Difference between revisions of "Part:BBa K606035:Experience"

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First we launch cells from the overnight's tube without IPGT. Then we wash the cells and relaunch them with glucose to inhibit out gene expression.
 
First we launch cells from the overnight's tube without IPGT. Then we wash the cells and relaunch them with glucose to inhibit out gene expression.
  
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|[[Image:T7 autoloop ctrl-26.jpg|450px|thumb|center|E.coli T7 autoloop without terminator at 37°C  (gfp image)]]
 
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===Positive Control===
 
===Positive Control===

Revision as of 17:09, 23 October 2011

Characterization

This construct was transformed into BL21 strains expressing the T7 polymerase under IPTG induction.

Fluorescence kinetics

The measurements have been carried out on a spectrophotometer, at 37°C under transient shaking. The experiment lasted 4h, we tested several colonies and several IPTG concentrations. The OD 600nm and the fluorescence of the GFP (exc: 470nm / meas:515 nm) was measured every 5 min, and the ratio of the two was calculated.

All values were normalized by substracting the fluorescence/OD value of the well with 0 mM IPTG at time 0. The values given are in arbitrary units.

Fig1: Growth curves for BL21 strain carrying the part

After 2 hrs of induction, we see a clear increase of the fluorescence proportional to the IPTG concentration (that is to say with the quantity of T7 polymerase induced in the cell). After 4 hrs, the expression of GFP under the pT7 is still not saturated

Fig2: Comparison of the Fluo/OD ratio for transcription

Here, we plot the ratio of induction of the T7 polymerase dependant construct for the different concentrations of IPTG at a given time (4 hrs), taking the well with 0 IPTG at time 0 as the reference.


Microscopy Characterization

Here we used a simple protocol to make sure that we have nice positive and negative controls. Glucose is used as an inhibitor of our system.

Negative Control

First we launch cells from the overnight's tube without IPGT. Then we wash the cells and relaunch them with glucose to inhibit out gene expression.

T7 autoloop in E.coli at 37°C
E.coli T7 autoloop without terminator at 37°C (trans image)
E.coli T7 autoloop without terminator at 37°C (gfp image)

Positive Control

irst we launch cells from the overnight's tube. Cells are induced with IPGT. Then we wash the cells and relaunch them with IPTG. This way we are sure that the gene will be expressed.


Mixed strategies

1-First we launch cells from the overnight's tube without IPGT. Then we wash the cells and relaunch them with IPTG.

2-First we launch cells from the overnight's tube. Cells are induced with IPGT. Then we wash the cells and relaunch them with glucose.


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