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 #1 (C) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
  • Q04740 white (pink) colony #4 (D) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
  • sequencing results: red colony had IS5 element jump in after the hairpin / before the prefix; white colony, when grown up/prepped/sequenced had a population with the insertion element in the same spot and a population without IS5
    • i.e.: this isn't what we want
• 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: trial library (mnt)

• Day 1: 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
  • 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)
  • PCR cleanup
• Day 1.5 (July 17): did overnight double digest (XP) on PCR reactions
• Day 2 (July 18):
  • ran digest on a gel, did gel extraction - eluted into 30 ul EB total
  • performed a 40-ul ligation reaction using all of the high range mut freq digest + 1 ul cut SP1.0
  • dialyzed - after dialysis, total volume was 13 ul
    • used 6 ul for a transformation that arced
    • used 3 ul for a transformation that worked (~4 ul left over)
    • dialyzed 40 ul of positive control (2 ng/ul?) - took 1 ul of the diaylzed plasmid and transformed it
    • did a negative control electroporation with CW2553/pJat8
  • grew in 1 mL SOB for 1.25 hrs
  • plated 50 ul of the experimental (AG), positive control (A, could have used AG in CW2553/pJat8?), and negative control (AG) on antiobiotic plates
  • made a 1:1e5 dilution using 1 ul of the transformation and plated 10 ul of the 1:1e5 (+90 ul H20) on LB only plates - not sure if this is right, I was supposed to do a 1:1e6 dilution...next time do more serial dilutions, also figure out what you're supposed to do
  • put the rest of the transformation 1 mL - 65 ul into 9 mL of supplemented M9 in buffeted (?) flasks around 7:30 or 8 p.m.
  • put plates in the warm room around 9 p.m.
• Day 3 (July 19):
  • the 10 ml culture grew ~15-16 hours overnight - end OD = 0.27
  • set up 25 ml experimental cultures at 0% and 1e-4% arabinose
    • added 1 ml or 0.5 ml of overnight (4 flasks total)
  • plate results:
    • + control:
      • 1:1e7 dilution on no antibiotic = 11 colonies
      • 1:20 dilution on AG = lawn of cells --> redo with a 1:1e5 , 1:1e4
    • - control: no cells on either plate
      • diluted too much for no antibiotic --> try 1:1e5, 1:1e4
    • Q04720 library: 1 colony from 1:20 on AG, no colonies on LB only (dilution too high)
      • replate LB only at 1:1e4

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

restriction digest (XP) of Q04720 library

July 19, 2006: inverter libraries

• Day 1:
  • mutagenic PCR
    • start with 20 ng target DNA
    • use VF2-VR
    • inverters:
      • Q04400
      • Q04720 (also try BioBricks primers I ordered)
      • Reshma QPI C2002
      • Reshma QPI C2003
    • BioBricks primers didn't work -- melting temp too high? ran at 45 C, ~20 C below Tm
  • restriction digest

July 20, 2006

• everything except GFP only control (and negative control) in SP1.0
• grew overnight, diluted back 50x around 2 p.m., grew ~6 hrs
  • at this point, the first 3 penI cultures (1,2,3) gave an OD of 0.00 (used 50 ul), the fourth (4) 0.05 (10 ul)
  • Q04400 (40) OD = 0.01 (50 ul)
  • Q01400 (14) OD = 0.16 (3.125 ul)
• put experimental cultures in at 9 p.m.
• Q04740 (from a plate from original glycerol) (no induction and 1e-4%)
  • colony 1 - innoculated at ??? no OD reading - grew 16.5 hrs
  • colony 2 - innoculated at ??? no OD reading - grew 16.5 hrs
  • colony 3 - innoculated at ??? no OD reading - grew 16.5 hrs
  • colony 4 - innoculated at 0.0001 - grew 16.5 hrs
• tetR inverters (all 6 induction levels)
  • Q04400 - innoculated at 0.0001 - grew 13.5 hrs
  • Q01400 - innoculated at 0.0001 - grew 13.5 hrs
• GFP only control (I13522) - started overnight ~5 p.m. - harvest at 10:30 a.m.
• CW2553/pJat8 negative control - started overnight ~5 p.m. - harvest at 10:30 a.m.