Coding

Part:BBa_M10088:Design

Designed by: David de Renzy   Group: Berkeley BioE140L - S09   (2009-05-06)

{˂TshA!} | N-terminal displaying Tsh Autotransporter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 856
    Illegal PstI site found at 1201
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 856
    Illegal PstI site found at 1201
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 856
    Illegal PstI site found at 1201
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 856
    Illegal PstI site found at 1201
    Illegal NgoMIV site found at 1342
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part was designed for attachment to the C-terminal end of a desired exoprotein passengers, therefore it does not contain a start codon, but does contain a stop codon. Also, it does not include the necessary N-terminal Prepro sequences for SEC secretion across the perplasmic membrane.

Source

Pubmed nucleotide sequence AF218073 or protein AAA24698 from E. Coli Avian APEC genomic DNA. Selected coding sequence includes amino acid 855 to 1358.

Made by PCR based assembly from synthetic sources.

Construction of Tsh Autotransporter Part 

PCR Odd001F/Odd002R on E. Coli plasmid pAPEC-1 (AF218073)	(1603 bp, EcoRI/BamHI)
Sub into pBca9495KC-Bca1144#5					(EcoRI/BamHI, 2974+910 bp, L)
Product is pBca9495KC-M10040					{<TshA!}
---------------------------
Odd001F	Forward EcoRI oligo for TshA	ccataGAATTCatgAGATCTacacttttacctgtatacg
Odd002R	Reverse BamHI oligo for TshA	ctgatGGATCCtcagaatgaataacgaatattagcg

References

PMID: 15546669
Jacob-Dubuissona, F., R. Fernandez, and L. Coutte, Protein secretion through autotransporter and two-partner pathways. Biochimica et Biophysica Acta 1694, 2004: p. 235-257