Difference between revisions of "Part:BBa K4247023"
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'''Result:''' SDS and Western Blot the lysate purification from BL21(DE3) cells induced with 0.1 mM IPTG overnight. As it can be observed, in the SDS tyrosinase (31.56 KDa) and orf438-cofactor- (16.48 KDa) are being produced. Mfp151 cannot be observed in the stain-free SDS but as it can be observed in the western blot it’s also there. | '''Result:''' SDS and Western Blot the lysate purification from BL21(DE3) cells induced with 0.1 mM IPTG overnight. As it can be observed, in the SDS tyrosinase (31.56 KDa) and orf438-cofactor- (16.48 KDa) are being produced. Mfp151 cannot be observed in the stain-free SDS but as it can be observed in the western blot it’s also there. | ||
| − | [[File: sdst.jpeg| | + | [[File: sdst.jpeg|700px|]] |
| − | [[File: wbt.jpeg| | + | [[File: wbt.jpeg|700px|]] |
'''Conclusion:'''As observable in the Western Blot, we lost proteins in the ft and washes, however, it proves we managed to produce mfp and mfp-catcher in co-transformation with tyrosinase and orf438. The cofactor is not clearly visible in the Western Blot lot but is clear in the SDS possibly because the 6HisTag is not well exposed. | '''Conclusion:'''As observable in the Western Blot, we lost proteins in the ft and washes, however, it proves we managed to produce mfp and mfp-catcher in co-transformation with tyrosinase and orf438. The cofactor is not clearly visible in the Western Blot lot but is clear in the SDS possibly because the 6HisTag is not well exposed. | ||
Revision as of 10:09, 3 October 2022
Tyrosinase [Streptomyces antibioticus]
This basic part codes for the tyrosinase enzyme of Streptomyces antibioticus. Orf438 (part BBa_K4247022) 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 this 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.
Characterization
Aim: Show that the co-transformation of mfp (BBa_K4247018-BBa_K4247021) and the pRSETa containing tyrosinase (BBa_K4247023) and orf438 (tyr-cofactor) (BBa_K4247022).
Result: SDS and Western Blot the lysate purification from BL21(DE3) cells induced with 0.1 mM IPTG overnight. As it can be observed, in the SDS tyrosinase (31.56 KDa) and orf438-cofactor- (16.48 KDa) are being produced. Mfp151 cannot be observed in the stain-free SDS but as it can be observed in the western blot it’s also there.
Conclusion:As observable in the Western Blot, we lost proteins in the ft and washes, however, it proves we managed to produce mfp and mfp-catcher in co-transformation with tyrosinase and orf438. The cofactor is not clearly visible in the Western Blot lot but is clear in the SDS possibly because the 6HisTag is not well exposed.