Difference between revisions of "Part:BBa K802002"
Line 2: Line 2:
 
<partinfo>BBa_K802002 short</partinfo>
 
<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 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/>
+
This part is used to determine if the promoter P<sub>lac</sub> 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>
−
<br/>
+
 
 
== Characterization ==
 
== Characterization ==
 
<html>
 
<html>
  
  
−
<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/>
+
<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/>
 
<br/>
  
−
<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/>
+
<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/>
  
−
<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/>
+
<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/>
 
<br/>
 
<br/>
  
−
<p>With and without IPTG addition the NM522 <i>E.coli</i> grows so there is no toxic effect of the inducter.
+
<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
+
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/>
−
pressure. The different concentrations have no effect on the bacteria growth.</p><br/>
+
 
<br/>
 
<br/>
  
−
<p>Concerning the ratio fluorescence/OD a spike is observed at 7h30 which is the time where the amount
+
<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/>
−
of GFP is the highest, in other words when the promoter is the most active. Then because of the
+
−
instability of the GFP, it is reduced.<p><br/>
+
  
−
<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>
+
<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>
 
<br/><br/><br/>
 
<br/><br/><br/>
  
−
<big><b>Conclusion:</b></big>
+
<big><b>Conclusion :</b></big>
 
<br/>
 
<br/>
−
<p>IPTG is an inductor of <i>Plac</i> as the in silico test says. The hypothesis can be approved but no influence
+
<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/>
−
of the concentration is observed.</p><br/>
+
  
  

Revision as of 13:58, 26 September 2012

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

This part is used to determine if the promoter Plac works or not and especially how it works when it is activated by different concentration of the inducer IPTG. Within this part there is Plac from Bacillus subtilis which is the promoter that induces production of XylR to form a positive biofilm. Then 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

Modelling of Plac is made in silico. It is said and shown that this promoter needs an inducer to be activated. This inducer is IPTG so to approve this in silico data, biological experiments are made with different amount of inducer.



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.


With regards to the protocol 200µL of IPTG 1mM in LB and 2µL of the saturated NM522 culture containing Plac-RBS-GFP are added in each well. Thus, bacteria are inoculated to the hundredth. Then OD600 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.



With and without IPTG addition, E.coli 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.



Concerning the ratio fluorescence/OD600, 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.


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 :

IPTG is an inducer of Plac as predicted in the in silico test. The hypothesis can be approved but no influence of the concentration is observed.






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