Difference between revisions of "Part:BBa K606035:Experience"
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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.
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===Characterization===
  
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=== Growth measurements ===
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In order to characterize the pT7 promotor, we used the construct: pT7-RBS-GFP-T7ter. This construct was transformed into BL21 strains expressing the T7 polymerase under IPTG induction.
  
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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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'''Fluorescence kinetics'''
  
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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>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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[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
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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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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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[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
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=== Comparison of the growth with the traduction saturated cells ===
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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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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.
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription]]
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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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<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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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.
 
  
 
===User Reviews===
 
===User Reviews===

Revision as of 00:37, 22 September 2011


Characterization

In order to characterize the pT7 promotor, we used the construct: pT7-RBS-GFP-T7ter. 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.

===User Reviews=== BBa_K606035 StartReviews BBa_K606035 EndReviews