Difference between revisions of "Part:BBa K542008"
 
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<partinfo>BBa_K542008 short</partinfo>
 
<partinfo>BBa_K542008 short</partinfo>
  
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Made by assembling [https://parts.igem.org/wiki/index.php?title=Part:BBa_K542004 BBa_K542004] with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K542005 BBa_542005].
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Made by assembling [https://parts.igem.org/wiki/index.php?title=Part:BBa_K542004 BBa_K542004] with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K542005 BBa_K542005].
  
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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).
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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.
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<br><br>
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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).
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<br><br>
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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.
  
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Because the fluorescent proteins are positively tagged (ie. arginines) and the Lumazine Synthase is mutated with a negative interior ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K249002 BBa_249002] 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.
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Datasheet for Part BBa_K542008 in <i>E. coli</i> strain DH5alpha. You may also wish to refer to the "Experience" page. <br>
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[[image:uoflECdatasheet.png|150px]]  
  
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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.
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===References===
  
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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 during formation?)
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(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.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 17:25, 28 September 2011

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.
UoflECdatasheet.png

References

(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.

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]