From 2012.igem.org

Revision as of 13:25, 5 July 2012

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

July 1st-7th

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.

Because no transformations from our previous competent freezer stocks were successful, Dylan decided to make a starter culture of Shewanella strain JG 700 in preparation for making new competent stocks using the PNNL Protocol. Swati and Tina used this starter culture to complete the preparation.