Difference between revisions of "Part:BBa K802002"

Latest revision as of 22:19, 26 September 2012

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

This part has been designed in order to determine the P_lac 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_lac 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 P_lac promoter. These results helped refine the parameters for the P_lac 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

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 <partinfo>BBa_K802002 short</partinfo>
-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 inducer IPTG. Within this part there is P<sub>lac</sub> from <i>Bacillus subtilis</i> which is the promoter that induces production of XylR to form a positive biofilm. Then 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>
+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>
 == Characterization ==
 <html>
+<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/>
-<p>Modelling of P<sub>lac</sub> is made <i>in silico</i>. It is said and shown that this promoter needs an inducer to be activated. This inducer is IPTG so to approve this <i>in silico</i> data, biological experiments are made with different amount of inducer.</p><br/>
 <br/>
+<center>
-<p> A 96-well plate test is made with a saturated NM522 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 addition of IPTG. A control of fluorescence is also made with a NM522 culture.</p><br/>
+<img src="https://static.igem.org/mediawiki/parts/9/9e/Graphe_coll%C3%A9.JPG" width=900/>
+<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>
-<p>With regards to the protocol 200µL of IPTG 1mM in LB and 2µL of the saturated NM522 culture containing P<sub>lac</sub>-RBS-GFP are added in each well. Thus, bacteria are inoculated to the hundredth. Then OD<sub>600</sub> and fluorescence are automatically recorded during 24 hours at 30°C with a 10-second agitation every 10 minutes. To be more specific, the excitation wavelength is 485 nm and the emission wavelength is 530nm for the fluorescence measurements.</p><br/>
+</center>
-<br/>
-<p>With and without IPTG addition, <i>E.coli</i> NM522 grows so there is no toxic effect of the inducer.
-However, the addition of IPTG induces a growth delay which is normal because of the selection pressure. The different concentrations have no effect on bacteria growth.</p><br/>
 <br/>
-<p>Concerning the ratio fluorescence/OD<sub>600</sub>, a spike is observed after 7.5 hours, when the amount of GFP is the highest, in other words when the promoter is the most active. Then. due to instability of the GFP, it is reduced.<p><br/>
+<p>Our results show a spike of fluorescence after 7.5 hours.<p><br/>
 <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>
@@ Line 27: / Line 21: @@
 <big><b>Conclusion :</b></big>
 <br/>
-<p>IPTG 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/>
+<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/>