Part:BBa_K1489000
pBAD-RBS-Bovine Galectin-1-B0012 Terminator
His-tagged soluble beta-galactoside binding protein (galectin) originally from Bos taurus. Bovine galectin 1 is expressed both intracellularly and extracellularly, where they serve to binding various types of beta-galactosides and initialize signal cascades. BtGal1 is 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. This indicates that our biobrick is properly expressed. As BtGal1 has previously been successfully expressed in E. coli, it is highly likely that the function of the biobrick has been preserved.
Protein purifcation of Bovine Galectin 1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal XhoI site found at 499 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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