Team:Aachen/Notebook
Lab Book
To express a photocaged protease in our case subtilisin E, we developed three lab strategies. Their milestones can be seen below in three different timelines, one for every sub-division. Details about reaching these milestones will become visible, if you click on them. On the page “Project Idea” the theory beneath our lab work is described and on the subpage “Protocols & Methods” under “Lab” the exact protocols can be seen, if you would like to repeat the experiments.
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Research on screening system. Decision to work with kit from iGEM Team Austin, Texas 2014
Definition of incubation conditions
Arrival and Preparation of Testkit with DNA from Texas
Recovery of plasmids in TRIS buffer pH 8.5, followed by transformation in BL21 DE3 gold, LB solid plates with Gentamycin 30µg/ml.
Determining concentration of antiobiotcs in LB
The growth of E.coli cells transformed with plasmids wild type tyrosine -tRNA/synthetase and oNBY-tRNA/synthetase on LB solid as well as in LB liquid is not affected by concentrations of gentamycin up to 30µg/ml, whereas a wild type BL21 DE3 gold is affected already at 5µg/ml. Hence, appropriate concentration for gentamycin plates and cultures is 30µg/ml. This observation is also valid for 50µg/ml Kanamycin tested with transformed reporter plasmids in BL21 DE3 gold.
Furthermore the combination of the two antibiotics is evaluated. Since BL21 DE3 gold showed a proper growth rate in LB with 30µg/ml gentamycin + 50µg/ml Kanamycin when transformed with a reporter plasmid and a synthetase plasmid the assessed concentrations are kept.
Determining concentration of atibiotics in M9 minimal medium
Competent cells already containing the reporter plasmid pFRY are beeing transformed with the PCR product from Site Saturated Mutagenesis 3 onto M9 solid with different antibiotics concentration and afterwards picked in overnight cultures with the same antibiotics concentrations. A gel electrophoresis as well as a colony PCR is made to detect, if the concentration of both antibiotics is high enough to have a appropriate pressure on the cells, but is low enough, so that cultivation of cells is after all possible in minimal medium. The growth on microtiter plates is as well evaluated via plate reader.
Gentamycin 5µg/mg + Kanamycin 50µg/ml is the only concentration where E.coli is growing and reliably keeps both plasmids. As after transformation on M9 solid with Gentamycin 5µg/ml some wild type BL21 colonies grew, gentamycin concentration in M9 solid was increased: Transformatiom onto Gentymacin 10µg/ml + Kanamycin 50µg/ml and subsequent picking in liquid M9 cultures with Gentymacin 5µg/ml + Kanamycin 50µg/ml resulted in practically no growth of BL21 wild type but in appropriate growth rates of cells transformed with two plasmids.
- Gentamycin 10 µg/ml
- Kanamycin 50 µg/ml
- Gentamycin 5 µg/ml and
- Kanamycin 10 µg/ml
