IGEM:Caltech/2007/Progress

Week of 2007/06/11

2007/06/13 Wednesday – Transformed the BioBricks into bacteria. Titered lambda phage into concentrations of 10, 100, and 1000 colonies per plate.
Used BioBricks:
E0040 GFP
J23100 and J23116 constitutive promoter
B0015 terminator
P0440 tetR gene
P1010 on 2K3 plasmid

2007/06/14 Thursday – Checked results of titered phage (concentration of initial phage sample seemed to be off). Picked one colony from the transformed bacteria and cultured overnight.

2007/06/15 Friday – Purified the plasmids from the transformed bacteria colony using a prep kit, added restriction enzymes and buffer solution. Plan to integrate the terminator, constitutive promoter, and the tetR gene system (RBS, tetR gene, and terminator) BioBrick parts onto the 2K3 plasmid.
[Part name; Enzymes used; Buffer]
P0440; Xba/Pst; Eco Buffer & BSA
B0015; Eco/Pst; Eco Buffer & BSA
P1010; Eco/Pst; Eco Buffer & BSA
constitutive promoter (J23100, J23116); Spe/Pst; NEB & Buffer 2

Week of 2007/06/18

2007/06/18 Monday - Gel electrophoresis of J23100, J23116, 2K3(P1010), P4040, and B0015
Cut DNA bands from gel, purified DNA using Qiagen kit
Prepared ligations for:
A. Insert B0015 into Vector 2K3 (two reactions, from two digest bands)
B. Insert P0440 into Vector J23100
C. Insert P0440 into Vector J23116

2007/06/19 Tuesday - Appearance of colonies on ligation plates, no colonies on control plates. Set up PCR reactions for 6 colonies (cell suspensions) on each of the ligation plates (A1, A2, B, C).
Set up PCR for overlap extensions (lambda c1 60, GFP, etc) of DNA template + premade primers.
Upon gel analysis of PCR reactions, found that all except the ptet reaction did not have bands matching expected base lengths. Reset the PCR primer reactions for N, Q, cro, and GFP, using controls (the cro, N, and Q mut primers).
Cultured colonies for reactions A1-6, A2-2, A2-3, A2-4, B1, B4, C1, C2.

2007/06/20 Wednesday - Set up quick gel for the overlap PCR reactions (primers of GFP, cro + cro mut, N + N mut, Q + Q mut). The quick gel appeared accurate and precise, with bands of approximately correct length. Gel extraction was performed on another gel with same samples.

2007/06/21 Thursday - Overlapping Extension PCR performed again on N, Q, Cro, and Ptet. Upon running the results on an agarose gel, lanes were found to be impure, and gel extraction performed. The extracted DNA was digested with EcoR1/Spe1.

2007/06/22 Friday - Digests PCR purified. The result was run on agarose gel. N and Cro appeared to have the same length--mistake made somewhere in the procedure.

Week of 2007/07/09

2007/07/08 Sunday - Set up EX digests for vectors S03724 and B0015-2K3, ES for vector 2K3, and SP for vector B0015-2K3. Set up ES digests for inserts pTet-spacer-cro/N/Q (the cro/N/Q construct) and XP for insert S03706.

2007/07/09 Monday - Gel extracted vectors (and insert S03706), PCR purified the inserts.
Set up ligation reactions for:
N insert in 2K3, B0015-2K3 vectors
cro insert in 2K3, B0015-2K3, S03724 vectors
Q insert in 2K3, B0015-2K3, S03724 vectors
S03706 insert in B0015-2K3
Transformed ligations into DH10B cells, plated onto LB Kan Agar plates
Practiced titering with cI phage on D1210 cells

2007/07/10 Tuesday -