Difference between revisions of "Part:BBa K1893017:Experience"

(→‎Applications of BBa_K1893017)
(→‎Characterisation of BBa_K1893017)
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Without any arabinose in the cell, araC forms a homodimer, binding to operator sites upstream of the pBAD promoter, generating an inhibitory loop in the DNA that prevents access by the RNAP holoenzyme. In the absence of glucose, and the presence of L-arabinose, the dimer dissociates from the DNA, allowing transcription of GFP.  
 
Without any arabinose in the cell, araC forms a homodimer, binding to operator sites upstream of the pBAD promoter, generating an inhibitory loop in the DNA that prevents access by the RNAP holoenzyme. In the absence of glucose, and the presence of L-arabinose, the dimer dissociates from the DNA, allowing transcription of GFP.  
  
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We added varying concentrations of arabinose (optimum concentration ranges were obtained from (sloveniaon igem characterisation), and recorded fluorescence in a BMG plate reader.  
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We added varying concentrations of arabinose (optimum concentration ranges were obtained from [https://parts.igem.org/Part:BBa_I0500:Experience 2010 iGEM team Slovenia characterisation]), and recorded fluorescence in a BMG plate reader.  
  
 
[[File:AraC_TOP10.png|700px|center|]]
 
[[File:AraC_TOP10.png|700px|center|]]
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Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. (BBa_K1893017).Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation
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Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. [https://parts.igem.org/Part:BBa_K1893017 (BBa_K1893017)].Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation
  
 
[[File:Transfer_curve_arac.png|700px|center|]]
 
[[File:Transfer_curve_arac.png|700px|center|]]

Revision as of 15:42, 27 October 2016


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Characterisation of BBa_K1893017

Without any arabinose in the cell, araC forms a homodimer, binding to operator sites upstream of the pBAD promoter, generating an inhibitory loop in the DNA that prevents access by the RNAP holoenzyme. In the absence of glucose, and the presence of L-arabinose, the dimer dissociates from the DNA, allowing transcription of GFP.

We added varying concentrations of arabinose (optimum concentration ranges were obtained from 2010 iGEM team Slovenia characterisation), and recorded fluorescence in a BMG plate reader.

AraC TOP10.png

Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. (BBa_K1893017).Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation

Transfer curve arac.png

Figure 2: Transfer function of normalised GFP fluorescence against concentration of L-arabinose

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