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Team:Cornell/Notebook/Salicylate reporter/June24
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The salicylate reporter is analogous to the arsenic reporter, but with a salicylate-sensitive promoter. That is, in the presence of salicylate it will produce MtrB, completing the Mtr pathway and allowing Shewanella to produce current in our biosensor. In combination with the nah operon, this reporter will be able to detect naphthalene levels. Back to salicylate reporter week view
June 24th-30th
June 29th, Friday
- Vent PCR at 11:00 (DPW)
- Amplifying both previous Phusion PCR band and original p21 template
- Dylan's magic triple anneal method (55/60/63)
- Gel purified PCR product from Phusion template (~1:20 pm) (DPW)
- Quantified product at 22.4 ng/uL
- Set up digestion of p21 PCR product with EcoRI-HF and AscI (~9:00 pm).
- 22 ng/uL --> 45.5 uL sample for 1 ug digest
- Buffer 4
- Ran digestion on gel. (~11:00 pm)
- Sliced out relevant band on gel, stored overnight at -20.
- Miniprepped overnight cultures of PL14-PL20 (~1:00 pm, STC)
- Using C1015 rotor, 6666 x g, the Corning culture tubes only fit in the centrifuge with the lids off
- Made LB, 3x 60 mL in 100 mL bottles (~ 3:00pm, SS)
- Made LB Agar, 4 x 250 mL LB Agar in 500 mL bottles (~3:00, CS)
- Autoclaved LB, LB Agar, and milliQ (~3:30 pm, SS)
- Made LB plates with Kan (~6:30 pm, CMR)
- CUGEM movie outing at 8:00 pm.
- Phusion PCR at 10:00 pm (DPW)
- Dylan's magic triple anneal method (57/65/70, 35 cycles total)
- Amplifying nah operon from Gibson 1
- Appending BioBrick cutsites for ligation into pSB3C5
June 30th, Saturday
- Took PCR out of thermal cycler at 9:00 am (DPW)
- Set up gel using NEB 100bp and 2-log ladders (10:00am)
- Gel extracted PCR product, quantified at ~10ng/uL
- Set up new Phusion PCR using Gibson 1 as template
- Dylan's magic three-anneal method (57.6/65/72)
- Extension time of 3 min.
- Continued gel extraction of p21 PCR digest from previous day (SS)
- Set up ligation of p21 PCR digest extraction and dephosphorylated pBBRBB+mtrB (11:11am, SS)
- Desalted ligation using Millipore membrane paper
- Transformed 2 electrocompetent DH5alpha stocks at 5:30 pm and 5:50 pm, respectively.
- Observed time constants for electroporation of 4.38 ms and 4.24 ms, respectively
- Let cells recover for 1 hour, plated on LB + Kan.
- Set up two ligations of pSB3C5 into PNNL electrocompetent Shewanella strain JG700 (6:30 pm, Sp.C and St.C)
- First transformation performed at usual PNNL voltage of 0.75 kV (time constant of 9.30 ms)
- Second transformation performed at Myers and Myers specification of 0.55 kV (time constant of 9.34 ms).
