Difference between revisions of "Part:BBa K606014:Experience"

 
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
  
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===Applications of BBa_K606014===
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This part has been characterized under the control of the pT7 T7 RNA polymerase promoter (part K606035) in E. coli BL21 strain.
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'''Fluorescence kinetics'''
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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 (excitation: 470nm / emission:515 nm) was measured every 5 min, and the ratio of the two was calculated.</p>
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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]]
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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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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription]]
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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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</html>
  
 
===User Reviews===
 
===User Reviews===
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Latest revision as of 07:16, 27 October 2011


This part has been characterized under the control of the pT7 T7 RNA polymerase promoter (part K606035) in E. coli BL21 strain.

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 (excitation: 470nm / emission: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

UNIQcbf9394378bc78c6-partinfo-00000002-QINU UNIQcbf9394378bc78c6-partinfo-00000003-QINU