User:University of Washington/13 August 2008

From 2008.igem.org

Contents

Conjugation (Scott)

experiment from Aug. 7 results

experiment #BacteriaYeastYeast counttransconjugation efficiency
1250uL250uL 662.64E-5
2200uL300uL 642.13E-5
3100uL400uL 328.00e-6
450uL450uL 184.00e-6

LuxR from AraC and TetR(Faifan)

QuikChange Approach

- Overnight QuikChange#4 culture from Amp plate (reaction1 x 4, reaction2 x 2) in Tsy+Amp

- Sequence of DNA from QuikChange#3 came back ==> No mutation.

Elowitz

-Made Glycerol Stock of BBa_Elowitz x 2

-Restriction Digest of BBa_Elowitz, BBa_E0240(GFP), BBa_I0462(LuxR), BBa_P1010(on pSB3K3)

  • mix
dH2O(ul)Buffer(ul)BSA(ul)DNA(ul)E(ul)X(ul)S(ul)P(ul)
BBa_Elowitz32.5EcoRI, 50.5101-1-
LuxR32.5NEB3, 50.510-1-1
GFP32.5NEB3, 50.510-1-1
P101032.5EcoRI, 50.5101--1
  • incubate 37 degree Celsius, 1.5 hours
  • 15 mins water bath 80 degree Celsius
  • nanodrop at 230 nm
ng/ul260/280260/230
BBa_Elowitz76.21.120.34
LuxR43.50.980.52
GFP40.71.000.46
P101050.80.780.17
  • store in 4 degree Celsius

Lambda Red Recombineering of RP4 (Bryan)

Attempted the following transformations RP1 + pKD78, RP4 + pKD78, and pKD78 alone. Selected with cam/tet, cam/tet, and cam, resepectively. None of the O.N. cultures grew. Possible problems are imprecision in micropipetting due to extremely small volumes <0.5 ul of vector DNA or not enough cells (1 mL of culture per electroporation).

AHL Expression in Yeast (Bryan)

Transformed C0161/YEp426 ligation produce from yeseterday into XL-1 Blue. O.N. culture grew in amp, indicating successful transformation. Miniprepped and made glycerol stock.



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