Difference between revisions of "Part:BBa K4247021"
(→Mfp151_Snoopcatcher) |
(→Characterization) |
||
| Line 22: | Line 22: | ||
=== Addition of SnoopCatcher to Mfp151 === | === Addition of SnoopCatcher to Mfp151 === | ||
| − | The tyrosine residues in the Mfp151 protein undergo post-translational modifications (PTMs) to become 3,4-dihydroxyphenyl-alanine (Dopa) which provides the Mfp151 proteins with their adhesion properties. Since E.coli is not capable of performing PTMs, this can be overcome by co-expressing another plasmid producing tyrosinase to modify tyrosine residues to Dopa in vivo. Mfp151 and Mfp151_Snoopcatcher were expressed. Further, they were also co-expressed with tyrosinase to facilitate the post-translational modification of tyrosine to Dopa to make the proteins adhesive. | + | '''Aim:'''The tyrosine residues in the Mfp151 protein undergo post-translational modifications (PTMs) to become 3,4-dihydroxyphenyl-alanine (Dopa) which provides the Mfp151 proteins with their adhesion properties. Since E.coli is not capable of performing PTMs, this can be overcome by co-expressing another plasmid producing tyrosinase to modify tyrosine residues to Dopa in vivo. Mfp151 and Mfp151_Snoopcatcher were expressed. Further, they were also co-expressed with tyrosinase to facilitate the post-translational modification of tyrosine to Dopa to make the proteins adhesive. |
| Line 28: | Line 28: | ||
| − | In the SDS-gel, it is difficult to visualise the Mfp151 protein. So, a western blot was done on the above SDS-gel. | + | '''Result:'''In the SDS-gel, it is difficult to visualise the Mfp151 protein. So, a western blot was done on the above SDS-gel. |
| Line 34: | Line 34: | ||
| − | Thus, we can see the expression of Mfp151 (25kDa), Mfp151_Snoopcatcher (37kDa) along with tyrosinase (30kDa) in the co-expression cultures. | + | '''Conclusion''' Thus, we can see the expression of Mfp151 (25kDa), Mfp151_Snoopcatcher (37kDa) along with tyrosinase (30kDa) in the co-expression cultures. We cannnot see orf438 (cofactor) because it ran out of the gel. In the next experiment however it's visible. |
| + | |||
| + | |||
| + | ===Co-transformation with tyrosinase and tyr-cofactor=== | ||
| + | '''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. | ||
| + | |||
| + | [[File: sdst.jpeg|700px|]] | ||
| + | |||
| + | [[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 but it is clear in the SDS possibly because the 6HisTag is not well exposed. | ||
Revision as of 10:16, 3 October 2022
Contents
Mfp151_Snoopcatcher
This part codes for MFP151_Snoopcatcher, a chimeric protein composed of MFP151 and Snoopcatcher. This is a composite part consisting of the following basic parts: BBa_K4247018 (mfp151_first-half), BBa_K4247019 (mfp151_second-half) and BBa_K4247009 (SnoopCatcher).
This part is one of a collection of compatible mussel foot protein parts: BBa_K4247018 (mfp151_first-half), BBa_K4247019 (mfp151_second-half) and BBa_K4247020 (mfp151).
Usage and Biology
Mussels have the ability to attach themselves to various surfaces underwater by permanent adhesion. This adhesion is facilitated by their byssus, which is secreted from their foot. The byssus comprises a bundle of threads and at the end of each thread, there is an adhesion plaque containing a water-resistant adhesive that enables the mussel to anchor itself to surfaces.
Several types of foot proteins have been characterised and each of them have a different function as per their location in the byssus. Of these, MFP3 and MFP5 are found in the distal end of the byssus. Post-translational modification of tyrosines yields L-3,4-dihydroxyphenylalanine (DOPA) and these DOPA groups are associated with the adhesion strength of MFPs. MFP3 and MFP5 are known to have the highest DOPA content among MFPs and hence, these intrinsically disordered proteins enable the adhesion mechanisms of the byssus. MFP1 forms the outer coating of the byssus and it has a lower DOPA content compared to MFP3 and MFP5.
Recombinant production of MFP5 wasn’t very successful and there were several bottlenecks in terms of cell growth and protein purification since the sticky nature of the protein makes it difficult to purify. In order to overcome these limitations, a hybrid protein called MFP151 was constructed and produced. This hybrid protein consists of six M. galloprovincialis MFP1 decapeptide repeats added to the N- and C-terminus of M. galloprovincialis MFP5. So, the protein consists of 6 MFP1 repeats followed by a MFP5 sequence and 6 MFP1 repeats again. MFP151 was found to have comparable adhesion characteristics to recombinant MFP5, could be produced with greater yields and could be purified easily.
Characterization
Addition of SnoopCatcher to Mfp151
Aim:The tyrosine residues in the Mfp151 protein undergo post-translational modifications (PTMs) to become 3,4-dihydroxyphenyl-alanine (Dopa) which provides the Mfp151 proteins with their adhesion properties. Since E.coli is not capable of performing PTMs, this can be overcome by co-expressing another plasmid producing tyrosinase to modify tyrosine residues to Dopa in vivo. Mfp151 and Mfp151_Snoopcatcher were expressed. Further, they were also co-expressed with tyrosinase to facilitate the post-translational modification of tyrosine to Dopa to make the proteins adhesive.
Result:In the SDS-gel, it is difficult to visualise the Mfp151 protein. So, a western blot was done on the above SDS-gel.
Conclusion Thus, we can see the expression of Mfp151 (25kDa), Mfp151_Snoopcatcher (37kDa) along with tyrosinase (30kDa) in the co-expression cultures. We cannnot see orf438 (cofactor) because it ran out of the gel. In the next experiment however it's visible.
Co-transformation with tyrosinase and tyr-cofactor
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 but it is clear in the SDS possibly because the 6HisTag is not well exposed.