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Results

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Introduction

Our Results page details our attempts to build our GFP (BBa_K5111004) and ComR (BBa_K5111002) parts in the lab. Unfortunately, as a high school team, we are dependent on others for lab space, so we only had a week in the lab. In that time, we attempted to construct our parts twice, both of which were unsuccessful. Please see the Notebook page for more details.

However, our supervisor in the lab, Dr Anatoily Markiv, continued to work on the parts, and successfully constructed both parts, although there appears to be a frameshift issue in the ComR part. Analysis of sequencing is ongoing as of 2 October, but further information will be available at the Jamboree, and following the Wiki thaw, sequencing documents will be uploaded.

Results

Gel results from PCR amplification of IDT constructs. Left to right: 100bp ladder, ComR (j), GFP (j), ComR (r), GFP (r), GFP (a), ComR (a). As the ComR insert is slightly shorter, it has moved further.

Parts were successfully amplified through PCR.

First attempt at transformation

Red colonies indicate ligation of the plasmid backbone to itself without the insert. We decided not to sequence these colonies.

Second attempt at transformation

Some red colonies indicate that the original plasmid was not fully digested; unfortunately this round resulted in the recombination of the plasmid backbone and the bacterial chromosome. Sequencing documents will be uploaded following the thaw, as analysis is ongoing.

Third transformation - completed by Dr Anatoily Markiv

Green colonies can be seen in those containing the GFP part. Sequencing of these colonies confirmed that the correct sequence was present. The expression of GFP in these colonies suggests that there is room for further suppression of the gene through synthesis of further ComR. This is promising, and presents an opportunity for future iGEM teams to explore how transformation of high copy plasmids containing the ComR CDS can further suppress expression of genes downstream of the binding site.

The ComR colonies in both backbones contained the part. However, the part in the psB1C3 plasmid appears to have a frameshift mutation. We are analysing the sequencing to find the potential source of this error, and further information will be provided when available.

Sequencing documents will be provided following the wiki thaw or at the Jamboree if requested. This is because analysis is still ongoing.