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

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===Characterization===
  
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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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This construct was transformed into BL21 strains expressing the T7 polymerase under IPTG induction.
  
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=== Growth measurements ===
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'''Fluorescence kinetics'''
  
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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.
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<p>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.</p>
  
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The offset values for these curves was adjusted for better visualisation. The values given are in arbitrary units.
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<p>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.</p>
  
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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]]
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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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<p>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</p>
  
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=== Comparison of the growth with the traduction saturated cells ===
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription]]
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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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<p>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.</p>
  
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription saturated and non saturated cells]]
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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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===User Reviews===
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Latest revision as of 12:03, 24 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.