Difference between revisions of "Part:BBa K802002"

Revision as of 21:44, 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 response of this construct to IPTG induction, we have monitored the fluorescence under different The kinetic of activation of this construct has been tested in 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.

Modelling of P_lac 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.

With regards to the protocol 200µL of IPTG 1mM in LB and 2µL of the saturated NM522 culture containing P_lac-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 P_lac as predicted in the in silico test. The hypothesis can be approved but no influence of the concentration is observed.

Sequence and Features