. Increases the resistance of organisms to the toxic effects of nitrocompounds.

This sequence encodes for a protein (with an attached His (x6) Tag to allow for purification) and nothing else. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimized for expression in E. coli.

BBa_K1398002 : NemA (N-ethylmaleimide reductase)

N-ethylmaleimide reductase, created for use as a Trinitrotoluene and Nitroglycerine degrading protein. NemA is a flavoprotein that primarily catalyses the reduction of N-ethylmaleimide (NEM), which is toxic to cell growth.

However, it is also involved in the degradation of other toxic compounds for their reuse in nitrogen metabolism. Some of these compounds include PETN, quinones and chromate. It increases the resistance of organisms to the toxic effects of these compounds.

This sequence only encodes for the protein, an attached His (x6) Tag to allow for purification and a double-STOP codon. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimised for expression in E. coli.

BBa_K1398005 : NemR Upstream Intergenic Region

This sequence is found upstream of several NemR regulated genes. It was created for use as a TNT-detection mechanism.

It contains the bases from X+ to the start codon, which include a promoter and RBS. This region should allow regulation of gene products through the detection of TNT. If it is not present NemR will bind to a specified sequence of bases and inhibit transcription. If TNT is not present NemR will not bind and transcription will complete.

BBa_K1398008 : NemR Recognition Promoter

This sequences combines a high level constitutive promoter with the NemR binding box, which allows NemR to bind to DNA when no TNT is present in the cell. It was created for use as a TNT-detection mechanism. It combines BBa_J23100 with the NemR box to create a promoter that will theoretically have high levels of transcription when TNT is not present.

Composite Parts

BBa_K1398001 : XenB (Inducible Construct)

A construct created to degrade TNT and nitroglycerin. The construct contains the coding sequence for XenB (BBa_1398000), an enzyme with the capability to degrade nitro- groups in chemicals.

The construct also contains a Lactose inducible promoter (BBa_R0010), a strong RBS (BBa_R0034) and a double terminator of BBa_B0010 and BBa_B0012. The protein has been codon-optimised for expression in E. coli.

BBa_K1398003 : NemA (Inducible Construct)

A construct created to degraded TNT and nitroglycerin. The construct contains the coding sequence for NemA (BBa_1398002), 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_R0034) and a double terminator made up of BBa_B0010 and BBa_B0012. The protein has been codon-optimised for expression in E. coli.

BBa_K1398004 : NemR Intergenic Reporter

A construct created to test the effectiveness of the NemR upstream intergenic region (BBa_1398005).

The construct contains the entire NemR upstream region (BBa_1398005), which contains a promoter and RBS. It is followed by the fluorescent reporter iLOV (listed in the repository as BBa_K660004), a double STOP codon and a double terminator made up of BBa_B0010 and BBa_B0012.

See Detection of Xenobiotics for the results of testing.

BBa_K1398007 : NemR Promoter Reporter

A construct created to test the effectiveness of the NemR recognizing promoter (BBa_1398008).

The construct begins with the synthetic promoter NemR, which combines a high-expression promoter (BBa_J23100) with the NemR recognition box (BBa_K1398008). It is followed by a strong RBS (BBa_R0034), the fluorescent reporter iLOV (BBa_K660004), a double STOP codon and a double terminator made up of BBa_B0010 and BBa_B0012

See Detection of Xenobiotics for the results of testing.

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