Notebook : July 3

Summary:

FNR regulator system:

• Continued what we started yesterday. Observed the Petri dish, selected the colonies. 4 colonies in total, they were 2 include FNR+plasmid in PSB1C3 and 2 from the control.
• Used 4 primers: VF2, VR, Pfnr_up, Pfnr_down for the verification test. They were designed to cut 4 special sites for creating 3 different regions on plasmid chain: VF2/VR, VF2/PFNR_down, PFNR_up/VR. After the amplification, those PCR products had been put on electrophoresis gel for the verification.

Lab work

• A.aero/anaerobic regulation system

• 2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
• BioBrick RBS+amilCP+terminator in plasmid PSB1C3

Observation

After the incubation overnight, we observed the Petri dishes.

colonies Normal concentration High concentration control 11 60

Fnr in plasmid PSB1C3	0	2

We picked up 4 colonies for further test (2 include FNR+plasmid in PSB1C3 and 2 from the control) PCR et Primer VF2, VR, Pfnr_up, Pfnr_down are 4 primes that we used for plasmid restriction. The primers, if the promoter fnr entre the plasmid successfully will amplify 3 sub-pieces with specific size.

We had prepared 4(colonies)*3(amplification) = 12 PCR tubes.

Dream Taq(5µg/µl)……………………..2µl Buffer (Dream Taq) 10X………………10µl dNTP……………………………………………2µl Primer (F/R;F/fnr_R;fnr_F/R)……….2µl+2µl H2O……………………………………………..82µl Total…………………………………………….100µl (volume for 4 tubes, so 25µl for each)

PCR was programed for 95°C for 3mins and 30 cycles of 94°C 30s + 58°C 30s + 72°C 30s. The PCR products was put on a gel of agarose 1.5% with BET (1.5%), migrated during 30 min at 100V. the picture analysis was be delayed to the next day. Culture confirmation We performed another colonies seeding. 2 colonies selected from each Petri dish were seeded in liquid medium with their corresponding antibiotic at 37°C under stirring during 1 night.