User:Kchang17/notes

February 28, 2006

• characterization of penI QPI in I13537.pSB1A3 with three replicates (replicate 1) (concatenated) (MATLAB analysis)
• rough measurement of original (unmutated) tetR inverter, Q04400 (one promoter SP1.0) (two promoter SP (I13537))

March 15, 2006

• first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 (MATLAB analysis)

June 22, 2006

• tested everything in SP1.0 at 6 arabinose levels (0%-10^-4%)
• Q04400.007 innoculated at OD 0.0005 and grew for ~10 hrs (picture) (MATLAB analysis)
  • note: 10 hrs probably wasn't enough time for it to reach steady state esp. at high induction, assuming inverter was working better on July 11
• Q04740 exptl cultures grew too dense - performed an extra dilution and redid the innoculation - innoculated at OD 0.0005 and grew for ~14 hrs
• I14103 - grew very slowly - innoculated at OD 0.001 and grew for ~14 hrs (grew a little too dense)

June 29, 2006

• devices in SP1.0, six arabinose levels (0%-10^-4%)
• Q04720 - innoculated at 0.0003 - grew ~15 hrs (too dense) (picture)
• Q04730 - innoculated at 0.0003 - grew ~16 hrs (too dense) (picture)
  • during construction, a point mutation occurred in the ORF of the repressor protein (C0073) resulting in a change from Phe to Leu
• Q04740 - innoculated 0.0003 - grew ~16 hrs (too dense) - weirdness, where did GFP and RFP go?
• I14103 - innoculated at 0.0005 - grew ~15 hrs (too dense) (picture)
• I14104 - having trouble cloning this, have to wait on testing

July 6, 2006

• picture
• induced ~7 hrs at no and high induction
• the single red colony on a plate (probably got mixed with some white colony) from Q04740 in SP1.0 glycerol - got high RFP signal and GFP tracked induction - streaked this culture for July 11 testing
• one white colony from the same plate - GFP showed this time - background RFP
• empty SP1.0 - showed GFP and RFP

July 11, 2006

• grew to stationary, then diluted back 100x, grew for ~8 hrs (cultures were all very light except for the empty SP and Q04400.007), innoculated at ~11:30 p.m.

  

  Colony PCR of Q04740 colonies, E0040F-E1010R. Two red and two white colonies. Region should be ~1.6 kb. Red colonies ~3 kb (transposable element jumped in). White colonies ~1.6 kb.

• devices in SP1.0, six arabinose levels (0%-10^-4%)
• Reshma's constructs:
  • B0030.C2002.B0015.p8 (A) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
  • B0030.C2003.B0015.p8 (B) - innoculated at 0.0001 - grew ~15 hrs (picture)
• clones from July 6's penI red no induction culture, streaked on plate:
  • Q04740 red colony (C) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
  • Q04740 white (pink) colony (D) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
• empty SP1.0 (E) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
• Q04400.007 (F) - innoculated at 0.0001 - grew ~14.5 hrs (picture) (concatenated) (MATLAB analysis)

July 14, 2006

• trying out mutagenic PCR on Q04720.pSB1AK3 (mnt) - the prep for this was taken from MC4100, so pJat8 isn't in there and I know how much DNA I'm adding

  

  mutagenic PCR, lanes left to right: 188 ng, 75 ng, 12.5 ng target DNA, 1.1 kb control (100ng)

• Stratagene's GeneMorph® II Random Mutagenesis kit
• using VF2-VR (24 pmol each per 50 ul reaction)
• three 50 ul PCR reactions: (note, I goofed, meant to do 4x these amts so I could cover low-med-high mutation frequencies but forgot to take the vector into account)
  • ~188 ng target DNA (medium range mutation freq)
  • ~75 ng target DNA (medium range mutation freq)
  • ~12.5 ng target DNA (high range mutation freq)