Difference between revisions of "Part:BBa K802002"
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== Characterization ==
 
== Characterization ==
 
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<p> To evaluate the behavior of this construct to IPTG induction, we have monitored the fluorescence under different in a 96-well plate test assay using three different IPTG concentrations: 1mM, 0.5mM and 0.1mM. Negative controls included the <i>E. coli</i> host without plasmid or with the empty vector.</p><br/>Fluorescence was recorded over 24 hours at 30°C with a 10-second agitation every 10 minutes.</p><br/>
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<p> To evaluate the behavior of this construct to IPTG induction, we have monitored the fluorescence of the different constructs using three different IPTG concentrations: 1mM, 0.5mM and 0.1mM in a 96-well plate test assay. Negative controls included the <i>E. coli</i> host without plasmid or with the empty vector.</p><br/>Fluorescence was recorded over 24 hours at 30°C with a 10-second agitation every 10 minutes.</p><br/>
 
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<img src="https://static.igem.org/mediawiki/parts/9/9e/Graphe_coll%C3%A9.JPG" width=900/>
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<div style="font-size:12px">Figure 1: Influence of different concentrations of IPTG on the promoter P<span style="font-size:8px">lac</span> in 500µm cuves</div>
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<p>Our results show a spike of fluorescence after 7.5 hours.<p><br/>
 
<p>Our results show a spike of fluorescence after 7.5 hours.<p><br/>
  
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<big><b>Conclusion :</b></big>
 
<big><b>Conclusion :</b></big>
 
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<p>Our results confirm the expected behavior for the <i>B. subtilis</i> P<sub>lac</sub> promoter. These behaviorIPTG is an inducer of P<sub>lac</sub> as predicted in the <i>in silico</i> test. The hypothesis can be approved but no influence of the concentration is observed.</p><br/>
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<p>Our results confirm the expected behavior for the <i>B. subtilis</i> P<sub>lac</sub> promoter. These results helped refine the parameters for the P<sub>lac</sub> used in the <i>in silico</i> model.</p><br/>
  
  

Latest revision as of 22:19, 26 September 2012

Plac(B. subtilis)-RBS(E. coli)-GFP

This part has been designed in order to determine the Plac promoter kinetic parameters needed for our model, and especially to characterize its association kinetics with its inducer : IPTG. This part is composed of the Plac from Bacillus subtilis which is the promoter that we have used to induce the production of XylR to form a positive biofilm. A RBS from E. coli is added to finally enable production of GFP which is an easy measurable parameter using the amount of fluorescence.

Characterization

To evaluate the behavior of this construct to IPTG induction, we have monitored the fluorescence of the different constructs using three different IPTG concentrations: 1mM, 0.5mM and 0.1mM in a 96-well plate test assay. Negative controls included the E. coli host without plasmid or with the empty vector.


Fluorescence was recorded over 24 hours at 30°C with a 10-second agitation every 10 minutes.



Figure 1: Influence of different concentrations of IPTG on the promoter Plac in 500µm cuves

Our results show a spike of fluorescence after 7.5 hours.


If you have any question on the following experiments, don’t forget that all the information concerning our strains, plasmids and protocols are on our wiki notebook.


Conclusion :

Our results confirm the expected behavior for the B. subtilis Plac promoter. These results helped refine the parameters for the Plac used in the in silico model.






Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 762