Paris/July 24
From 2007.igem.org
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]
