Difference between revisions of "Part:BBa K1489000"

Revision as of 03:37, 17 October 2014

pBAD-RBS-Bovine Galectin-1-B0012 Terminator

His-tagged 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 protein is his-tagged in order to ease protein purification and western blot recognition assays.

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_K1489000 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.

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