Summer Intern Project 2010: Andrew Johnston

Project: Mutating a fluorescent protein to overcome the diffraction limit


Supervisor: Dr Guy Moss


The aim of the project undertaken over a six week period was to mutate a fluorescent protein so that its properties could be exploited in the context of the aforementioned STED technique.  For example, by mutating a protein that could be switched between dark and bright states (photoswitching) imaging could be performed via STED-like methods. Attempts were also made to model the parameters affecting a laser beam, which would be influential in the possible applications of STED-like methods.

Initially, three separate mutants were attempted to be introduced into the fluorescent protein. These were mutations that had previously been identified as potentially having an influence on the photoswitching ability of the protein. The mutations were introduced into the existing DNA by firstly using overlap extension PCR. Subsequently, the mutated DNA was ligated into bacterial plasmids using restriction endonuclease enzymes. The resulting plasmids were transformed into bacteria, which were then grown on agar plates in order to harvest sufficient quantities of the plasmids containing the mutation. Once these new plasmids had been extracted and purified they were transfected into cells, to enable observation of whether the desired characteristics had been expressed. 

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On comparing the fluorescence of the successfully mutated protein to its unmutated counterpart, there appeared to be the opposite of the desired differences in photobleaching/photoswitching, as shown in Figure 1A.  Namely, the recovery of the mutated FP was slower than the wild type. As said, the possibility of photoswitchable fluorescent proteins opens up new perspectives for overcoming the diffraction limit by STED-like methods.



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