Team:Yeshiva NYC/Notebook

Faige's Work:

May 26-June 2: Made LB-media agar plates with different antibiotics (Cam, Kan, and Amp); learned techniques

June 3: Received primers for leader sequences

June 4: Purified genomic XL1-Blue DNA

June 5-10: Unsuccessful PCR with 3 primers; digestion, ligation, and transformation (DLT) of 3 ccdB plasmid DNA

June 11: Miniprepped ccdB

June 12-19: Continued toggling with annealing temperatures for all 8 primers, 2 were successful!

June 22-24: DLT of ccdB with two successful leader sequence PCR reactions; DLT of RBS; DLT of promoter

June 25: Three-way DLT of RBS and promoter into ccdB plasmid

June 29: Received 6 long primers (for the 6 unsuccessful ones) and ran PCR with them, 4 were successful!

June 30-July 9: Resumed tweaking conditions for two remaining unsuccessful primers; DLT of 4 recently successful leader sequence PCR reactions, and three-way DLT of each of first 2 successful leader sequences with RBS/promoter into ccdB; DLT of RFP DNA; received RFP primers

July 10-14: Three-way DLT of the 4 later successes with RBS and promoter; three-way DLT of cytoslac with 2 earlier leader sequence/RBS/promoter

July 15: Ran PCR with RFP primers; tweaked conditions for unsuccessful ligation reactions

July 16-21: Three-way DLT of RFP and 1 earlier leader sequence/RBS/promoter; DLT of RFP DNA [as a simple Biobrick(BB)]; continued tweaking of previously unsuccessful DLTs; gel extraction of cytoslac digests (to remove resistance)

July 22-23: Transformation of RFP BBs into BL21 DE3 cells; Three-way DLT of RFP with other 5 successful leader sequence/RBS/promoter combinations; DLT of pure cytoslac with 4 recently successful leader sequence/RBS/promoter combinations; transformed 2 successful cytoslac/leader sequence/RBS/promoter combos into BL21 DE3 cells

July 24: Made 2XYT-media liquid cultures of BL21 DE3 cells and stimulated with IPTG; Also added IPTG to plates of BL21 DE3 cells. No expression, unfortunately.