Team:Shenzhen SFLS/Design
Design
Theoretical design:
In order to improve our protein, we did research to derive methods of manufacturing and improve our protein.
First, we discover that dihydroxyphenylalanine (DOPA), a compound derived from tyrosine, is crucial to the adhesion characteristic of mfp proteins. [1]
Fig 1: The effect of DOPA oxidation
However, the component chemicals of bacterial expressed protein lack DOPA and only has tyrosine, which needs to be catalyzed to form DOPA. The tyrosine needs an enzyme, known as tyrosinase to aid its oxidation to produce DOPA. Therefore, we acquired the sequence of tyrosinase[4] and express the enzyme using E. coli. Eventually, we add the enzyme to the protein to catalyze tyrosine to produce DOPA. [1]
Fig 2: The relationship between DOPA and tyrosinase (abbreviated as tyr in the picture)
Secondly, we researched through the mussel foot protein family and find that one of the recombinant mussel foot proteins, Mgfp-5, exhibits a large capacity of DOPA [2], which makes it the core protein of our project. We decided to express this protein first and evaluate its production as well as its function.
Thirdly, during our exploration of enhancing the strength of the protein, we encountered numerous difficulties, such as the failure of utilizing amyloid beta protein, a protein that exhibits self-assemble phenomenon but can cause amyloid angiopathy. Nevertheless, we came across spider silk protein through the iGEM Southern China Meetup at Shenzhen University. Exploring this protein, we discovered that despite its well-known strength, it also exhibits high bio-compatibility. In 2018, Pulakesh Aich and co-authors researched into the properties of a fused protein of mfp3 and their recombinant spider silk protein. Their results indicate that the fused protein MS “formed well-organized fibres making it significantly more elastic than Mfp3.” [3]
Fig 3: The assembling effect of the two proteins (sourced from passage [3])
Fig 4: The elasticity and adhesion figure of the fused protein MS (sourced from passage [3])
Their research and results inspired our design as we later decide to design our own fused protein. In designing this protein, considering that Mgfp-5 carries more DOPA, which will significantly boost the viscidity of the resultant protein, we fusion Mgfp-5 and MaSp-1 instead of Mfp-3 and MaSp-1. We expect the fused protein to self-assemble into a well-defined supramolecular structure so that we can further improve it to develop a new type of recombinant protein-based adhesive biomaterials [3]. With this strong adhesion force, we are able to ensure the fixation of the wound to prevent further rupture.
Fourthly, to promote wound healing, we decide to add rhEFG (recombinant human Epidermal Growth Factor) as well as Aloe vera extract into our protein. As for EGF, it is a wound healing mediator and Aloe vera stimulates the proliferation and activity of fibroblast [3]. This ultimately takes us to our goal of manufacturing a waterproof, biocompatible, healing promoting band-aid.
Experimental design
In order to successfully manufacture and validate the proteins discuss above, we designed these experiments.
First, we design parts consisting of genes regulating this protein and load the parts into pET28a plasmid backbone. (Discussed in parts overview later).
Secondly, we perform transformation to integrate the plasmids into the metabolism system of E. coli BL21 and culture the bacteria.
induction to it to stimulate the expression of the protein.
Fourthly, we conduct protein purification and sds-page to validate the protein produced and to measure the molecular weight. In order to have the protein successfully purified, we attach 6 X his-tags on the protein sequence and use a his-tag purification resin to capture the protein. Sds-page is an effective method to separate the proteins using electrophoresis. With the sds-page gel and protein strips record on it, we can readily determine whether we have produced the targeted protein sequence or not.
Parts overview
Our project involves four parts. These parts work separately to produce different proteins that we later either fuse or add together to enhance the quality of our band-aid.
BBa_K197018
The first part is the mgfp5 part. First created by 2009 Berkeley Wetlab team and registered in parts.igem.org under the code of BBa_K197018, this part sourced from the recombinant of part of the genomics of mussel, this part regulates the expression of the protein mgfp5 in bacteria. We conducted transformation (in the bacteria BL21), Iptg induction, his-tag purification and sds-page to further characterize this part.
BBa_K3148001
The second part is the Tyr part, which is our original design. Registered in parts.igem.org under the code of BBa_3148001, this part controls the expression of tyrosinase in bacteria. Tyrosinase, an enzyme, plays an important role in our project due to the fact that it enhances the strength of adhesion strength of mgfp5. The sequence of tyrosinase [4] is sourced from a part of the genomic of V.spinosum, a bacteria whose genetic sequence has long been adopted to express tyrosinase in E.coli. We conducted transformation (in the bacteria BL21), Iptg induction, his-tag purification and sds-page to further characterize this part.
Visit http://parts.igem.org/Part:BBa_K3148001 for more information and the sequence.
BBa_K3148002
The third part is the mgfp5-masp1 part, which is also our original design. Registered in parts.igem.org under to code of BBa_3148002, the part is a fused protein of the existed BBa_K197018 and the protein Masp1, which sourced from the recombinant of part of the genomics of spider [3]. This part regulates the production of our final product: the adhesive medium in band-aids.
Visit http://parts.igem.org/Part:BBa_K3148002 for more information and the sequence.
BBa_K1583011
The fourth part is the mfp3 part. Created first by iGEM15_TU_Delft team and registered in parts.igem.org under the code of BBa_K1583011. This part regulates the production of mfp3 protein in E. coli. This protein is aimed to enhance the viscidity of our protein.
Reference:
[2]: Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli
Dong Soo Hwang,1,2 Hyo Jin Yoo,2 Jong Hyub Jun,3,4 Won Kyu Moon,3,4 and Hyung Joon Cha1,2*
[3]: Self-assembled adhesive biomaterials formed by a genetically designed fusion protein†
Pulakesh Aich,‡a Jaeyeon An,‡b Byeongseon Yang,‡c Young Ho Ko,a Junghyun Kim, a James Murray,a Hyung Joon Cha, *c Joon Ho Roh,*a Kyeng Min Park *a and Kimoon Kim *ab
[4]: Unusual Stability of a Recombinant Verrucomicrobium spinosum Tyrosinase to Denaturing Agents and Its Use for a Production of a Protein with Adhesive Properties
A. S. Axambayeva1 & L. R. Zhaparova1 & Zh. S. Shagyrova1 & E. M. Ramankulov2 & A. V. Shustov1
[1]: Mini-review: The role of redox in Dopa-mediated marine adhesion
Sascha C.T. Nicklisch a & J. Herbert Waite a
a Marine Science Institute & Department of Molecular , Cell & Developmental Biology, University of California , Santa Barbara , CA , 93106 , USA Published online: 28 Aug 2012.