Difference between revisions of "Team:Aix-Marseille/Composite Part"
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| + | The [https://www.ebi.ac.uk/ena/data/view/CCD33152 HmaS gene] that codes for the mandelate synthase from ''Amycolatopsis orientalis'', an enzyme which catalyzes phenylpyruvate + O2 => (S)Mandelate + CO2 was made as [http://parts.igem.org/Part:BBa_K2718010 BBa_K2718010]. We used [https://www.uniprot.org/uniprot/G4V4S7 protein sequence] to optimize synthesis by ''E.coli'' with [https://eu.idtdna.com/CodonOpt IDT tool]. We used [http://parts.igem.org/Part:BBa_B0030 BBa_B0030]like RBS and [http://parts.igem.org/Part:BBa_R0011 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 [https://www.sciencedirect.com/science/article/pii/S2214030115300018?via%3DihubPugh,] | ||
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==BBa_K2718022== | ==BBa_K2718022== | ||
Our part [http://parts.igem.org/Part:BBa_K2718022 BBa_K2718022] is made by fusing the promoter RBS in part | Our part [http://parts.igem.org/Part:BBa_K2718022 BBa_K2718022] is made by fusing the promoter RBS in part | ||
Revision as of 18:42, 17 October 2018
Composite Parts
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.