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Experimental Layout
From 2010.igem.org
University of Aberdeen - ayeSwitch
Troubleshooting pRS414
Aim
Following the analysis of the two promoters using N4 and N25 the next experiments planned would start to characterise the interactions of both constructs (pRS414 and pRS415) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or any CFP in our calibration samples.
Hypothesis
Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).
The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.
Protocol
A series of experiments were set up in order to determine the functionality of various parts of pRS414 in view to repair the construct.
1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP
2. Confirming the CFP sequence is functional
3. N-GFP Swap Experiment
4. Replacing the CUP1 promoter in pRS414 with the CUP1-2 promoter from the N4 construct
Conclusion
The second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the Cup1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.
Our data suggests that the reason why pRS414 is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjuncture with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR as a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.
Our experiments do not rule out however the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion to fluorescence due to improper folding.
