Composite
OmpA-BtGal

Part:BBa_K1489004

Designed by: Iowis Zhu   Group: iGEM14_UMaryland   (2014-10-06)
Revision as of 23:51, 16 October 2014 by HHeLiBeBCNOFNe (Talk | contribs)

pBAD-RBS-OmpA-Bovine Galectin-1-B0012 Terminator

A fusion protein of OmpA (BBa_K1489002) fused to Bovine Galectin-1 (BBa_K1489001). Used to bind sugars at the outer cell membrane. Under pBAD regulation.

Expression Test: BBa_K1489004 was transformed into chemically competent DH5-alpha cells and plated overnight with chloramphenicol challenge. One colony was incubated in 5 mL LB media + 34 ug/mL chloramphenicol and grown to mid-log phase at 37oC. Arabinose was added to a final concentration of 0.2% to initiate protein production and cells were re-incubated at 37 oC for 21 hours. Cells were then pelleted and lysed with 10% SDS solution. Cell lysate was centrifuged at 13200 rpm in a tabletop centrifuge for 1 hour to separate soluble and insoluble fractions. Both fractions were loaded on an SDS-PAGE gel for western blot analysis using anti-His antibodies.

OmpA BovineExpression.jpg

The presence of bands in both the induced and uninduced cells indicates leaky expression of the protein. The molecular weight corresponds with the weight of OmpA-Bovine. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal XhoI site found at 971
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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