Team:Aix-Marseille/Composite Part
BBa_K2718006
Methionine gamma-lyase (MGL) catalyzes the conversion of l-methionine to methanethiol, the oxidation of methanethiol then produces first DMDS (Dimethyl disulfide) and then DMTS ( Dimethyl trisulfide). The composite part BBa_K2718006 is made from BBa_K2718005, an RFC 10 compatible part that we derived from BBa_K1493300 using a PCR to add a C-terminal histidine tag, preceded by the BBa_R0011, an IPTG inducible promoter part. To test the activity of BBa_K2718006 we produced and purified the his-tagged protein and used DTNB to measure the enzyme activity. DTNB reacts with the released thiol group [1].
BBa_K2718010
The HmaS gene that codes for mandelate synthase from Amycolatopsis orientalis, an enzyme which catalyzes the conversion of phenylpyruvate and O2 into (S)Mandelate and CO2, was made as BBa_K2718010. We used the protein sequence to optimize synthesis by E.coli with IDT tool. We used BBa_B0030 as an RBS. This part uses the RFC 10 standard. We test activity by Mandelate measurment by HPLC [3]
BBa_K2718011
The HmaS gene that codes for the mandelate synthase from Amycolatopsis orientalis, an enzyme which catalyzes phenylpyruvate + O2 => (S)Mandelate + CO2 was made as BBa_K2718010. We used protein sequence to optimize synthesis by E.coli with IDT tool. We used BBa_B0030like RBS and BBa_R0011 like inducible promotor . Part used RFC 10 standards. We purified with ion exchange column and we test activity by Mandelate measurment by HPLC [4]
BBa_K2718022
Our part BBa_K2718022 is made by fusing the promoter RBS in part BBa_J04500 to our chitinase part with a C-terminal oligo-histidine tag BBa_K2718021. We tested the enzyme activity using Schales' method[2] we also tested production of the protein and used the his-tag to purify the protein.