Difference between revisions of "Team:Aix-Marseille/Basic Part"

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This part is the same as the chitinase [http://parts.igem.org/Part:BBa_K2718020 BBa_K2718020] from ''Beauveria bassiana'' but we added a C-terminal oligo-histidine tag to the protein to facilitate purification and detection using a western blot. The part used the RFC 25 standard. We test enzyme activity of this part, and the availability of the his-tag for purification as [http://parts.igem.org/Part:BBa_K2718020 BBa_K2718020].
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Chitinase [http://parts.igem.org/Part:BBa_K2718020 BBa_K2718020]is a fungus (''Beauveria bassiana'') enzyme that degrads chitin. It's endochitinase wich hydrolyses 1-4 beta link between N-acetyl-D-glucosamine monomere  . Sequence comes from [https://www.uniprot.org/uniprot/Q8J1Y3 uniprot], we used peptidic sequence between 41 to 330 AA, that's correspond  to [https://www.ebi.ac.uk/interpro/protein/Q8J1Y3 catalitic domain]. We improve  nucleotidic sequence with [https://eu.idtdna.com/CodonOpt IDT tool]  for synthesis by ''E.coli'', and there is his tag in C-ter. Part is RFC 25 standards. We test activiy with Schales' procedure[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975300/pdf/1754-6834-7-37.pdf].
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Revision as of 16:30, 17 October 2018

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Basic Parts

We made three basic parts during this years iGEM competition coding for 2 different enzymes.

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BBa_K2718005

Methionine gamma-lyase (MGL) catalyzes the conversion of l-methionine into methanthiol, then the oxidation of methanethiol produces DMDS (Dimethyl disulfide) and DMTS ( Dimethyl trisulfide). BBa_K2718005 is an RFC 10 compatible part that we derived from BBa_K1493300 using PCR to add a his tag. To test activity we used the compositive part BBa_K2718006

--Aix-Marseille--Shcema_MGL_parts.jpg

BBa_K2718020

The Chit1 gene that codes for the endochitinase from the fungus Beauveria bassiana, an enzyme that degrads chitin, was made as BBa_K2718020. It's endochitinase wich hydrolyses 1-4 beta link between N-acetyl-D-glucosamine monomeres the sequence comes from uniprot, we used the protein sequence between amino acid 41 to 330, that corresponds to the catalitic domain. We improved the DNA sequence with the IDT tool to optimize it for synthesis by E.coli. Part used the RFC 25 standard. We tested the activiy using Schales' method[1] with the part BBa_K2718022

BBa_K2718021

This part is the same as the chitinase BBa_K2718020 from Beauveria bassiana but we added a C-terminal oligo-histidine tag to the protein to facilitate purification and detection using a western blot. The part used the RFC 25 standard. We test enzyme activity of this part, and the availability of the his-tag for purification as BBa_K2718020.