Composite

Part:BBa_K808030

Designed by: Marie Burghard, Henrik Cordes, Jascha Diemer, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling   Group: iGEM12_TU_Darmstadt   (2012-09-02)
Revision as of 21:02, 24 September 2012 by Arne (Talk | contribs) (→‎Usage and Biology)

RBS-PhoA-His6tag-pNBEst13-Myctag-EstA

RBS-PhoA-His6tag-pNBEst13-Myctag-EstA is a long name for a long protein construct. It is a fusion protein which is secreted into the peri plasma in order to be integrated into the outer membran. When the phoA signal sequence is cut off, the N-terminus is formed by our pNB-Est13. This esterase shows actitivty towards PET and is anchored onto the cell by its inactive EstA membrane anchor.

Annotation: This site is about the construct and composition of our chimeric protein BBa_K808030. For any information about its expression rate or enzyme activity please visit BBa_K808032


Usage and Biology

Composition

Our fusion protein BBa_K808030 is highly chimeric. It consists of parts from three different bacteria, which are the following (starting from N-terminus to C-terminus):

  • PhoA (BBa_K808028) is responsible for the peri plasmatic expression of our chimeric protein. This is highly important because once expressed into peri plasma, our construct will self-install into the outer membrane. PhoA is native to E.coli
  • pNB-Esterase 13 (BBa_K808026) is the the catalytical domain of BBa_K808030. It derives from an esterase of Bacillus licheniformis.
  • EstA (BBa_K808027) serves as a membrane anchor but is an inactive and membrane bound esterase mutant deriving from Pseudomonas aeruginosa

Visualization

The composition and folding of BBa_K808030. Yellow:pNB-Esterase 13 (BBa_K808026)after the N-terminal cut-off of PhoA. Gray: inactive catalytical part of EstA (BBa_K808027). Blue: Transmembrane helix of EstA, connecting its inactive catalytical domain and membrane anchor. Red: Anchor domain of EstA (BBa_K808027)






























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 1174
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1700
    Illegal NgoMIV site found at 1838
    Illegal NgoMIV site found at 2378
    Illegal NgoMIV site found at 2747
    Illegal AgeI site found at 1245
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/enzyme
//cds/membrane/extracellular
//function/degradation
Parameters
protein