Team:Groningen/Notebook/tasA Transformation t16
Blue Bio Energy
tasA Heat Shock Transformation in E. coli (t16)
In this experiment tasA in the RFP backbone is transformed in E. coli.
Our goal is to overexpress TasA by adding a second copy of the tasA gene under the control of a salt inducible promoter. This overexpression will lead to a stronger biofilm when the biofilm is exposed to salt water.
The heat shock transformation was performed and colonies were visible after growing overnight.
Heat shock transformation
00:00, 21 May 2015 - 00:00, 21 May 2015
The mixture was incubated at room temperature for 2 hours. During this time ampicillin plates were poured (100 µg/mL). Afterwards the heat shock transformation was carried out using the following components.
10 µL ligation mixture in competent DH5 alpha
Competent cells Harm (E.coli (1)) (CompH)
Competent cells Clement (CompC)
The transformed cells were plated on LB+amp plates.
90% CompH
10% CompH
90% CompC
10% CompC
The plates were put in the incubator overnight at 37 °C.
00:00, 22 May 2015 - 00:00, 22 May 2015
The transformation was checked. There were 5 colonies visible on the LB+amp plates. 4 colonies on 90% compH and one colony on 10% CompH. Both plates were stored in the fridge at 4 °C.
00:00, 26 May 2015 - 00:00, 26 May 2015
The five transformed colonies were inoculated overnight in liquid LB+amp (30 µL amp per 3 mL LB (1/100)). This should have been 3 µL per 3 mL amp, so also this concentration was made and used. The culture of the colony from the 10% plate was labeled sample 1 and the cultures of the four colonies from the 90% plate were labeled sample 2, 3, 4 and 5. The cultures were incubated at 37 °C, at 250 rpm.