Part:BBa_K3738031

MlrA N-terminal 6XHistidine Tag and C-Terminal and Anionic Tag (Composite)

MlrA, or microcystinase, cleaves the peptide backbone of MC-LR cyanotoxins produced by Microcystis aeruginosa cyanobacteria. This crucial step results in the peptide losing the cyclic structure, and therefore the majority of its toxicity. Further degradation continues in an enzymatic pathway by interactions with other peptidolytic enzymes from the microcystinase gene cluster. This part originally comes from the Aalto-Helsinki 2016 iGEM team (Part BBa_K1907001), but was originally codon-optimized for yeast. We have modified this part by codon-optimizing it for use in E. coli. MlrA is known to be taken up into the periplasm with aid of transport protein MlrD (Saito et al., 2003), however for the mlrA sequence from Sphingomonas sp. USTB-05 the mlrD sequence is currently unavailable. Thus we have added both a histidine tag and anionic tag for purification and MS2-encapsulation respectively.

Saito T, Okano K, Park H-D, Itayama T, Inamori Y, Neilan BA, et al. Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes. FEMS Microbiology Letters. 2003;229(2):271-6. doi: 10.1016/S0378-1097(03)00847-4.

Jiang Y, Shao J, Wu X, Xu Y, Li R. Active and silent members in the mlr gene cluster of a microcystin-degrading bacterium isolated from Lake Taihu, China. FEMS Microbiology Letters. 2011;322(2):108-14. doi: 10.1111/j.1574-6968.2011.02337.x.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 229
  Illegal BglII site found at 970
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 912