At this stage we have both the FOFr and CheZ* with XbaI and SteI restriction sites. Our next goal will be to insert each part into a vector backbone so they will be ready for addition of the ToxR and Ctx genes. Once these two fusion products are made, we hope to transform and test whether local motion can be inhibited with CheZ and the receptor combo
August 2-
Gel electrophoresis and purification
Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify
Ligation at 16 degrees Celsius overnight
We added both CheZ* and FGFR each into its own vector and if the ligation just performed works we will have 2 new biobricks. The phosphorylation was done to prevent ligase from reannealing the X and S site on the vector. This means only the phosphate on the insert can be used to connect the 2 DNA sequences.
August 3-
Transformation of FOFR and CheZ*, DH5α, 50 µL 2 µL DNA at 37 degrees Celsius
August 4-
Incubation in 3 mL and chlora at 37 degrees Celsius and 220 rpm
Plating CheZ (K283006) and ToxR (J07009)
Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify
August 5-
Making glycerol stock
Miniprep
Incubation in 3 mL LB carb at 37 degrees Celsius and 220 rpm overnight
Test digestion ( 1 hour and 30 minutes)- about 200 ng of DNA
Gel electrophoresis
Observations: i2.15.16 looks positive, the rest are negative
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