Team:Calgary/27 July 2009

• Finished writing modeling paper that summarizes what we will be doing once the circuit is completed. The experiments should not take long to accomplished and these results can be tabulated in Matlab quickly.
• Plated the week-long ligation of the promoter library in both XL Gold Ultra competent cells and Top 10 cells, but there is no growth.
• A reason that we thought of that might have caused minimal colonies is the phosphatase step. The 32 primers that we have synthesized does not have 5' phosphates. If we phosphatase the vector, that might result in no ligation.
• Re-started restriction enzyme digestion on vector pCS26 with XhoI and BamHI at 37^oC for 4 hours. Then I ligated the promoter with the vector with Quick Ligase for 5 minutes. Then I transformed the plasmids into both XL Gold Ultracompetent cells and Top 10 cells. Incubate at 37^oC for 16 hours.

Protocol

MM1 (6x) (μL) MM2 (6x) (μL) 10X PCR buffer minus MgCl2 30 30 10mM dNTPs 6 6 50mM MgCl2 9 9 F primer 6 (BBK CP F) 6 (LuxPQ F) R primer 6 (BBK CP R) 6 (LuxOU R) ddH2O 241.8 241.8 pTaq 1.2 1.2

Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3

PCR conditions

1. of cycles Temp (ºC) Time

1 94 6 min 36 94 30sec 55 45sec 72 6min 20sec 1 72 10min 4 hold

Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.

Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers

Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)

Protocol

MM1 (8x) (μL) MM2 (6x) (μL) 10X PCR buffer minus MgCl2 40 40 10mM dNTPs 8 8 50mM MgCl2 12 12 F primer 8 (LuxPQ F) 8 (BBK CP F) R primer 8 (LuxPQ R) 8 (BBK CP R) ddH2O 322.4 322.4 pTaq 1.6 1.6

Positive control = ∆LuxPQ in psB1AK3

PCR conditions

1. of cycles Temp (ºC) Time

1 94 6 min 36 94 30sec 55 45sec 72 6min 20sec 1 72 10min 4 hold

Result: Of the 6 colonies, 5 had bands of the correct size (~3.9kb).

Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)

Purpose: isolate and measure concentrations of pure plasmids.

DNA 260/280 260/230 Conc. [ng/μL] ∆LuxPQ in AC3 C1 1.83 2.23 146.8 ∆LuxPQ in AC3 C2 1.81 2.19 112.6 ∆LuxPQ in AC3 C3 1.80 2.27 145.8 ∆LuxPQ in AC3 C4 1.78 2.09 96.6 ∆LuxPQ in AC3 C5 1.81 2.25 115.3 ∆LuxPQ in AC3 C6 1.79 2.17 44.8 PQ-B-R-OU-B in AC3 C1 1.76 1.86 29.4 PQ-B-R-OU-B in AC3 C2 1.59 1.44 20.1 PQ-B-R-OU-B in AC3 C3 1.70 2.27 16.3 PQ-B-R-OU-B in AC3 C4 1.71 1.79 17.0

Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Protocol

Recipient Recipient 1 Recipient 2 2μL REact 4 2μL REact 4 0.75 μL SpeI 0.75 μL SpeI 0.75 μL PstI 0.75 μL PstI 2 μL of ∆LuxPQ in AK3 C2 [112.6ng/μL] 2 μL of ∆LuxPQ in AK3 C5 [115.3ng/μL] 14.5 μL ddH2O 14.5 μL ddH2O

Insert 2μL REact 2 0.75 μL XbaI 0.75 μL PstI 2 μL of LuxPQ-B0015-R0040-LuxOU-B0015 in AK3 C1 [156.3ng/μL] 12.5 μL ddH2O

Put in the incubator at 37ºC overnight.