Team:Brown-Stanford/Lab/Notebook/Week15

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Brown-Stanford
iGEM

September 12, 2011

PowerCell

  • clean up Ana ES digest (from 9/7)
  • using Wizard PCR cleanup (w/ Andre’s protocol modifications)
  • -> 15 ng/μl (~50%)
  • ligation
  • cscB ES w/ Ana-pSB1C3 ES (both cleaned w/ wizard)
  • 2.4 μl (30ng) pSB1C3
  • 1.7μl cscB (3:1 molar ratio)
  • 1 μl T4 DNA ligase
  • .5 μl 10x ligase buffer
  • 4.4 μl PCR H20

REGOBrick

  • 50 μL batch PCR to develop more construct
    • Previously, Lynn indicated an interest in finding and harvesting a biobrick part from nature. She had a bunch of samples taken from extreme environments from aroud the world (some low, high pH, some high salinity, some high and low temperature samples). Samples were suspended in both LB and water
    • We looked for increases in pH: three samples showed increases: Magada, Kenya (grown in LB), Thermals in Bolivia (grown in LB), and Laguna Verde, Bolivia (grown in Water)

September 13, 2011

PowerCell

  • miniprep Ana liq cult
  • 84 ng/μl
  • make BG11 plates
  • 24 BG11 + neo50
  • 21 BG11 + 5% LB + 2% agar
  • liq cultures
  • 6 cscB candidate colonies
  • 2x2 PowerCell colonies
  • 1 ea. pRL25, pRL25->623

REGOBrick

  • Ran gel of previous day’s PCR... unsuccessful. Ran a new PCR testing the PCR template sources we have. Turns out the template source we had used for the last PCR did not work, so we will have to use other minipreps/template sources to do subsequent PCR reactions.
    • Did work concerning the extremolphiles Lynn gave us; did PCR of 10 samples taken from around the world with universal archaebacteria primers. Two samples, one from Nymph river (Yellowstone) and Magada, Kenya showed signs of archaebacteria, and the bands were distinct. Archaebacteria have already been described in Nymph river; but there is little or no literature on the site in Kenya.

September 14, 2011

PowerCell

  • miniprep
  • 6 cscB candidates (100-280 ng/μl)
  • 2 colonies of PowerCell final construct (duplicates) (300-250 ng/μl)
  • pRL25 (150 ng/μl)
  • pRL25+623 (111 ng/μl)
  • send in csc candidates for sequencing
  • ES digest of PowerCell (40 μl)
  • cleanup of diges
  • -> 32 ng/μl
  • planning
  • ordered new primers for PowerCell construct
  • rev primer modified to add in BamHI site after PstI for ligation into pRL25

REGOBrick

  • 50 μL PCR batch run using “good” template (template that showed good results in previous PCR runs
    • Ran gel of this PCR, showing an unsuccessful reaction. The problem was traced back to old dNTPs, which will need to be replaced
    • Made new Urease plates for the maker faire. In case we don’t have it in here already, here is the recipe:
      • Urea: 20 g/L
      • NaCl: 5 g/L
      • KH2PO4: 2 g/L
      • Peptone: 1 g/L
      • Dextrose: 1 g/L
      • Phenol red: .012 g/L
  • Add ingredients into 100 mL water, pH to 6.8 ± .2, filter sterilize. Autoclave 900 mL water w/ 15 g agar. Combine and plate.
    • Plated flight specimens from yesterday...
    • Results:
      • All specimens that were contained in Whirlpak bags exhibited ureolytic activity (Outside the payload, inside the payload, and ground control.
      • All specimens not contained in Whirlpak bags did NOT exhibit ureolytic activity
      • All specimens, both inside and outside the whirlpak bags, were plated on Bang media, and all showed growth on the plates. All these growths looked similar, but it seems as if the bacterial growth came from contamination. All specimens were subjected to air from the outside environment, and therefore could have been contaminated. The data concerning growth of the samples is thus unusable.

September 15, 2011

REGOBrick

  • Restriction of Urease construct at X, P sites (Knight lab protocol)
    • Cleanup of restriction product and storage for ligation using Promega Wizard SV DNA purification system

September 16, 2011

PowerCell

  • Ligation of construct with pSB1C3
    • Transformation of ligation product into K12 compotent E. coli


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