Team:SBS SH 112144/Improve


Golden Medal fulfillment

Improved mlrA: BBa_K1378001 to BBa_K2888010

Previous Parts: mlrA BBa_K1378001

MlrA is a 28kDa protease found in Sphingomonas sp. which can cleavage microcystins(MCs). MlrA is one part of the gene cluster responsible for the ability of MC degradation. The cluster includes four ORFs, mlrA, mlrB, mlrC and mlrD, which can hydrolyze MCs and facilitate absorption of the products as carbon source. MlrA is sometimes referred as a metalprotease by inhibitor studies. MlrA can cleavage the Adda-Arg bond and causes ring opening. The first-step linearized product shows much weaker hepatoxin compared with MCs. In the experiment of mouse bioassay, up to 250 mg/kg of linearized MC-LR shows no toxicity to mouse, much higher than 50% lethal dose 50mg/kg of cyclic MC-LR. Furthermore, the linearization also raise the median inhibition concentration to 95nM, around 160 times higher than original 0.6nM.

Our Improved Parts: tagged mlrA BBa_K2888010

Description: This BioBrick is an improvement on the one created by the Peking 2014 team (BBa_K1378001). Our composite part within prefix and suffix consists of the most commonly used promoter, RBS, 6x His tag and terminator, as well as our functional coding sequence --- mlrA gene. This composite part is assembled through Gibson Assembly; then, it is fused with linearized vector, pSB1C3 backbone, via infusion. Ultimately, we successfully extract plasmids which contain our target gene from transformed bacteria. Furthermore, we improved mlrA parts BBa_K1378001 by purposely adding 6x His tag into our sequence in order to improve the efficacy and efficiency of expression and purification; it will also strengthen effects of binding to Ni-NTA column, making it possible to immobilize proteins and lyse cyanobacteria repeatedly in our prototype device. In addition, we find that the attachment of SUMO protein can enhance the protein stability and expression system as an N-terminal fusion partner. And SUMO tag can be easily cleaved by a SUMO-specific protease in vitro during purification process.

Length: 1248 bp

Our experience and application of improved parts

Silver Medal Fulfillment: Our New Parts

Fused Lysozyme Gene

Fused Tagged Lysozyme Gene

Cyanophage Lysozyme Gene