Part:BBa_K802002

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