Expected results
Due to laboratory based difficulties, discussed later, we could not clone our parts, and therefore could not implement either system. Our project is based on two systems, using fluorescence for the quantitative analysis. There is a base level of fluorescence which will be used for comparison.Our expectation for the upregulation construct would be an increase of fluorescence from the baseline, this is due to the increased amount of RNA polymerase (RNAP) recruitment. RNAP recruitment is increased because the standard promoter is acting in tandem with the Cas9-sspA fusion therefore more GFP is being expressed.
For the downregulation system, we expected a decrease in the fluorescence from the baseline, as the Cas9 is bound to the DNA disrupting the binding of RNAP, this will result in it lower transcription lowering the expression of GFP.
What could have gone wrong
There are two major possibilities as to why we could not clone any synthesised part into a backbone, both are design based;- We Designed our biobricks with the prefix and suffix on the very borders of the sequence, limiting the effectiveness of the restriction enzymes. This would mean that when we tried to restrict the parts, they did not have the complementary sticky ends in order to ligate into the backbone.
- A problem with the calcitonin promoter sequence is that it may have been ordered without the prefixes or suffixes, this would obviously mean that the DNA ordered could not be used, unless we could order PCR primers that overlap with the ends of the sequence, and build the necessary cut sites onto the ends of the part.
