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Main purpose

• Colony PCR of AMO, HAO, cytochromes and Nir transformants

Who was in the lab

Julia, Kristian, Gosia

Procedure

Colony PCR

Colony PCR was performed according to standard method in order to check if transformed cells which grew on medium contain desired insert in pZA21 plasmid. Constructs for transformations contained one gene in plasmid pZA21 and were created using USER reaction. Inserted genes are as follows: HAO, AMO, cycAX (cytochromes), Nir operon.

Several colonies were chosen from each plate to be checked. Names and numbers of colonies as well as plates names and primers are:

• 1,2 - HAO in pZA21 USER, primers 18a, 18b
• 3-10 - AMO in pZA21 USER, primers 17a, 17b
• 11-24 - cycAX in pZA21 USER, primers 16a, 16b
• 25-28 - Nir in pZA21 USER, primers 15a, 15b
• RFP 1 - RFP 20 - RFP in pZA21, primers 14a, 14b

PCR programms based on standard PCR programm with differences in annealing temperatures and extension time as it was done on 12-07-2013.

Gel electrophoresis

Samples after colony PCR were checked on 1% agarose gel today and most will be checked tomorrow.

M9

Made 3L of M9 minimal medium the the following recipe for 1L of medium:

• 10 mL Casamino Acid (from stock solution)
• 2 mM MgSO4 (1mL from 2M liquid stock)
• 0.1 mM CaCl2 (0.1mL from 1M liquid stock)
• 40g glucose (then the final solution will be 0.4 weight/vol)
• 200 mL M9 salts x5
• Fill to 1 L with sterilized water

1L x5 stock of M9 salts:

• 64 g Na2HPO4*7H2O = 31.2 g Na2HPO4
• 15 g KH2PO4
• 25 g NaCl
• 5 g NH4Cl

Conclusion

USER transformant from last Saturday didn't seem very good and we speculate that this could be because of membrane stress caused by all of the membrane proteins that we are suddenly incorporating. We will try to get around this by making a vector with an arabinose inducible promoter. Then there will be no damaging protein expression before we apply arabinose.

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