Hello,
While I was preparing for the experiment that I will show you guys in a few days, I wanted to show you an experiment that we had to carry out in a project we had last year. This project included plasmids (which can carry DNA information) and electroporation (a process that allows the cell to "absorb" the plasmid with the message encoded within).
It was one of the most interesting projects I had in my first year. The task was to integrate certain transposon into a plasmid (pGLO). We integrated a transposon in the pGLO plasmid and trhough electroporation we inserted the pGLO plasmid in E.coli bacteria. Electroporation is needed to make sure that the bacteria can receive the pGLO-plasmid.
The objective was to determine were the transposon inserted itself in the plasmid. This happens randomly. The bacteria are grown on a special agar media which allows only the growth of the bacteria whom received the pGLO plasmid. We had to determine wheter the transposon placed itself in the GFP gene, araC gene, AmpR gene (which codes for ampicillin resistance) or even in the origin of replication (which is the starting point of repliction of the plasmid).
After a lot of work and adjustments, we came to the conclusion that the transposon had inserted itself in the GFP gene and the AmpR gene. It did not place itself in the araC gene.
This is the shortest version that I can give you guys right now about the project. The goal was to integrate the transposon into a plasmid. Thereafter we had to determine were the transposon was placed in the plasmid by looking at the features that the E.coli had obtained from the plasmid (the plasmid codes e.g. for ampicillin resistance and other functions).
This is one of the results we got. We can see in this picture that the colonies that fluoresce do not have the transposon integrated in the GFP or araC region. Because the araC region is supplying the GFP with important proteins that the GFP needs to fluoresce under UV-light and the GFP region is needed to excecute the proces to fluoresce. Even with the transposon in the araC region, the colonies will still fluoresce under UV-light, but not so much as seen in the picture.
It was a lot of work. Sure we had to do some things over and over again, but we got the results that we needed and the team and I were very happy about it. It was a fun project and I hope we can replicate the proces that we took in other bacteria which are more special than the standard E.coli bacteria we use on the lab.
If you have any questions you can ask them and I will see you guys in a couple of days. I will be wourking on my official first post on one of the experiments that I am preparing for you.
Have a nice week guys ;)