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

(→‎Comparison of the growth with the traduction saturated cells)
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We hae characterized this part transforming it into a BL21 strain. This strain carries insite the chromosome a T7 polymerase under the control of an IPTG inducible promoter. This strain is mostly used for protein expression for protein purification application.
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We have characterized this part by transforming it into a BL21 strain. This strain carries inside the chromosomal gene for a T7 polymerase under the control of an IPTG inducible promoter.
  
 
=== Growth measurements ===
 
=== Growth measurements ===
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The offset values for these curves was adjusted for better visualisation. The values given are in arbitrary units.
 
The offset values for these curves was adjusted for better visualisation. The values given are in arbitrary units.
  
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[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part, in the presence of cloramphenicol]]
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[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
  
 
First we see an inflexion in the curve, that is due to the stong influence of the IPTG on the metabolism of the cells. Then, this loss it taken up and the bacteria start growing again. We see a clear increase of the fluorescence with the IPTG concentration, that is to say with the quantity of T7 polymerase in the cell
 
First we see an inflexion in the curve, that is due to the stong influence of the IPTG on the metabolism of the cells. Then, this loss it taken up and the bacteria start growing again. We see a clear increase of the fluorescence with the IPTG concentration, that is to say with the quantity of T7 polymerase in the cell
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=== Comparison of the growth with the traduction saturated cells ===
 
=== Comparison of the growth with the traduction saturated cells ===
  
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As a positive control we have saturated a strain with a lot of IPTG. After 1h50 of growth, we compare the fluorescence of the gradient of IPTG with the saturated cells. We see a clear increase of the fluorescence wereas the saturated strains is quite stable.
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As a positive control we have saturated a strain with a lot of IPTG. After 1h50 of growth, we compare the fluorescence of the gradient of IPTG with the saturated cells. We see a clear increase of the fluorescence whereas the saturated strains show quite stable measures.
  
 
[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription saturated and non saturated cells]]
 
[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription saturated and non saturated cells]]

Revision as of 20:45, 21 September 2011


We have characterized this part by transforming it into a BL21 strain. This strain carries inside the chromosomal gene for a T7 polymerase under the control of an IPTG inducible promoter.

Growth measurements

The measurements had been carried on in a TECAN i-control machine, at 37°C under transcient shaking, for 4h, for several colonies and several range of IPTG concentration. 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.

The offset values for these curves was adjusted for better visualisation. The values given are in arbitrary units.

Fig1: Growth curves for BL21 strain carrying the part

First we see an inflexion in the curve, that is due to the stong influence of the IPTG on the metabolism of the cells. Then, this loss it taken up and the bacteria start growing again. We see a clear increase of the fluorescence with the IPTG concentration, that is to say with the quantity of T7 polymerase in the cell

Comparison of the growth with the traduction saturated cells

As a positive control we have saturated a strain with a lot of IPTG. After 1h50 of growth, we compare the fluorescence of the gradient of IPTG with the saturated cells. We see a clear increase of the fluorescence whereas the saturated strains show quite stable measures.

Fig2: Comparison of the Fluo/OD ratio for transcription saturated and non saturated cells

The offset of the curves is here renormalize at the inflexion point of the growth, when the cells start have passed the IPTG stress and are growing again.

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