Difference between revisions of "Part:BBa K542010"
 
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<partinfo>BBa_K542010 short</partinfo>
 
<partinfo>BBa_K542010 short</partinfo>
  
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The part was synthesized by [http://www.biobasic.com/ Bio Basic Inc.] into the pET28a plasmid vector with an N-terminal His-tag. The Enhanced Lumazine Synthase (ELS) without the His-tag was moved into the pSB1C3 plasmid vector by the Lethbridge 2011 team.
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Lumazine synthase (LS) from <i>Aquifex aeolicus</i> forms icosahedral microcompartment (MC) assemblies of 60 or 180 monomeric units that self assemble and are capable of isolating proteins from their local environment. As shown in previously published work (1), the LS protein has been mutated so that the interior of the MC is negatively charged; the UL 2009 iGEM team has submitted the mutated LS gene to the parts registry (BBa_K249002). A negatively charged interior allows for preferential compartmentalization of positively charged molecules, which can easily be engineered through the addition of a poly-arginine tag to a target protein (1). The size of the cavity of the Lumazine Synthase microcompartment was enlarged and the negative charge was increased via directed evolution (2). The compartment formed by this polypeptide has a larger cavity and a larger net negative charge, thus allowing a larger loading capacity into the cavity of the compartment.
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<br><br>
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The part was synthesized by Bio Basic Inc. into the pET28a plasmid vector (which allows for expression of ELS with an N-terminal His-tag). The Enhanced Lumazine Synthase (ELS) was moved into the pSB1C3 vector by the Lethbridge 2011 team.
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<br><br>
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Datasheet for Part BBa_K542010 in <i>E. coli</i> strain BL21 (DE3). You may also wish to refer to the "Experience" page.
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<br>
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[[image:uoflELSdatasheet.png|200px]]
  
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The size of the cavity of the Lumazine Synthase microcompartment was enlarged via directed evolution (Wörsdörfer ''et al.,'' 2011). The compartment formed by this polypeptide has a larger cavity than the Lumazine Synthase microcompartment submitted by the Lethbridge 2009 team ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002]). A larger interior allows for the option of localizing larger and/or more proteins into the cavity of the compartment.
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===References===
  
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The His-tagged ELS in pET28a was transformed into ''E. coli'' BL21 (DE3) and overexpressed.
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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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<br>
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[[Image:OverexpressELS.jpg|x200px]]<br>
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(2) Wörsdörfer, B., Woycechowsky, K.J., and Hilvert, D. (2011). Directed Evolution of a Protein Container. Science. 331: 589-592.
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'''Figure 1: 12.5% SDS-PAGE of His-tagged ELS in BL21 (DE3) Overexpression.''' Samples were taken at 1 hour increments after IPTG induction. Lane 1: zero hours; Lane 2: 1 hour; Lane 3: 2 hours; Lane 4: 3 hours. Clear overexpression observed near ~25.0 kD marker.
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Reference:<br>
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Wörsdörfer, B., Woycechowsky, K.J., and Hilvert, D., (2011). Directed Evolution of a Protein Container. ''Science.'' 331: 589-592.
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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 17:26, 28 September 2011

Enhanced Lumazine Synthase (ELS)

Lumazine synthase (LS) from Aquifex aeolicus forms icosahedral microcompartment (MC) assemblies of 60 or 180 monomeric units that self assemble and are capable of isolating proteins from their local environment. As shown in previously published work (1), the LS protein has been mutated so that the interior of the MC is negatively charged; the UL 2009 iGEM team has submitted the mutated LS gene to the parts registry (BBa_K249002). A negatively charged interior allows for preferential compartmentalization of positively charged molecules, which can easily be engineered through the addition of a poly-arginine tag to a target protein (1). The size of the cavity of the Lumazine Synthase microcompartment was enlarged and the negative charge was increased via directed evolution (2). The compartment formed by this polypeptide has a larger cavity and a larger net negative charge, thus allowing a larger loading capacity into the cavity of the compartment.

The part was synthesized by Bio Basic Inc. into the pET28a plasmid vector (which allows for expression of ELS with an N-terminal His-tag). The Enhanced Lumazine Synthase (ELS) was moved into the pSB1C3 vector by the Lethbridge 2011 team.

Datasheet for Part BBa_K542010 in E. coli strain BL21 (DE3). You may also wish to refer to the "Experience" page.
UoflELSdatasheet.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.
(2) Wörsdörfer, B., Woycechowsky, K.J., and Hilvert, D. (2011). Directed Evolution of a Protein Container. Science. 331: 589-592.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]