IGEM:MIT/2007/Notebook/2007-7-24

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Contents

AGENDA

  • Plate the Mer/I13500 3A transformation after overnight ligation (and grow)
  • Check if sequence results are available from Monday (length was correct)
  • Second polystyrene assay (JH and TTP?)
    • CPX, PBS with different concentrations and controls
    • Suggested: one person per plate, pipet to the edge, more washes, harder aspirations
  • Sequence the FhuA (pHIE) with CORRECT primers (which already exist?)
    • Prepped plasmid exists (AL, 7/23)
  • EC_20 requests
    • Harass the people AK e-mailed OR design and order?
  • If T7 antibody comes in...test!

Transformation of Mer/I13500 after overnight ligation (in TK's DH5alpha)

  • Added 5µL of ligation reaction to 100µL of DH5a
  • Incubation in 37 degree room for one hour and forty-five minutes (Amp and Tet resistant)

Sequencing of FhuA (picss8)

  • Used FhuA, 10 µl to make primers (top and bottom)
    • 1 µl of FhuA, 10 µl + 2.12 µl water = 3.2 primer
    • 1 µl of primer + 2 µl DNA + 9 µl water = 12 µl total sequencing mixture
    • DNA = "picss8" with nanodrop of 99.6
  • Strip of 8 PCR tubes labeled "Liying Huang, 8201" sent in for sequencing at 3 PM
    • 1 = top primer
    • 2 = bottom primer

Transformation of CPX (A+,Cm+), FhuA (K+) and pUC18 (A+) into Barry's BL21 cells (more protein!)

  • Obtained pUC18 and BL21 courtesy of Barry
  • BL21 Cell volume: 50 µl
  • DNA mass to add: 50 to 60 ng
  • For spreading on plate: 300 µl and let dry
  • NOTE: to help plates absorb liquid bacteria, leave plates out overnight after making them before putting them in fridge (dries them out)
  • "BL21 trans. w/ pUC18 (A+)": pulled out 55 µl of pUC (at 1 ng/µl of stock) and added to cells
  • "BL21 trans. w/ FhuA (K+)": diluted 1 µl of stock into 2 µl total; pulled out 1.5 µl and added to Bl21
  • "BL21 trans. w CPX (Cm+ A+)": diluted 1 µl of stock into 4 µl total; pulled out 1.5 µl and added to BL21

Polystyrene Assay

  • Used CPX induced with AHL at concentrations: 10E (0,-5,-6,-8,-8.5)
  • Performed three wash steps for 15 minutes each (washed with 150 ul PBS)
  • Results:
    • Marked difference in growth between 10E -5 CPX and negative control (DH5-alpha without transformed CPX)
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