Part:BBa_K1486002

Arabinose promoter + sfGFP CpxR (Nterm)

Purpose of the Biobrick

This construct aimed to evaluate the expression of our CpxR construct, and the function of the arabinose promoter in E coli by fusing a superfolder GFP protein to the N terminus of CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations.

An experiment on both possible CpxR - sfGFP orientations was launched to determine whether the proteins were well expressed and folded, and if the arabinose promoter worked well. The experiment conditions can be found here. The basics were the following: increasing concentrations of arabinose as shown in Table 1 and results of a plate reader with excitation and emission wavelengths of 490 nm and 510 nm respectively.

The results of this experiment show that CpxR is well expressed, as GFP can be seen. Also, the results show that the arabinose promoter works as expected.

Experiment 1: CpxR dimerization & Dimerization Orientation

CpxR is the relay protein in the CpxAR two component regulatory system. It has been shown through Protein Complementation techniques that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in E.Coli. To determine this, we built four constructs with the various possible orientations as seen under the associated Biobricks section bellow. As seen in the graph bellow, we successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. Thanks to the fusion of the split IFP1.4, our biobrick allows fine tuned spatiotemporal analysis of the activation of CpxR in vivo. This finding is important as CpxR is part of the highly conserved OmpR/PhoB subfamily - especially for their C-terminus. This system could be used to study various other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.

As seen in the graph, induction of the signal was done at minute 24 (marked via a vertically spoted line). It is to be noted that the signal is immediate (3 fold increase in 2 minutes) and that the signal overall increased 30-fold.

Experiment 2: Signal induction by various concentrations of KCl & signal shutdown by centrifugation

Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change. To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we then centrifuged our cells and replaced the medium with PBS.

As seen in the figure above, we successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system.

Associated Biobricks

In the context of the same experiment, we designed another construct with the sfGFP moiety attached to the C terminus of CpxR: https://parts.igem.org/Part:BBa_K1486005. </UL>

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal XhoI site found at 2027
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 2438
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  Illegal SapI.rc site found at 1235