Composite
pBAD-BtGal

Part:BBa_K1489000

Designed by: Iowis Zhu   Group: iGEM14_UMaryland   (2014-10-06)
Revision as of 20:37, 16 October 2014 by HHeLiBeBCNOFNe (Talk | contribs)

pBAD-RBS-Bovine Galectin-1-B0012 Terminator

Soluble beta-galactoside binding protein (galectin) originally from Bos taurus. Believed to expressed both intracellularly and extracellularly, where they serve to binding various types of beta-galactosides and initialize signal cascades. Believed to be a dimer in its active form, as is true with most mammalian galectins.

This biobrick is under the pBAD promoter (BBa_K206000) with attached RBS (BBa_B0034), and utilizes an additional RNA polymerase terminator (BBa_B0012).

UMaryland 2014 is interested in utilizing this part to investigate whether E. coli can recognize and bind to surface carbohydrates on a marine pathogen. Other uses of this part lie in the recognition of various carbohydrate ligands and potential activation of signal transduction pathways.

Expression Test:

BBa_K148900 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.

BovineGalectinExpression.jpg

Clear band of correct size can be seen in the soluble induced fraction, while no band is seen in the uninduced cells.


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 499
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None