Week 6

August 15-

PCR of ptetR, RBS, LuxI, term

Gel electrophoresis and purification (insert 2 pictures here)

Glycerol stock and miniprep

Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm

Test digestion for 1 hour and 30 minutes at 37 degrees Celsius

Gel Electrophoresis

Test Digestion Observation: Lane 5 gave positive results for FGF-R: 2 bands observed at predicted bands size. Lane 4, 7, 9 were all negative bad results because EcoRI was used but only 1 band showed. This suggests that FGFR was not inserted. Lanes 10, 11, 12 were CheZ* and all matched negative control. Results are bad for CheZ*- will be regrown/miniprep/ligated.

Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm

August 16-

August 17-

Digestion: 1 hour and 30 minutes at 37 degrees Celsius

Plating agar stab of ToxR

Dephosphorylation for 1 hour at 37 degrees Celsius

PCR purify

Ligation- incubated at 16 degrees Celsius overnight

Summary: Digestion of positive result sample of previous FGFR biobrick was performed with EcoRI to verify if true/false positive results. In parallel, a digestion of Alfredo’s phototaxis receptor and eYFP was performed using Alfredo’s backbone XbaI and SpeI. This was done to take it out of the native back bone and eventually ligate into new vector backbone (pSBIC3). First the part was dephosphorylated and then ligated into new vector. At previous FGFR and CheZ* (digested parts) were also ligated into the vector backbone pSBIC3 in parallel procedure (will incubate overnight). ToxR, which did not grow previously, was re-inoculated and incubating overnight.

August 18-

Digestion: 1 hour and 30 minutes at 37 degrees Celsius

Plating agar stab of ToxR

Dephosphorylation for 1 hour at 37 degrees Celsius

PCR purify

Ligation- incubated at 16 degrees Celsius overnight

Summary: Digestion of positive result sample of previous FGFR biobrick was performed with EcoRI to verify if true/false positive results. In parallel, a digestion of Alfredo’s phototaxis receptor and eYFP was performed using Alfredo’s backbone XbaI and SpeI. This was done to take it out of the native back bone and eventually ligate into new vector backbone (pSBIC3). First the part was dephosphorylated and then ligated into new vector. At previous FGFR and CheZ* (digested parts) were also ligated into the vector backbone pSBIC3 in parallel procedure (will incubate overnight). ToxR, which did not grow previously, was re-inoculated and incubating overnight.