Biobricks

Part:BBa_K1489002 | Lpp-OmpA in new backbone

We moved an existing biobrick, BBa_K103006, from an old plasmid backbone, pSB1A2, into the current backbone, pSB1C3, via RE cloning.

Lpp-OmpA-Linker (pSB1C3)

Outer membrane protein A (OmpA) is a common outer membrane protein found in bacteria that serves various purposes. For our purposes, the beta-barrel transmembrane domain can be utilized to anchor otherwise soluble proteins in the outer cell membrane. Lpp serves to direct the fusion protein, while a unstructured linker (GGGSGGGS) serves to separate OmpA from its cargo in order to avoid interactions between the two proteins.

The expressed protein is structurally identical to BBa_K103006. This version of the biobrick has been moved into the new backbone, pSB1C3, which contains the gene for chloramphenicol resistance. This resistance gene contains an internal SacI site, making the SacI site at the end of the gene almost unusable for RE cloning. BBa_K1489003 mutagenizes this SacI site into a KasI in order to avoid this issue.

 

Part:BBa_K1489000 | pBAD-RBS-Bovine Galectin 1-B0012 Terminator

A soluble his-tagged galectin from Bos taurus, expressed under Arabinose induction

pBAD-RBS-Bovine Galectin-1-B0012 Terminator

Soluble beta-galactoside binding protein (galectin) originally from Bos taurus. It is believed to expressed both intracellularly and extracellularly, where they serve to binding various types of beta-galactosides and initialize signal cascades. It is 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.

 

 

Part:BBa_K1489004 | pBAD-RBS-Lpp-OmpA-Bovine Galectin 1-B0012 Terminator

His-tagged bovine galectin 1 attached to the c-terminus of OmpA, expressed under arabinose induction

 

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.

 

Part:BBa_K1489003 | Revised Lpp-OmpA

Biobrick 1, with a SacI site at the C-terminus replaced by a KasI site. This improvement was made in order to re-enable RE cloning; a SacI site exists in the chloramphenicol resistance gene.

Lpp-OmpA-Linker with KasI site

Outer membrane protein A (OmpA) is a common outer membrane protein found in bacteria that serves various purposes. For our purposes, the beta-barrel transmembrane domain can be utilized to anchor otherwise soluble proteins in the outer cell membrane. Lpp serves to direct the fusion protein, while a unstructured linker (GGGSGGGS) serves to separate OmpA from its cargo in order to avoid interactions between the two proteins.

The expressed protein is almost structurally identical to BBa_K103006, with the terminal serine on the linker replaced by an alanine. This change was made in order to convert the previous SacI site (GAGCTC), which was unusable for RE cloning due to a second SacI site being present in the plasmid backbone, into a KasI site (GGCGCC). The KasI site codes for Gly-Ala, while the SacI site codes for Gly-Ser. This amino acid substitution is unlikely to impact the function of Lpp-OmpA-Linker in a major way.

 

 

Part:BBa_K1489001 | Bovine Galectin-1

This is the Bovine Galectin which should interact with beta-galactosides and intialize a signal cascase

Bovine Galectin-1

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

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.