Difference between revisions of "Part:BBa K802002"
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<partinfo>BBa_K802002 short</partinfo>
 
<partinfo>BBa_K802002 short</partinfo>
  
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This part is used to determine if the P<sub>lac</sub> promoter works or not and especially how it works when it is activated by different concentration of the IPTG inductor. Within this part, there is P<sub>lac</sub> from <i>Bacillus subtilis</i> which is the promoter that induces a XylR production to form a positive biofilm. Then a RBS from <i>E. coli</i> is added to allow it and finally the GFP, which is an easy measurable biomarker for its amount of fluorescence.<br/>
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This part has been designed in order to determine the P<sub>lac</sub> promoter kinetic parameters needed for our model, and especially to characterize its association kinetics with its inducer : IPTG. This part is composed of the P<sub>lac</sub> from <i>Bacillus subtilis</i> which is the promoter that we have used to induce the production of XylR to form a positive biofilm. A RBS from <i>E. coli</i> is added to finally enable production of GFP which is an easy measurable parameter using the amount of fluorescence.<br>
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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 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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<p>Modelling of <i>Plac</i> is made in silico. It is said and shown that this promoter needs an inductor to be activated. This inductor is IPTG so to approve this in silico data, biological experiments are made with different amount of inducter.</p><br/>
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<p> A 96 wells plate test is made with a NM522 saturated culture containing the plasmid and three different IPTG concentrations: 1mM; 0.5mM and 0.1mM. The control is the same saturated NM522 culture with the plasmid but without the IPTG addition. A control of fluorescence is also made with a NM522 culture.</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>With regards to the protocol 200µL of IPTG 1mM in LB and 2µL of the saturated NM522 culture containing <i>Plac</i>-RBS-GFP are added in each well. Thus, bacteria are inoculated to the hundredth. Then the OD and fluorescence is automatically recorded for 24h at 30°C with a 10 second agitation every 10min. To be more specific< the OD is recorded at 600nm, the excitation wavelength is 485 nm and the emission wavelength is 530nm.</p><br/>
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<p>With and without IPTG addition the NM522 <i>E.coli</i> grows so there is no toxic effect of the inducter.
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However, the addition of IPTG induces a growth delay which is normal because of the selection
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pressure. The different concentrations have no effect on the bacteria growth.</p><br/>
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<p>Concerning the ratio fluorescence/OD a spike is observed at 7h30 which is the time where the amount
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<p>Our results show a spike of fluorescence after 7.5 hours.<p><br/>
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of GFP is the highest, in other words when the promoter is the most active. Then because of the
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instability of the GFP, it is reduced.<p><br/>
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<a href="http://2012.igem.org/Team:Lyon-INSA/protocol"/><font color="grey"><b>In you have any question on the following experiments, don’t forget that all the information relative to our strains, plasmids and protocols are on our wiki notebook.</b></font></a>
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<a href="http://2012.igem.org/Team:Lyon-INSA/protocol"/><font color="grey"><b>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.</b></font></a>
 
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<big><b>Conclusion:</b></big>
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<big><b>Conclusion :</b></big>
 
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<p>IPTG is an inductor of <i>Plac</i> as the in silico test says. The hypothesis can be approved but no influence
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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/>
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of the concentration is observed.</p><br/>
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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