Part:BBa_K542008

pLacI Regulated Lumazine Synthase and pBAD Inverse-Regulated Arg-tagged ECFP and EYFP

Made by assembling BBa_K542004 with BBa_K542005.

The intended purpose of the Lumazine Synthase microcompartment device is to specifically target proteins that have been tagged with a positively charged oligopeptide into the cavity formed by the microcompartment. Furthermore, multiple unique proteins may be targeted into the cavity (1) for the purposes of increasing the efficiency of a metabolic pathway, to name just one example. This test construct is designed to demonstrate that proteins with positively charged tags can be localized into the cavity of the microcompartment formed by the oligomerization of Lumazine Synthase monomers.

The expression of Lumazine Synthase and the fluorescent proteins are independently regulated by two separate promoters. Lumazine Synthase is regulated by the pLacI promoter and the fluorescent proteins are regulated by the pBAD inverter. Since the two are independently controlled, Lumazine Synthase microcompartments may be formed in the presence or absence of the fluorescent proteins (ie. absence or presence of arabinose, respectively).

Because the fluorescent proteins are tagged with a positively-charged poly-arginine tag and the Lumazine Synthase is mutated with a negative interior (BBa_K249002 and Lethbridge 2009 Modeling), enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) should be targeted into the microcompartments. ECFP and EYFP are a well known FRET pair.

Datasheet for Part BBa_K542008 in E. coli strain DH5alpha. You may also wish to refer to the "Experience" page.

References

(1) (1) Seebeck, F., Woycechowsky, K., Zhuang, W., Rabe, J., and Hilvert, D. (2006). A simple tagging system for protein encapsulation. Journal of the American Chemical Society. 128: 4516-4517.

Characterization By

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 961
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 901
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]