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 under different in a 96-well plate test assay using three different IPTG concentrations: 1mM, 0.5mM and 0.1mM. 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.

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 behaviorIPTG 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

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