Team:CSU Fort Collins/Notebook/Breakdown/Jul

JULY

Tuesday, July 1 - Wednesday, July 16

Researched which promoters, primers, sequences, and assembly techniques to use and developed a plan of action.

Thursday, July 17

Designed primers to connect biobrick lab promoter and the FadD gene from E. coli.

Friday, July 18

With competent cells, used the transformation protocol to add our two different lac promoters to E. coli and grew overnight at 37 °C. Used lac promoters from the biobrick distribution kit (2013 iGEM Distribution Kit, Plate 5, Well 1D (ampicillin resistance) and 2014 iGEM Distribution Kit, Plate 3, Well 4G (chloramphenicol resistance)).

Sunday, July 19

Removed plates from the incubator and stored at 4 °C.

Monday, July 21

The plate using the promoter from 1D had no growth. The plate with the promoter from 4G had two small colonies. We replated the colonies using the rest of our transformed competent cells. Incubated again overnight at 37 °C.

Tuesday, July 22

The plate using the promoter from 1D still had no growth. The plate with the promoter from 4G had a lawn. Streaked the 4G plate to create individual colonies. Incubated overnight at 37 °C.

Wednesday, July 23

The 4G plate had good growth with many individual colonies. We created two 2 mL overnight cultures using 1 mL LB/1 μL CM and a colony from our streaked plate. Incubated overnight at 37 °C and 225 rpm.

Thursday, July 24

Removed the tubes from the incubator and stored at 4 °C.

Friday, July 25

The primers for FadD gene came in. Created a 100 μM stock solution. Diluted by 10X to make a working solution. Following the PCR protocol outlined in the Cloning a Gene into a Plasmid protocol, PCRed 4 replicates. Used a 68 °C annealing temperature and a 1 minute 30 seconds extension. Stored the PCR product tubes at 4 °C. Miniprepped 1 mL of overnight cultures to extract the DNA containing the desired lac promoter as directed in the Miniprep Protocol. The products of the miniprep were stored at -20 °C. Created 2 more overnight cultures using the same protocol as before, this time seeding them using what was left of the previous overnight cultures.

Tuesday, July 29

Used two gel electrophoresis protocols to determine if the PCR worked. Both gels failed.

Wednesday, July 30

Reran PCR using a different temperature for the extension step. Ran another gel to determine success of PCR. This gel also failed.

Thursday, July 31

Reran PCR using previously isolated E. coli genome and various extension temperatures in a gradient. Ran another gel, this gel also failed.

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