Difference between revisions of "Team:Aix-Marseille/Composite Part"
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| + | <!-- Pour chaque part il serait bien d'expliquer d'ou vien la sequence (les parts utilisé pour le faire), ce que c'est censé faire (pourquoi on la construit), comment on a testé ce que ca fait essentiellement copier coller du registry --> | ||
| + | ==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 [http://parts.igem.org/Part:BBa_K2718006 BBa_K2718006] is made from [http://parts.igem.org/Part:BBa_K2718005 BBa_K2718005], an RFC 10 compatible part that we derived from [http://parts.igem.org/Part:BBa_K1493300 BBa_K1493300] using a PCR to add a C-terminal histidine tag, preceded by the [http://parts.igem.org/wiki/index.php?title=Part:BBa_R0011 BBa_R0011], an IPTG inducible promoter part. | ||
| + | To test the activity of [http://parts.igem.org/Part:BBa_K2718006 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 <ref>[https://www.dovepress.com/activities-of-methionine-gamma-lyase-in-the-acidophilic-archaeon-ldquo-peer-reviewed-article-RRB]</ref>. | ||
| − | + | ==BBa_K2718010== | |
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| − | + | The [https://www.ebi.ac.uk/ena/data/view/CCD33152 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 [http://parts.igem.org/Part:BBa_K2718010 BBa_K2718010]. We used [https://www.uniprot.org/uniprot/G4V4S7 the 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] as an RBS. This part uses the RFC 10 standard. We test activity by Mandelate measurment by HPLC [https://www.sciencedirect.com/science/article/pii/S2214030115300018?via%3DihubPugh,] | |
| + | ==BBa_K2718011== | ||
| + | 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 the reaction phenylpyruvate + O2 => (S)Mandelate + CO2 was made as [http://parts.igem.org/Part:BBa_K2718011 BBa_K2718011]. | ||
| + | We used [https://www.uniprot.org/uniprot/G4V4S7 the protein sequence] and to optimize synthesis by ''E.coli'' we used [https://eu.idtdna.com/CodonOpt IDT tool]. As an RBS the part contains [http://parts.igem.org/Part:BBa_B0030 BBa_B0030] and [http://parts.igem.org/Part:BBa_R0011 BBa_R0011] as a IPTG inducable promotor. | ||
| + | The part uses RFC 10 standards. | ||
| + | We purified the protein with an anion exchange column. | ||
| + | We tested activity in cells by monitoring Mandelate production using analytical reverse phase HPLC [https://www.sciencedirect.com/science/article/pii/S2214030115300018?via%3DihubPugh,] | ||
| + | ==BBa_K2718022== | ||
| + | Our part [http://parts.igem.org/Part:BBa_K2718022 BBa_K2718022] is made by fusing the promoter RBS in part | ||
| + | [http://parts.igem.org/Part:BBa_J04500 BBa_J04500] to our chitinase part with a C-terminal oligo-histidine tag [http://parts.igem.org/Part:BBa_K2718021 BBa_K2718021]. | ||
| + | We tested the enzyme activity using Schales' method<ref>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975300/pdf/1754-6834-7-37.pdf]</ref> we also tested production of the protein and used the his-tag to purify the protein. | ||
| − | + | ==References== | |
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Latest revision as of 01:29, 18 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 the reaction phenylpyruvate + O2 => (S)Mandelate + CO2 was made as BBa_K2718011. We used the protein sequence and to optimize synthesis by E.coli we used IDT tool. As an RBS the part contains BBa_B0030 and BBa_R0011 as a IPTG inducable promotor. The part uses RFC 10 standards. We purified the protein with an anion exchange column. We tested activity in cells by monitoring Mandelate production using analytical reverse phase 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.