Results

PCR of nadD, nadE and PncB

The three aforementioned genes were PCR amplified initially without the iGEM prefix and suffix, which would enable the correct hybridisation of the primer to the E.coli genomic DNA to amplify the genes. The forward and reverse primer sequences for these genes are: (see table)

To make these genes into biobricks the iGEM prefix and suffix (underlined) were added to primers (called fusion primers) which can be seen in the table below. (see table)

These primers were used for PCR amplification, using the previous PCR products as the target of amplification rather than genomic DNA. NadD and NadE amplification were successful.

These primers were successful in amplifying these genes and was confirmed through gel electrophoresis. However as PncB has a Pst1 site which is incompatible with the biobrick system, internal primers were used which had a single base change (from a G to a C) to remove the restriction site. The Forward Internal and the Reverse Exterior primer were used for one PCR reaction and in another it was the Reverse Internal primer and the Forward Exterior primer. This resulted in two PCR products overlapping at the removed restriction site. This was confirmed by a gel. The forward and reverse exterior primers were then used to amplify these products into one PncB amplification and thus having a PncB without a restriction site. This was confirmed by digesting this PncB with Pst1 and resulted in one band, as can be seen in figure 1. The PCR product that was successful was purified and had concentrations of 8.2ng/µl on average. (see figure 1)

Transformation

gBlocks were ordered from IDT for NadD, NadE, PncB; with a promoter, RBS and TAT signal peptide sequence immediately upstream and the Kill Switch. This was because ccdA was not a part and it was more time efficient to synthesis the Kill Switch than to assemble all the individual components. The NadD, NadE and PncB were ordered as it allowed for the fusion of the TAT signal peptide and our genes of interest, which cannot happen via the 3A assembly system. These sequences contained the iGEM prefix and suffix to be compatible with the 3A system. These were first digested with EcoR1-H1 and Pst1 and ligated into pSBC3 backbone, then transformed into NEB competent E.coli cells. The negative control for the transformations was cells that are not chloramphenicol resistant, the positive control being RFP as it has a clear visual conformation of correct transformation, as can be seen in figure 2. The transformation for the IDT ordered parts did not grow, although the controls worked. This happened multiple times, with different team members performing the transformation. The protocol was altered to increase transformation efficiency but did not produce any notable change. This also occurred from the IDT ordered NadD, NadE and PncB without promoters, RBS or TAT signal peptide sequence as well as the Kill switch part which has the ccdB containing a stop codon. Thus no biobricks were created. ( see figure)

Discussion

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