Part:BBa_K2387055

Split mRFP controlled by inducible araC/pBAD promoter

This a split version of BBa_K2387054. The two halves of mRFP are fused to leucine zippers to induce the reassembly. Both parts are under the control of the araC/pBad promoter and each part is under the control of a stron RBS. The reassembly process is irreversible. This part can be used as a positive control for experiments using BiFC, as well as a source for the fragments of mRFP.

The mRFP has already been split for the iGEM Registry (BBa_I715022 & BBa_I715023), where the mRFP was split at the position 155. However, in these parts the mRFP is not split at the right site, and therefore does not produce fluorescence upon interaction. As observed by Guido et al. (2006), mRFP only is able to reassemble into a functional fluorescent protein when it is split in the position 168.

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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 2043
  Illegal AgeI site found at 2155
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Functional Parameters

In order to test the functionality of this split protein under the araC/pBad promoter, the absorbance and the fluorescence spectra of the full length protein and the split protein were measured after induction with 0.02% arabinose (Figure 1). The stability of the shape in both spectra for the full length protein and the split protein show that the split has been successful.

Figure 1: Absorbance and fluorescence spectra of full length mRFP (left) and split mRFP (right), measured after induction with 0.02% arabinose.

This protein is part of a collection of split proteins developed for BiFC analysis and tested with the CpxR system (BBa_K2387032). These proteins were characterized through multiple experiments using as interacting proteins antiparallel synthetic leucine zippers. The proteins of this collection are the ones found in Table 1.

Table 1: Split Proteins.
Protein	Part Number (Full length)	Part Number (Split Protein)
mRFP	BBa_K2387054	BBa_K2387055
eYFP	BBa_K2387003	BBa_K2387065
mVenus	BBa_K2387045	BBa_K2387046
sfGFP	BBa_K2387047	BBa_K2387048
mCerulean	BBa_K2387052	BBa_K2387053

References

Guido, Jach, et al. "An improved mRFP1 adds red to bimolecular fluorescence complementation." Nature Methods 13.8 (2006): 597-600.