MoClo-Library for future iGEM teams
As a contribution for future iGEM teams we established the Modular Cloning system for Leishmania tarentolae. We designed and cloned 19 partswhich can be put together according to your needs. We decided to design different tags for protein purification as well as detection. Because of that, future iGEM teams can use this system to express their desired protein in Leishmania tarentolae. The variety of tags we used allows purification and detection of the desired protein via fluorescence microscopy.
Troubleshooting for dam methylation problem
In the process of finishing our destination vector, we had a problem with cutting a plasmid with a restriction enzyme which was blocked by dam methylation. Because of that, we tried to transform our dam methylated plasmid into the GM48 E. coli strain which is dam-. To our surprise, the transformation did not work as planned, so we decided to try out different transformation conditions and did a lot of troubleshooting as seen in the table below.
Combinations of different transformation conditions and their outcome.
Our first thought was that the GM48 E. coli cells were very squeamish, so we raised the time for regeneration after heat shock and started to just pour the cells on preheated agar plates, without success. Applying heat for a shorter duration or lowering of the DNA concentration was also unsuccessful. Increasing the amount of inserted DNA about the ratio 130 to 20 µg showed the first successful transformation. To optimize the transformation results we increased the amount of DNA even more and tested if preheating of the agar plates or just pouring of the cells is necessary at all.
As a concluded experience for future iGEM teams we found that the biggest factor for transforming dam methylated plasmids into dam- E. coli strains is the amount of DNA you use for the transformation. The efficiency of transformating dam methylated plasmids into dam- E.coli strains seems to be more than 200 times lower then of transformations of the dam methylated plasmid into a dam+ E. coli strain like TOP10. Because of that we would advise future iGEM teams, who want to do similar transformations, to increase the amount of used DNA by a factor of 200.
Our contribution not only includes our whole library and the dam methylation troubleshooting, but also a very detailed protocol on "How to handle Leishmania" as well as a Step-by-Step instruction for generating MoClo parts.