Difference between revisions of "Part:BBa K2960002"

Revision as of 00:31, 21 October 2019

mlrB with TorA tat Sequence

This is a composite part consisting of a constitutively-active promoter sequence (J23100), a ribosome binding site (BBa_J61100), the mlrB gene, the TorA tat sequence, a 3x FLAG tag, and a terminator (BBa_B0015). MlrB is the second enzyme in the microcystin-degradation pathway.

A cassette of enzymes endogenous to Sphingopyxis sp. has been shown to sequentially break down microcystin-LR. The enzymes have been named mlrA, mlrB, mlrC, mlrD, mlrE, and mlrF. MlrB is a serine protease that catalyzes the hydrolysis of linearized MCs via the cutting of the peptide bond between alanine at position 1 and a variable amino acid (e.g. leucine) residue at position 2. This results in the formation of a tetrapeptide that is subsequently degraded by mlrC.

We have also included the TorA twin-arginine translocation tag in our biobrick. The twin-arginine translocation (Tat) pathway is responsible for the export of folded proteins across the cytoplasmic membrane of bacteria into the periplasmic space. The Tat pathway acts separately from the general secretory pathway, which transports proteins in an unfolded state. A specific signal peptide, which contains three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain, is necessary to initiate protein export by the Tat pathway.

We have included this tag with the goal of transporting the microcystin-degrading enzyme into the bacterial periplasmic space. This biobrick was designed for expression in bacteria contained in a bioreactor. Moving the enzymes closer to the periphery of the bacterial cell, and therefore closer to the flow of contaminated water passing through the bioreactor, increases the efficiency of the degradation.

As an additional note, we decided to utilize the Twin-Arginine Translocation pathway because of its ability to transport fully folded proteins. Many proteins (such as GFP), are unable to fold properly if exported unfolded into the periplasm. The periplasm is an oxidizing environment, which promotes the formation of aberrant disulfide bonds. This can cause proteins to misfold, aggregate, and become inactive. By exporting our enzymes fully-folded into the periplasmic space, we avoid this problem.

We have also included a 3X FLAG tag at the end of the sequence. This permits protein expression to be detected, quantified and/or purified by western blot, SDS-PAGE, and other methods.

Figure 1. Schematic of TorA twin-argenine translocation pathway altered to transport folded mlr proteins.

Sequence and Features