Difference between revisions of "Part:BBa K4247022"

(→‎Orf438 tyrosinase cofactor [Streptomyces antibioticus])
(→‎Usage and Biology)
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==Usage and Biology==
 
==Usage and Biology==
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The enzyme tyrosinase is an oxidase found across taxa, it contains copper and is well known for its tyrosine modifying step that gives melanin. Our project specifically focused on converting the tyrosines of mfp151 (parts BBa_K4247020, BBa_K4247021) into DOPA in E.coli, a post-translational modification that makes mfp sticky. A way to achieve this dopaquinone conversion is by first producing mfp in vitro and then expose it to tyrosinase, but it has the limitations of having to purify the enzymes and of not allowing the dopa modification to occur in all the tyrosines that are not exposed to the enzyme. For this reason, we focused on a co-expression system where E.coli would have mfp151 on a plasmid and tyrosinase with its copper cofactor (orf438) on another. By inducing with IPTG, the tyrosines incorporated in the mfp151 protein have been hydroxylated to DOPA.
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The enzyme tyrosinase is an oxidase found across taxa. It contains copper and is well known for its tyrosine modifying step which gives melanin. Our project specifically focused on converting the tyrosines of mfp151 (parts BBa_K4247020, BBa_K4247021) into DOPA in E.coli, a post-translational modification that makes mfp151 sticky. A way to achieve this dopaquinone conversion is by first producing mfp151 in vitro and then expose it to tyrosinase. But, this method has the limitations of having to purify the enzymes and of not allowing the DOPA modification to occur in the tyrosines that are not exposed to the enzyme. To overcome these limitations, we focused on developing  a co-expression system where E.coli would co-express 2 plasmids, one with mfp151 and another with tyrosinase along with its copper cofactor (orf438). Upon induction of both plasmids with IPTG, the tyrosines incorporated in the mfp151 protein would be hydroxylated to DOPA, thus making the mfp151 protein adhesive.
  
 
[[File: Tyrosinase.jpeg|px350|]]
 
[[File: Tyrosinase.jpeg|px350|]]

Revision as of 13:29, 30 September 2022

Orf438 - Tyrosinase Cofactor [Streptomyces antibioticus]

This basic part codes for the orf438 of Streptomyces antibioticus, a copper cofactor of the tyrosinase enzyme. Orf438 is indispensable for the functioning of S. antibioticus tyrosinase according to Bernan et al., 1985 and was used by Choi et al., 2012 to activate the same enzyme (BBa_K4247023) in a co-expression system.

Usage and Biology

The enzyme tyrosinase is an oxidase found across taxa. It contains copper and is well known for its tyrosine modifying step which gives melanin. Our project specifically focused on converting the tyrosines of mfp151 (parts BBa_K4247020, BBa_K4247021) into DOPA in E.coli, a post-translational modification that makes mfp151 sticky. A way to achieve this dopaquinone conversion is by first producing mfp151 in vitro and then expose it to tyrosinase. But, this method has the limitations of having to purify the enzymes and of not allowing the DOPA modification to occur in the tyrosines that are not exposed to the enzyme. To overcome these limitations, we focused on developing a co-expression system where E.coli would co-express 2 plasmids, one with mfp151 and another with tyrosinase along with its copper cofactor (orf438). Upon induction of both plasmids with IPTG, the tyrosines incorporated in the mfp151 protein would be hydroxylated to DOPA, thus making the mfp151 protein adhesive.

Tyrosinase.jpeg