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 expression of Lumazine Synthase and the fluorescence 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 positively tagged (ie. arginines) and the Lumazine Synthase is mutated with a negative interior (BBa_K249002 and [http://2009.igem.org/Team:Lethbridge/Modeling Lethbridge 2009 Modeling]), ECFP and EYFP should be targeted into the microcompartments. ECFP and EYFP is a well known FRET pair.

Fluorescence/Förster Resonance Energy Transfer(FRET) is a distance-dependent phenomenon in which the excitation of a donor fluorophore leads to emission by an acceptor fluorophore; this occurs if the two fluorophores are within a certain distance to each other. The distance is dependent on the FRET pair used. The emission spectrum of the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore for FRET to occur. FRET is explained in further detail on the [http://2009.igem.org/Team:Lethbridge/Project#FRET Lethbridge 2009 Wiki]. This phenomenon will allow for characterizing co-localization within the Lumazine Synthase microcompartment.

If FRET is observed, it is suggestive that both ECFP and EYFP were co-localized into the microcompartment. In the design of this "Co-localization Testing Construct" control experiments were also taken into consideration. In the presence of arabinose (cease fluorescent protein expression), no FRET should be observed because there are no fluorescent proteins. The removal of arabinose after microcompartment formation will show the dynamics of the microcompartment. (ie. Can fluorescent proteins migrate into the microcompartment after they have formed, or must they be targeted before/during formation?)

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]