Part:BBa_K638403

Arabinose inducible TorA-Reflectin A1-sfGFP generator

This construct is Reflectin A1 protein with a TorA signal peptide on the N terminus, and superfolder GFP (BBa_I746916) fused at the C-terminus. The reflectin A1 sequence is codon-optimized for E coli. The TorA signal peptide has been shown to allow export of GFP via the twin arginine pathway. The fusion gene is under the control of the pBAD promoter BBa_I0500, which is tightly controlled by two factors:

• L-arabinose monosaccharide taken up by the cell from the medium, which acts as an inducer.
• AraC protein included in the I0500 biobrick, which acts an a repressor.

Therefore, the araC-pBAD system offers regulatable control of gene expression in the presence of the inducer and highly repressed in the absence of the inducer. Read more about [http://2011.igem.org/Team:Cambridge/Experiments/Low_Level_Expression the pBAD and arabinose system in E.coli].

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 BamHI site found at 1369
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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

  Illegal SapI site found at 961
  Illegal SapI.rc site found at 2437
  

Usage and Biology

Best used in an E. coli chassis such as strain [http://cgsc.biology.yale.edu/Strain.php?ID=111773 BW27783], with constitutive expression of an Arabinose transporter. See our experience of pBAD for some issues with tuning expression levels.

Cambridge 2011 were unable to test export fully as of the wiki freeze (21st Sep). [http://2011.igem.org/Team:Cambridge/Experiments/Periplasmic_Export See our wiki] for more information on our experiments.

Safety

The protein coding sequence for reflectin originally came from cells of an edible squid. There have been no reported safety issues for reflectins, so we do not anticipate the need for extra precautions when using this BioBrick part. See our [http://2011.igem.org/Team:Cambridge/Safety safety page] for more information.