IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/07/17

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Lab

Floor Two

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.

Floor One

Made up 600 ml of LB media (no salt) - Difco Bacto tryptone, Difco yeast extract, glucose, distilled water and agar.

Performed transformations of E. coli 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 - 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.

Heat shock method - cells thawed on ice. An aliquot of 1 ul of DNA was added. Incubation period of 30 mins. Heat shock at 42°C for 90 sec. Incubation of ice, 4°C for 2 min. Added 850 ul 2 x yt. Incubation for 1 hour shaking (250 rpm) at 37°C.

Electroporation - DNA added to cells, mixed gently. Added to ice cold cuvette. Electroporation - EC2 setting. Added 850 ul 2 x yt. Incubated for 1 hr at 37°C shaking.

Plated on LB (-NaCl): + Apr (pAU3-45) Top10 + Apr + CM(Chloramphenicol) (pAU3-45) ETpuz + Hyg (pMS82) Top10 + Hyg + CM (pMS82) ETpuz



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