first accomplisment

My first attempt towards designing a proposal for my research thesis is to create a plasmid that carries a fusion proteins of 'enhanced cyan fluorescent protein - betagalactosidase - enhanced yellow fluorescent protein'. The plasmid has already been created but in order to make use of the plasmid, I have to transfer this fusion gene into a broad host range plasmid so that I can transform the plasmid into a large variety of bacterial species.

My approach for the transfer was to amplify the fusion gene by PCR, digest the amplified fusion gene and my broad host range vector, pMMB67EH, with restriction enzymes SmaI and XbaI, and clon the fusion gene into pMMB67EH. Initially I was having trouble with my PCR and it was solved after about a month. Then I couldn't get any positive transformants after I ligated the fusion gene into pMMB67EH. For the past 3 months, I've been changing one variable at a time, trying to find out the best conditions for the ligation. I was also trying different ways to obtain the DNA preparation without losing too much each step. But again and again, I've been getting white colonies on LB Amp-IPTG-Xgal.

Finally, I found a blue colonies! However, these colonies were initially white. But after spending last whole week screening these colonies, it has finally been confirmed that they do carry the plasmid that I have been aiming to construct. Phew!! This is a big step towards preparation for my research proposal. I finally have a thesis subject!!!

Now I'm looking forward to finding out more about the expression characteristics, which seem to be quite weak as I could not see any fluorescence under the microscope even after 6 hours of induction with IPTG. But knowing that they are blue when grown in the presence of IPTG and XGal, they are definitely expressing the gene. Woohoooo...I'm sooooo happy to achieve such accomplishment. At least it's a happy start of my PhD career.