Difference between revisions of "Part:BBa K802002"
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<partinfo>BBa_K802002 short</partinfo> | <partinfo>BBa_K802002 short</partinfo> | ||
| − | + | 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 inductor IPTG. Within this part there is pLac 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 parameter using the amount of fluorescence.<br/> | |
| + | <br/> | ||
| + | == Characterization == | ||
| + | <html> | ||
| + | |||
| + | |||
| + | <p>Modelling of pLac 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/> | ||
| + | <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>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 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/> | ||
| + | <br/> | ||
| + | |||
| + | <p>With and without IPTG addition the E coli NM522 grows so there is no toxic effect of the inducter. | ||
| + | However the addition of IPTG induces a growth delay which is normal because of the selection | ||
| + | pressure. The different concentrations have no effect on the bacteria growth.</p><br/> | ||
| + | <br/> | ||
| + | |||
| + | <p>Concerning the ratio fluorescence/OD a spike is observed at … which is the time where the amount | ||
| + | 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> | ||
| + | <br/><br/><br/> | ||
| + | |||
| + | <big><b>Conclusion:</b></big> | ||
| + | <br/> | ||
| + | <p>IPTG is an inductor of pLac as the in silico test says. The hypothesis can be approved but no influence | ||
| + | of the concentration is observed.</p><br/> | ||
| + | |||
| + | |||
| + | |||
| + | </br></br></br> | ||
| + | </html> | ||
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Revision as of 16:57, 25 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 inductor IPTG. Within this part there is pLac from Bacillus subtilis which is the promoter that induces a xylR production to form a positive biofilm. Then a RBS from E coli is added to allow it and finally the 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 inductor to be activated. This inductor is IPTG so to approve this in silico data, biological experiments are made with different amount of inducter.
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.
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 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.
With and without IPTG addition the E coli NM522 grows so there is no toxic effect of the inducter. However the addition of IPTG induces a growth delay which is normal because of the selection pressure. The different concentrations have no effect on the bacteria growth.
Concerning the ratio fluorescence/OD a spike is observed at … which is the time where the amount 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.
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.
Conclusion:
IPTG is an inductor of pLac as the in silico test says. The hypothesis can be approved but no influence of the concentration is observed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 762