Team:Cornell/Notebook/Salicylate reporter


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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.


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).


July 1st, Sunday

  • Set up gel for electrophoresis (9:50am, DPW + CMR)
    • 1% gel in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green(10:50, CMR)
      • Ran NEB 100 bp ladder, NEB 2-log ladder, Promega 1 kb ladder
      • Ran at 100 V.
    • 1% gel in Owl box using ethidium bromide (10:50, DPW)
      • Ran nah operon PCR product from previous night
      • Ran at 55 V.

Because we learned that our SYBR Green was causing ladder to run strangely, Dylan decided to redo a Vent PCR to amplify the salicylate reporter region out of p21.
Liquid culture of JG700.
Replated p21, p22, JG700, JG1220, JG1537, JG1219

July 2nd, Monday

Today, Dylan and Caleb ran a gel of a Vent PCR of p21 (the PCR product being the salicylate reporter) at 55 V. Additionally, Caleb decided to run a control gel at 100 V to determine whether higher voltage was a factor in our previous ladder problems (in addition to the SYBR Green stock). While we determined that the higher voltage did not cause our ladder issues, we did not see any bands from the p21 PCR.

Dylan prepared electrocompetent cells using the Myers and Myers protocol. Modified protocol using 2 5mL cultures @ 4000g for 10 min. Washed with 2 mL sorbitol, resuspended in 100 uL sorbitol. First electroporation ts = 2.32 ms, second ts = 2.02 ms. Added 1 uL of plasmid (575.5 ng) to each cuvette. First used .60 V, then 0.55 V, both with R = 400 ohms.

Mark and Danielle started liquid cultures of S1, S9, S10, S11, S15, S16, S18, S27 (See: Strain list)

July 3rd, Tuesday

Caleb miniprepped S16 (p15), S22 (p21), S9 (p8), S10 (p9), S11 (p10), S18 (p17), and S15 (p14). Instead of a single EB elution, Caleb did two elutions, 30 ul each. The double 30 ul elution turned out to be effective at recovering a usable amount of DNA in the second elution, so we're including it on our miniprep protocol for future minipreps.

The rerun of our Gibson sequencing failed again, so we tried a more roundabout method of determining whether our 3 Gibson transformants have complete nah operons or not. We digested them each with BamHI, expecting specific fragment lengths on a gel electrophoresis. We only saw one band on each, all of them at about 3.6kb.