Paris/July 24

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Contents

Minipreps

BBa_E0422 in pSB1A2 - Clone 1&2 - MP6.1' & MP6.2'
BBa_E0241 in pSB1A2 - Clone 1 - MP7.1'
BBa_J61047 in pSB1A2 - Clone 1&2 - MP9.1' & MP9.2'

Growth kinetics of w121 strain

We make an array to test growth of w121 on different growth media (LB, S0.2, S0.4, S0.6, S0.8), supplemented with different amount of DAP.

Two questions are addressed by the following assay:

-What is the growth behaviour of w121 (dapA- strain) at different concentrations of DAP? -How does w121 strain grow on filtrates of MG1655 growth medium; that is, does MG1655 secrete DAP during growth?

MG1655 was grown on LB medium and the growth medium was filtered free of bacteria at different DO (Optical Densities) during exponential growth phase:

S0.2 (at DO=0.2) S0.4 (at DO=0.4) S0.6 S0.8


W121 was grown on different media & Growth kinetics were measured:


LB line B in the array S0.2 (at DO=0.2) line C in the array S0.4 line D in the array S0.6 line E & F in the array S0.8 line G in the array

In the different columns, DAP was added to the indicated final concentrations (without taking into account DAP produced by MG1655 regarding the recycled growth media)


Kinetic Array :w121 kinetic as a function of DAP and supplemented medium (S0.x)
1 2 3 4 5 6 7 8 9 10 11 12
A H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20
B H20 LB+0µM DAP LB+20µM DAP LB+25µM DAP LB+30µM DAP LB+35µM DAP LB+40µM DAP LB+45µM DAP LB+50µM DAP LB+55µM DAP LB+60µM DAP H20
C H20 S0.2+0µM DAP S0.2+5µM DAP S0.2+10µM DAP S0.2+15µM DAP S0.2+20µM DAP S0.2+25µM DAP S0.2+30µM DAP S0.2+35µM DAP S0.2+40µM DAP S0.2+45µM DAP H20
D H20 S0.4+0µM DAP S0.4+5µM DAP S0.4+10µM DAP S0.4+15µM DAP S0.4+20µM DAP S0.4+25µM DAP S0.4+30µM DAP S0.4+35µM DAP S0.4+40µM DAP S0.4+45µM DAP H20
E H20 S0.6+0µM DAP S0.6+2µM DAP S0.6+5µM DAP S0.6+8µM DAP S0.6+11µM DAP S0.6+14µM DAP S0.6+17µM DAP S0.6+20µM DAP S0.6+23µM DAP S0.6+26µM DAP H20
F H20 S0.6+0µM DAP S0.6+2µM DAP S0.6+5µM DAP S0.6+8µM DAP S0.6+11µM DAP S0.6+14µM DAP S0.6+17µM DAP S0.6+20µM DAP S0.6+23µM DAP S0.6+26µM DAP H20
G H20 S0.8+0µM DAP S0.8+2µM DAP S0.8+5µM DAP S0.8+8µM DAP S0.8+11µM DAP S0.8+14µM DAP S0.8+17µM DAP S0.8+20µM DAP S0.8+23µM DAP S0.8+26µM DAP H20
H H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20


Ligation & transformation reactions

Ligations
Number Vector Vector Volume Insert Insert Volume Comments
L1 D9 (J61002 ready for insertion of a FI) D23 (AraC/pBad promoter FI)
L2 D18 (BB dig. lox66DapAE.coli) D22 (pSB1A2 Eco, Pst)
L3 D19 (BB dig. lox66DapAsubtilis) D22 (pSB1A2 Eco, Pst)

To do:

  • D23 (AraC/pBad promoter FI) in D9 (J61002 ready for insertion of a FI)
  • D18 (BB dig. lox66DapAE.coli) & D19 (BB dig. lox66DapAsubtilis) in D22 (pSB1A2 Eco, Pst)
  • D20 (Lox66-DapAE.coli BI) & D21 (lox66-DapAsubtilis BI) in D1 (pJ23100 BV) & D3 (pJ23107 BV)
  • D14 (Cre ORF) in D15 (B0030 BV)
  • D27 (Lox71-ftsZ BI) in D1 (pJ23100 BV) & D3 (pJ23107 BV)
  • lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)

Annealing of Lox66 and Lox71

Lox66 and Lox71 oligos were designed to form a double-stranded DNA. The extremities bear cohesive overhangs corresponding to digestion by EcoRI and SpeI. See oligos 14 + 15 and 16 + 17 here.

Annealing mix:

  • 8 μL of each of the concentrated primers
  • 4 μL of salt solution (10 mM NaCl)
  • 20 μL of water

Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.
More information [http://openwetware.org/wiki/Annealing_complementary_primers here]