Team:Aberdeen Scotland/WetLab/lacilatch/cloning

Cloning Strategy:
In order to construct the module in an effective and testable manner we had to take a number of things into consideration. The final product had to be easily compatible with the two other modules, in the sense that antibiotic resistance markers on the various plasmids should be different to allow straightforward selection for two or more plasmids following a transformation. With that in mind, we decided, in dialogue with the rest of our team, that the final version of this module should be on the medium copy Kanamycin plasmid pSB3K5. In addition to compatibility to the other modules, we decided that we wanted to be able to test aspects of the module before it was fully constructed. This was because we faced a rather large number of cloning steps and we wanted to make sure that we could test the intermediate parts, whenever possible. For this purpose we identified the BioBrick’s BBa_I13600, which consists of a Tet operon followed by a CFP coding gene on an Ampicilin Plasmid, and BBa_I13601, which is alike to BBa_I13600 apart from the having a Lac Operon instead of the Tet Operon.



Our first step in the cloning involved ligating K182004 (upstream) and S03518 (downstream) together into a Cholramphenicol vector, creating our first BioBrick named K182005. K182005 was then tested using BB_I13600, by transforming them both into a cell and observing the behaviour of the CFP expression.

The second step in our cloning strategy was the ligation of I732820 (upstream) and R0040 (downstream). This was also cloned into a Cholramphenicol vector. The result was our second BioBrick named K182001. The third cloning step involved the ligation of K182005 (upstream) and K182001 (downstream) into a Tetracycline vector. This construct could then have been tested using BBa_I13601 in essentially the same manner as BBa_I13600 was used in relation to K182005. However this cloning step was not successful and had to abandoned due to time constraints. The placeholder name of this clone is K182006.

The fourth and final step of this module would have been the addition of the Holin cassette downstream of K182006, at which point the whole cassette could have been tested on its own or in conjunction with other modules. It was important that the Holin cassette is added as the very last module as it would have killed the cell had it been present without the Tet Repressor.