Experiemtal Layout

Results

 

Title:

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

 

Conclusions

As a result ...