IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/07/17
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Today's experiments were not totally relevant to the project. But because of our suspicions that our enzymes are not working properly, we decided to test them on J04450 and another plasmid called pSB, while waiting for the sequencing results to arrive. We tested Pst1, Nde1 and Xba1 enzymes.
To continue protein expression half of the pellets (1a,1b,2a,2b) were resuspended (in 1X binding buffer), sonicated and centrifuged. The cells were then processed to give soluble and insoluble fractions of proteins. The samples were stored in the freezer.
Made up 600 ml of LB media (-NaCl).
Performed transformations of E. coli cells using heat shock and electroporation. Two different vectors were used, pMS82 and pAU3-45 with different resistance genes and insertion sites. Each plasmid contains the biosensor (our reporter construct from Floor 2 which consists of the AntGP promoter + Neomycin gene for kanamycin resistance). After E. coli is transformed, the plasmids will be conjugated into Streptomyces sp. isolated from the soil samples.
pMS82- Hygromycin (Hyg) resistance. pAU3-45 - Apramycin (Apr) resistance.
Top10 cells were transformed via heat shock and ETpuz cells were electro-competent and transformed via electroporation.
Plated on LB (-NaCl): + Apr (pAU3-45) Top10 + Apr + CM(Chloramphenicol) (pAU3-45) ETpuz + Hyg (pMS82) Top10 + Hyg + CM (pMS82) ETpuz