Part:BBa_K2960003
mlrC with TorA tat Sequence
This is a composite part consisting of a constitutively-active promoter sequence (J23100), a ribosome binding site (BBa_J61100), the mlrC gene (BBa_k2960008), the TorA tat sequence (BBa_K1012002), a 3x FLAG tag (BBa_K823034), and a terminator (BBa_B0015).
A cassette of enzymes endogenous to Sphingopyxis sp. has been shown to sequentially break down microcystin-LR (Figure 1). The enzymes have been named mlrA, mlrB, mlrC, mlrD, mlrE, and mlrF. MlrC is the third enzyme in the microcystin-degradation pathway. MlrC is assumed to be a metallopeptidase. It cleaves the tetrapeptide intermediate into an Adda amino acid and other smaller peptides. The exact smaller peptides that result are still unknown.
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-arginine translocation pathway altered to transport folded mlr proteins.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 722
Illegal BglII site found at 932
Illegal BamHI site found at 1467
Illegal XhoI site found at 1211 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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