Laboratory Notebook

13-08-2012 to 19-08-2012

We needed to address a problem with our genes and this was where our lack of experience finaly hit us. The transition from eukaryotic cells to bacterial cells has come with an untill now unforseen complication: the ribosomal binding site for eukaryotic cells(Kozak sequence), does not bind bacterial ribosomes, so we need to insert the bacterial version(Shine-Dalgarno sequence). Bacterial ribosomes bind to different sequences AGGAGG in a different distance, 6-7bp, from the coding sequence start codon ATG, while eukaryotic cells like plant cells have a ribosomal binding site CACC just in front of the start codon. Due to the original primers we designed for the coding sequences of both 1-FFT and 1-SST, both sequences still contained the Kozak sequences. We decided to let the genes hold on to the kozak sequence and just mutate the shine-dalgarno sequence hardcoded into the sequence between the restriction site and the start codon. This way our gene will be able to be transcribed in BOTH eukaryotic and prokaryotic cells. New primers were designed and ordered to accommodate the changes needed for the Shine-Dalgarno sequence in appropriate distance from the start codon for both genes. Meanwhile, we achieved high enough concentration for the one 1-FFT colony without the undesired EcoRI and sent it for sequencing along with 1-SST.