Paris/July 15
From 2007.igem.org
- Minipreps on the following ON cultures:
- clones 1 & 2 of DH5alpha transformed with biobrick pJ23100
- clones 1 & 2 of ... BB B0015
- Isolation of 10 colonies of the following transformants:
- BBa_pJ23107 (ON culture on LB Amp is launched for clones 1 & 2)
- BBa_I0500 (ON cultures on LB-Kan clones 1 & 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:
1) What is the growth behaviour of w121 (dapA- strain) at different concentrations of DAP?
2) 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 |
See results.
The 96 well plate is surrounded by H2O in order to maintain humidity during the assay. In each slot, 200µL of the growth medium (LB or S0.x) is mixed with 2µL of w121 culture grown ON, and with different amount of DAP (see table). The growth profile is measured over a period of 30H, a DO measurement is acquired each 6min10s.
Gel extraction of PCR products
- PCR products of 13/07/07 & 14/07/07
migration on 1% Agarose gel extraction of bands of interesst with gel extraction Wizard kit according to the manufacturer's instructions
