Paris/August 30
From 2007.igem.org
(→Growth kinetic on w121 strains) |
(→Digestions) |
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== Digestions == | == Digestions == | ||
| + | we digest: | ||
| + | |||
| + | * MP1.1 as Bw vector with Spe1 & Pst1 | ||
| + | * MP15 as Bw insert with Xba1 & Pst1 | ||
| + | * MP16 as Bw insert with Xba1 & Pst1 | ||
| + | |||
| + | Protocol: | ||
| + | ADN: 20µL<br> | ||
| + | BSA: 0.5µL<br> | ||
| + | NE Buffer 2: 5µL<br> | ||
| + | Enzyme 1: 2µL<br> | ||
| + | Enzyme 2: 2µL<br> | ||
| + | H2O: 20.5µL<br> | ||
== Growth kinetic on w121 strains == | == Growth kinetic on w121 strains == | ||
Revision as of 15:15, 30 August 2007
Contents |
Expression test of DapA promotor by differential DAP repression
Plasmids: MiniPrep Products
| Plasmids: MiniPrep Products | ||||
|---|---|---|---|---|
| Number | Name | Description | Products in use | Date |
| MP15 | Att B | Lambda Att B rev within pSB2K3 | August 30 | |
| MP16 | Att P | Lambda Att P within pSB2K3 | August 30 | |
Strains (glycerol stock)
| Strains glycerol stock | ||||
|---|---|---|---|---|
| Number | Name | Description | Verified clone | Date |
| S46 | MP15 | Lambda Att B rev within pSB2K3 | August 30 | |
| S47 | MP16 | Lambda Att P within pSB2K3 | August 30 | |
Digestions
we digest:
- MP1.1 as Bw vector with Spe1 & Pst1
- MP15 as Bw insert with Xba1 & Pst1
- MP16 as Bw insert with Xba1 & Pst1
Protocol:
ADN: 20µL
BSA: 0.5µL
NE Buffer 2: 5µL
Enzyme 1: 2µL
Enzyme 2: 2µL
H2O: 20.5µL
Growth kinetic on w121 strains
In order to precise the growth behavior/kinetics of our strain W121 in smaller DAP concentration, we fit those last ones in function of our last kinetics results (06/08/07).
But we will use this experiment to better understand the presence of the delay observed in low DAP conentration.
| 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+5µM DAP | LB+8µM DAP | LB+10µM DAP | LB+12µM DAP | LB+15µM DAP | LB+17µM DAP | LB+20µM DAP | LB+30µM DAP | LB+40µM DAP | H20 |
| C | H20 | S0.2+0µM DAP | S0.2+5µM DAP | S0.2+6µM DAP | S0.2+7µM DAP | S0.2+8µM DAP | S0.2+9µM DAP | S0.2+10µM DAP | S0.2+15µM DAP | S0.2+17µM DAP | S0.2+20µM DAP | H20 |
| D | H20 | S0.4+0µM DAP | S0.4+2µM DAP | S0.4+3µM DAP | S0.4+4µM DAP | S0.4+5µM DAP | S0.4+8µM DAP | S0.4+10µM DAP | S0.4+12µM DAP | S0.4+15µM DAP | S0.4+20µM DAP | H20 |
| E | H20 | S0.6+0µM DAP | S0.6+1µM DAP | S0.6+2µM DAP | S0.6+3µM DAP | S0.6+4µM DAP | S0.6+5µM DAP | S0.6+6µM DAP | S0.6+7µM DAP | S0.6+8µM DAP | S0.6+10µM DAP | H20 |
| F | H20 | S0.8+0µM DAP | S0.8+0,5µM DAP | S0.8+1µM DAP | S0.8+2µM DAP | S0.8+3µM DAP | S0.8+4µM DAP | S0.8+5µM DAP | S0.8+6µM DAP | S0.8+7µM DAP | S0.8+10µM DAP | H20 |
| G | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 |
| H | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 | H20 |
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 20H, a DO measurement is acquired each 4min10s.
In order to determine whether the delay observed would come from the growth of a mutant, we'll retake the broth with a 100X dilution. Will this delay appear again and the same way as first observed ?
