Part:BBa_K808030

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 (Figure 1) 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

Figure 1: schematic graphic of BBa_K808030, integrated into an biological membrane. The C-terminalEstA (BBa_K808027) consists of a C-terminal ß-Barrel froming the membrane anchor, and a N-terminal inactive esterase domain, carrying the passenger enzyme. In this case the passenger Enzyme is pNB-Esterase 13 (BBa_K808026).

Visualization

With special regards to our [http://2012.igem.org/Team:TU_Darmstadt/Project/Simulation Simulation lab] we show you the result of great computional work. Our dry lab made some homologies and edited these beautiful graphics of our chimeric protein. (Figure 2 & 3)

Figure 2: 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)

Figure 3: The composition and folding of BBa_K808030 when integrated into a biological membrane

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]