Difference between revisions of "Part:BBa K1489000"

Latest revision as of 15:42, 31 October 2014

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:

Cells were grown to OD of 0.3, then induced with 10% arabinose (final concentration 0.2% arabinose). Cells were induced for 6 hours at 37oC. Approximately 4 g of cell paste were harvested after centrifugation (4000xg for 15 minutes at 4oC). Cells were frozen at -80 oC overnight. Next day: 2 g of cell paste was resuspended in 40 mL of equilibration buffer (0.5 M NaCl, 25 mM Tris, 30 mM Imidazole pH 7.5) and lysed via French Press. Lysate was centrifuged at 22000xg for 1 hour; the supernatant was subsequently filtered and injected into an FPLC machine equiped with a His-Trap column charged with 0.1 M Cobalt acetate. Column was washed with equilibration buffer (1 mL/min), then eluted (1 mL/min) along a gradient between 30 and 400 mM Imidazole over 1 hr. 5 uL of selected fractions was run on an SDS-PAGE gel and stained overnight with Coomassie.

Bands appear to be correct size. Fractions were collected, concentrated, and dialyzed into 40 mM Tris, 0.1 mM EDTA pH 7.8, 0.1 mM DTT for binding assay

Fluorecence Binding Assay

Binding of BtGal1 to lactose decreases fluorescence of an internal tryptophan. Measurement of fluorescence percentage decrease with increasing [lactose] allows a binding curve to be generated and a binding constant, Kd, to be measured.

Sequence and Features