Week 5

August 8-

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 9-

Transformation of FGFR and CheZ* with pSB1C3 vector, 50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all chlora

August 11-

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

August 12-

Post- incubation observation: i2.17.4- through i2.17.10 all show positive result, i2.17.11- did not change

Results: All incubated colonies grew as expected. I2.17.4 tube seemed less dense than others. Negative control did not grow as expected.

August 13-

Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)

August 14-

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