Difference between revisions of "Part:BBa K4247024"
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| − | ===Orf438 and | + | ===Orf438 and Tyrosinase operon [Streptomyces antibioticus]=== |
| − | This composite part codes for orf438 (BBa_K4247022) and the tyrosinase enzyme (BBa_K4247023) of Streptomyces antibioticus. 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 this enzyme in a co-expression system. | + | This composite part codes for orf438 (<partinfo>BBa_K4247022</partinfo>) and the tyrosinase enzyme (<partinfo>BBa_K4247023</partinfo>) of Streptomyces antibioticus. 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 this enzyme in a co-expression system. |
==Usage and Biology== | ==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. | + | 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 <partinfo>BBa_K4247020</partinfo>, <partinfo>BBa_K4247021</partinfo>) 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|]] | ||
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| + | ==Characterization== | ||
| + | |||
| + | ===Co-transformation with tyrosinase and cofactor=== | ||
| + | |||
| + | '''Aim - ''' To show that the co-transformation of the pET24 (+) vector containing the mfp151 (<partinfo>BBa_K4247018</partinfo>-<partinfo>BBa_K4247021</partinfo>) and the pRSET A vector containing tyrosinase (<partinfo>BBa_K4247023</partinfo>) and orf438 (tyr-cofactor) (<partinfo>BBa_K4247022</partinfo>) works. | ||
| + | |||
| + | '''Result - ''' SDS and Western Blot was done on the purification fractions obtained from Ni-NTA purification of the protein 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. Since Mfp151 does not have any tryptophan residues, it is not possible to visualise Mfp151 proteins in an SDS-gel and hence, a western blot is needed. | ||
| + | |||
| + | [[File: sdst.jpeg|700px|]] | ||
| + | |||
| + | So, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins. Since the proteins were expressed with a 6x His-tag, we used mouse anti-hexa his primary antibodies and goat anti-mouse HRP-conjugated secondary antibodies for the western blot. | ||
| + | |||
| + | [[File: wbt.jpeg|700px|]] | ||
| + | |||
| + | '''Conclusion - '''As seen in the Western Blot, we lost proteins in the flowthrough and washes. However, it still proves we managed to produce mfp151 and mfp151_SnoopCatcher in co-transformation with tyrosinase and orf438. The cofactor is clearly visible in the SDS-gel but not so clear in the Western Blot, possibly because the 6x HisTag is not well exposed. | ||
Latest revision as of 12:40, 12 October 2022
Contents
Orf438 and Tyrosinase operon [Streptomyces antibioticus]
This composite part codes for orf438 (BBa_K4247022) and the tyrosinase enzyme (BBa_K4247023) of Streptomyces antibioticus. 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 this enzyme 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
Co-transformation with tyrosinase and cofactor
Aim - To show that the co-transformation of the pET24 (+) vector containing the mfp151 (BBa_K4247018-BBa_K4247021) and the pRSET A vector containing tyrosinase (BBa_K4247023) and orf438 (tyr-cofactor) (BBa_K4247022) works.
Result - SDS and Western Blot was done on the purification fractions obtained from Ni-NTA purification of the protein 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. Since Mfp151 does not have any tryptophan residues, it is not possible to visualise Mfp151 proteins in an SDS-gel and hence, a western blot is needed.
So, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins. Since the proteins were expressed with a 6x His-tag, we used mouse anti-hexa his primary antibodies and goat anti-mouse HRP-conjugated secondary antibodies for the western blot.
Conclusion - As seen in the Western Blot, we lost proteins in the flowthrough and washes. However, it still proves we managed to produce mfp151 and mfp151_SnoopCatcher in co-transformation with tyrosinase and orf438. The cofactor is clearly visible in the SDS-gel but not so clear in the Western Blot, possibly because the 6x HisTag is not well exposed.