16.5: Micro-report 4- Complementation analysis

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Figure: The figure at the heart of this micro-report is multi-panel figure showing replica plates of strains that have been transformed with overexpression plasmids. Refer to the guidelines for Micro-report 1 to see how to set up figures with multiple panels.

Materials and Methods: Provide information on the transformation and replica plating procedures, as well as the media used in the experiments. When possible, reference the lab manual, noting any changes that you made to procedures.

Strains and plasmids: See micro-reports 1 and 3.

Media: See micro-report 1.

Transformation: Someone trying to reproduce your results will need to know details about the transformation procedure, because transformation efficiencies vary widely and show a strong dependence on reagents and incubation conditions. You can reference the lab manual.

Replica plating: This is a standard procedure. You can also reference the lab manual.

Results and Discussion: Your figure with the scanned plates is the focal point of this section. Include a single data table that with the calculated transformation efficiency with each plasmid and the ability of transformed strains (Y/N or +/-) to grow on the various replica plates.Transformation efficiencies should be expressed in number of transformed cells/μg plasmid DNA.

The R&D section should tell a story of how the replica plates allowed you to decide if your
Met protein is conserved between the two yeast species. You may or may not have observed complementation. Failure to observe complementation is not necessarily due to experimental error. (Which plates serve as a control against experimental error?) Complementation is a functional assay that depends on both the expression of the fusion protein and the ability of the fusion protein to catalyze a reaction in Met biosynthesis. The fusion proteins have large C-terminal extensions that might affect their normal enzymatic functions. Negative results can be just as important as positive results in advancing scientific understanding.

If you did not observe complementation, discuss possible reasons that this may have happened and propose future experiments that could help to answer these questions. You may want to suggest new plasmid constructs for additional experiments. You may also want to bring in information from your BLASTP analyses. (What is the E-value?) Be sure to include enough justification that your proposed experiments will provide useful data.