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From 2013.igem.org

Post-HK Design

Throughout the year we’d been trying to assemble our DCA-degrading pathway in a single Gibson Assembly reaction. An approach with which we had found some troubles. We believe that the strong constitutive expression of our operon, especially some of our larger parts such as the cytochrome p450,-ferredoxin-reductase cluster may actively harm the cells or be metabolically taxing, effectively create a positive selection pressure for misassembled Gibson inserts. With this one-shot approach, we had left no flexibility in the face of failure: because we’d designed our gBlocks to assemble all at once, when this failed we couldn’t assemble the genes in our pathway from parts.

Having learned the virtues of modularity and flexibility the hard way, upon returning from the igem2013 Asia jamboree we designed replacement gBlocks for the start and end of each gene with the BioBrick prefix and suffix respectively. These new gBlocks, along with our old intermediate gBlocks could be gibson assembled with the Registry’s shipping vector, pSB1C3 separately. Because we still suspected that our pathway, or parts-of, might be toxic in E.coli we designed an inducible promoter system consisting of the Tetracycline resistance operon promoter preceded by a constitutive tetracycline repressor protein generator.

This approach would allow us to:

• use the more time-consuming but flexible of [http://parts.igem.org/Assembly:Standard_assembly conventional BioBrick assembly].
• identify which, if any, genes present a toxic or metabolic burden to E. coli.
• characterise each gene individually to inform our model.
• optimise the order of genes in our pathway.
• submit all of our genes as individually characterised parts that might be used by others in any other projects or context.

Post-HK Results

Gibson Assembly

When assembled individually, we observed colonies from transformation of Gibson assembly produces for all our genes:

• ([http://www.ncbi.nlm.nih.gov/protein/2660722 aldehyde dehydrogenase A] from Xanthobacter autotrophicus (aldA).
• [http://www.ncbi.nlm.nih.gov/protein/91700989 cytochrome p450]-[http://www.ncbi.nlm.nih.gov/protein/91700987 ferredoxin]-[http://www.ncbi.nlm.nih.gov/protein/91700988 reductase] from Polararomonas JS666 (CYP450)
• Our Tet-based inducible promoter system.
• Not adh1b2 because we forgot to order it! Oops.

The cytochrome p450,-ferredoxin-reductase cluster transformants yielded comparatively fewer colonies than the other two. This may be in-part the result of a more complex assembly (five blocks compared to two or three) and in-part a result of the suspected high metabolic load the cluster places on cells. To circumvent the low yield of clones we performed successive round of transformations from the gibson reaction product.

We genotypically screened around 40 colonies for each construct initially by junction PCR, followed by diagnostic digests of plasmid preparations of promising colonies.

Cloning

We Cloned our inducible promoter system in front of each transcriptional part (aldA and CYP450) and genotypically screened colonies by junction PCR and plasmid digest.

Characterisation

CYP450 catalyses the degradation of 1,2-Dichloroethane (DCA) to chloracetaldehyde, releasing chloride ions. We performed a chloride assay on successfully screened clones of CYP450 under control of our promoter system in the presence and absence of Tetracycline to test the activity of CYP450 on DCA

With thanks to: