Troubleshooting the GFP-tagging gene knockout (GGKO) method for the Leptosphaeria maculans effectors AvrLm6 and AvrLm4-7

Sammanfattning: An attempt was made to GFP-tag the effector proteins of AvrLm6 and AvrLm4-7 using the GFP-tagging gene knockout method (GGKO) developed by Saitoh et al. (2008) in order to determine whether or not they are secreted. Successful pETHG-(target)KO vectors were not generated. The protocol was examined for potential errors. Fatal errors were pinpointed to the ligation reaction and the transformation required to generate and propagate the desired vector pETGH-(target)KO. The Downstreams Flanking Region inserts were evidently successfully ligated into the pETHG vector but for the Upstreams Flanking Region inserts the results were highly ambiguous. Due to a mistake, too high vector:insert ratios were used; altering them to the recommended 1:1 – 1:5 is hence expedient. It was reasoned that the bacteria possibly absorbed empty pETHG vectors instead of putative insert-carrying pETHG-(target)KO vectors during the transformation. The procedure could be improved by digesting pETHG twice prior to ligation as well by separating linearised and uncleaved pETHG by gel extraction. The latter could also be performed on the ligation product. Suggested general improvements include: Sequencing the PCR product and purifying it of potential restriction enzyme inhibitors, use the maximal incubation time for the restriction enzymes, expand the colony screening, increase the spectinomycin concentration and test different bacterial strains in the transformation.