Part:BBa_K3755029

cfa-c-mfp5 CDS-his tag

Produce 3\*mfp5 CDS by splicing−ligation with part:BBa_K3755030

Usage and Biology

Mussels are magical creatures using their byssus to stick tightly to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms^[1]. We found that mussel foot protein (Mfp) is the key to the mussel's remarkable adhesion ability. The adhesion of mussel foot proteins is related to several molecular forces, among which the most important are the H-bond, π−π stacking interactions, and metal complexation between the 3,4-dihydroxyphenylalanine(dopa) group in the protein and the hydrophilic group on the material surface^[1,2,7].

Mfp is attractive not just for its great adhesion in water conditions, but for its superior biocompatibility as well^[3]. Up to now, Mfp has been well documented as an excellent wound healing dressing by many articles^[2,4,5]. Our project plans to add Mfp to the hydrogel to increase its mechanical strength and to link broken bone pieces. Since the product needs to be put into the internal environment, which takes high requirements for human safety, Mfp is exactly the best bioglue we can find. By comparison, in the Mfp family, Mfp-5 has the highest dopamine content(∼26 mol %) and the strongest adhesion^[1,6,7]. Therefore, we take the Mfp-5 as the best choice for our adhesion system.

In order to obtain high yield Mfp5 efficiently and conveniently, we chose to express recombinant Mfp5 in E.coli. Luckily, Dong Soo Hwang's team from Pohang University has isolated the Mfp5 sequence from an M. galloprovincialis foot cDNA library and successfully expressed the recombinant Mfp5 in E.coli BL21 in 2003^[8]. Later in 2018, Eugene Kim's team from Washington University in St. Louis referred to Dong Soo Hwang's research, using synthetic DNA with codons optimized for expression and making more amazing designs for Mfp5^[9]. The above results provide us a significant basis for us to produce Mfp5 with high yield and high viscosity.

The composite part is a part of 3 repeats of Mfp5 expression system, which needs to work with cfa-n-mfp5-2part:BBa_K3755030, through splicing−ligation reactions to connect mfp5 protein with 2 repeats of mfp5 protein to form 3 repeats of mfp5 protein. The sequence contains cfa-c(part:BBa_K3755005), Mfp5 CDS(part:BBa_K3755002) and His tag(part:BBa_K3755020).

Experiment and Results

Due to time constraints, we only constructed the plasmid and did not express the protein to complete further characterization. And since we constructed the plasmid of pET28a-cfaC-mfp5-1(this part) and pET28a-mfp5-2-cfaN(part:BBa_K3755030) together, and experiment data of the two would be both presented here.

Plasmid Construction

We constructed a pET28a-cfaC-mfp5-1 plasmid and a pET28a-mfp5-2-cfaN plasmid(Fig.1a, Fig.1b) , expressing in E.coli. We linearized the pET28a backbone and gene segment to do the homologous recombination. The agarose gel electrophoresis of Mfp5-2-CfaN and CfaC-Mfp5-1 are shown in Fig.1c, linearized pET28a is shown in Fig.1d.

Fig.1a: Construction of pET28a-cfaC-mfp5-1

Fig.1b: Construction of pET28a-mfp5-2-cfaN

Fig.1c: Linearized segments of Mfp5-2-CfaN and CfaC-Mfp5-1

Fig.1d: Linearized plasmid backbone pET28a

References

[1]Carvajal-Vallejos P, Pallissé R, Mootz HD, Schmidt SR. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem. 2012 Aug 17;287(34):28686-96. doi: 10.1074/jbc.M112.372680. Epub 2012 Jun 28. PMID: 22753413; PMCID: PMC3436554.
[2]Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW. Design of a Split Intein with Exceptional Protein Splicing Activity. J Am Chem Soc. 2016 Feb 24;138(7):2162-5. doi: 10.1021/jacs.5b13528. Epub 2016 Feb 8. PMID: 26854538; PMCID: PMC4894280.
[3]Kim E, Dai B, Qiao JB, Li W, Fortner JD, Zhang F. Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces. 2018 Dec 12;10(49):43003-43012. doi: 10.1021/acsami.8b14890. Epub 2018 Nov 27. PMID: 30480422.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 355
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]