Coding

Part:BBa_K1518000

Designed by: Ian Valentine McDermott   Group: iGEM14_UCC_Ireland   (2014-09-28)

Thread Biopolymer Filament Gamma TBFG_EPTST

Thread biopolymer filament subunit gamma (Eptatretus stoutii)

This part is intended to be produced as a component of an intermediate filament construct. TBFG codes for the γ protein subunit of the Hagfish thread biopolymer. It is intended to assemble as part of a coiled-coil heterodimer of an α/γ intermediate filament.

Cloning of codon-optimized hagfish slime intermediate filament genes

E.coli are the bacteria of choice for the production of useful proteins, which may be difficult to harvest from nature and have been used for example in the production of human insulin. Here we cloned the hagfish genes coding for the alpha and gamma subunits of the slime intermediate filament into plasmids that will facilitate their production at high levels in E. coli.

Codon Optimization

The protein sequence of alpha and gamma subunit proteins were retrieved from the protein database. Rather than using the natural hagfish DNA sequence coding for these proteins we generated a DNA sequence by optimizing the codon usage of the DNA sequence to better match that found in highly expressed E. coli genes. This was performed using an online bioinformatics tool called OPTIMIZER (Puigbo et al., 2007). This means that the bases coding for the certain amino acids were changed. This makes no alterations to the actual protein itself, but increases the chance of the bacteria being able to translate the mRNA sequence efficiently into a protein. Double stranded DNA sequences corresponding to the codon optimized alpha and gamma genes were then ordered from Gen9.

Cloning into the pSB1-C3 Biobrick vector

Forward and reverse oligonucleotides containing the standard biobrick prefix and suffix were used to amplify the codon-optimized alpha and gamma genes by PCR. Following digestion with EcoRI and SpeI enzymes these DNA fragments were cloned into the pSB1-C3 vector that had been cut with the same enzymes. These genes have been supplied to the Registry with parts numbers BBa_K1518000 and BBa_K1518001.

                   <img src="Seadna_cloning_of_biobrick_vector_psB1-C3.png" />

Fig. 6 Restriction digest of hagfish IF alpha and gamma genes cloned into the pSB1-C3 plasmid.Plasmid DNA was digested with the indicated enzymes and analysed by agarose gel electrophoresis. The alpha gene (2kb) is almost identical in size to the vector backbone and so appears as one band. Gamma (1.8kb) is slightly smaller can just be distinguished from the vector backbone. A single digest yields a band of 4kb confirming he presence of the hagfish genes.

Cloning to into the pCDF-Duet bacterial expression vector

To produce and purify the proteins that constitute hagfish slime we cloned the codon-optimized alpha and gamma genes in the pCDF Duet expression vector (Merck-Millipore). This is a low copy plasmid that is specifically designed to facilitate expression of multiple genes either from one plasmid or from separate plasmids maintained in the same cells. The pCDF Duet vector encodes a poly histidine tag that will be fused to the amino terminal end of each protein. The primary reason for this modification is to facilitate the rapid purification of the proteins by affinity chromatography. The His-tag allows the protein to be removed from all other proteins in the bacteria by use of a Ni-NTA pro-bond resin column (column chromatography).

Cloning was achieved by digesting the pCDF-duet plasmid with two restriction enzymes; EcoRI and HindIII. The hagfish subunit DNA as synthesised was flanked by MfeI and HindIII restriction enzymes, allowing it to be compatible with the EcoRI and HindIII restriction enzyme sites in the pCDF-duet plasmid. Gel electrophoresis was performed on these samples and the DNA was subsequently extracted from the gel. A ligation reaction was then performed followed by a bacterial transformation into E. coli DH5α. Cells then cultured on a streptomycin plate. Colonies obtained were screened by colony PCR and positive colonies picked and cultured in liquid broth to prepare minipreps of plasmid DNA. Plasmid mini preps were performed individually to isolate the plasmids from the DH5α cultures. From this we obtained an isolated plasmid pCDF-Duet with an alpha subunit, and a plasmid pCDF-Duet with a gamma subunit. To verify that the isolated plasmids had the correct insert we performed a restriction digest. This DNA was also checked by sequencing of the inserts.

                   <img src="Seadna_Cloning_of_alpha_and_gamma_subunit_genes_into_pCDF_vector.png" />

Fig. 7 Cloning of alpha and gamma subunit genes into pCDF vector. A) Agarose gel showing digested pCDF vector. B) and C) Results of colony PCR to verify the presence of the alpha and gamma genes in pCDF vector in colonies following ligation and transformation. D) and E) Image of plasmid DNA digested with BamHI and HindIII to verify presence of the alpha and gamma genes.

Expression and purification of recombinant hagfish slime intermediate filament proteins

For protein expression the BL21 E. coli strain was used and expression induced using IPTG which binds to the lac operator to induce transcription. Following cell lysis purification of the intermediate filament alpha and gamma proteins was performed under a variety of conditions.

Protein Expression

Transformation of the plasmids described above into E. coli BL21 cells was performed and cells plates on spectinomycin plates. An overnight culture from a single colony from both the alpha and gamma plates was grown and the next morning that culture volume was increased to 500ml with LB Broth (plus antibiotic). Gene expression was induced by the addition of 1mM IPTG at OD600 of 0.4 and for an incubation period of 3 hours. Induction of expression in log phase allows for robust transcription and translation of the target gene. Cultures were shaken at 200rpm for this period to provide sufficient aeration. Whole protein samples taken at 0 and 3 hours were analysed for protein expression by SDS PAGE. Expression of both proteins at the expected molecular weight was observed (see figure in next section).

                   <img src="Seadna_alpa_and_gamma_subunit_in_BL21_cells.png" />

Fig. 7 Hagfish intermediate filament alpha and gamma expression vectors transformed into BL21 E. coli cells for protein expression.

Purification of soluble hagfish intermediate filament proteins

Pelleted cells were lysed by sonication and a cleared lysate (supernatant) obtained by centrifugation. This was subjected to purification using a Ni-NTA column, washed extensively and bound proteins were eluted using imidazole. The purification fractions were analysed by SDS PAGE. Although strong expression of both protein was achieved these proteins were not obviously visible in the cleared lysates for either protein. No significant protein bands were seen in the elution fractions for either protein. This lead us to conclude that when expressed individually both proteins are insoluble and so are not present in the cleared lysate and cannot be purified.

                   <img src="Seadna_Expression_and_purification_of_hagfish_intermediate_filament_%28IF%29_alpha_and_gamma_proteins.png" />

Fig. 8 Expression and purification of hagfish intermediate filament (IF) alpha and gamma proteins. . Whole protein samples taken before (T0) and after (T3) induction of protein expression were analysed by SDS PAGE alongside various fractions from the Ni column purification. Red arrows indicate the expression of the position of the expressed alpha and gamma subunit proteins in the T3 samples at the expected molecular weights. These proteins could not be purified however. CL = cleared lysate, FT = flow through, W = wash, E1 and E2 = imidazole elutions 1 and 2

This experiment was also carried out at 30 ̊C to aid in the correct structural formation of the proteins as they are being expressed. It is thought that lower temperatures help some protein expression as the cell is under a mild stress conditions causing increased synthesis of chaperone proteins to help protein folding. Also a reduced temperature lowers the rate of protein synthesis reducing the risk of this protein aggregating and not folding properly or killing the host cell. The protein remained insoluble at 30 ̊C. Co-expression of the alpha and gamma subunits was also attempted but did not yield significant amounts of soluble protein (data not shown).

Purification of insoluble hagfish intermediate filament proteins

Since the hagfish intermediate filament proteins were expressed but seemed to be insoluble we attempted to purify them under denaturing conditions. Following induction, cells were disrupted by sonication in lysis buffer containing 8M Urea. The His-Tagged proteins were then purified in the presence of urea using Ni-NTA probond resin. The results of the expression and purification were evaluated by SDS-PAGE for both the alpha and the gamma subunits. Both proteins were purified to apparent homogeneity using this approach.

                   <img src="Seadna_Purification_of_hagfish_intermediate_filament_%28IF%29_alpha_protein_under_denaturing_conditions.png" />

Fig. 9 Purification of hagfish intermediate filament (IF) alpha protein under denaturing conditions. Various fractions from the Ni column purification of insoluble proteins in the presence of 8M urea were analysed by SDS PAGE. Red arrows indicate the position of the alpha subunit at the expected molecular weight. CL = cleared lysate, FT = flow through, W = wash, E1, E2 etc = imidazole elutions 1, 2 etc 

                   <img src="Seadna_SDS_PAGE_of_Gamma_Induced_e_coli.png" />

Fig. 10 Purification of hagfish intermediate filament (IF) gamma protein under denaturing conditions. Various fractions from the Ni column purification of insoluble proteins in the presence of 8M urea were analysed by SDS PAGE. Red arrows indicate the position of the gamma subunit at the expected molecular weight. CL = cleared lysate, FT = flow through, W = wash, E1, E2 etc = imidazole elutions 1, 2 etc

Summary of protein expression and purification

We successfully both cloned and expressed both subunits of the hagfish thread intermediate filaments. Both subunits were insoluble, but could be purified under denaturing conditions. It is possible that with further optimization soluble expression of the proteins could be obtained. While soluble protein would be preferable, we decided to proceed with the protein purified under denaturing conditions to see if it could be assembled into filaments.

Assembly of recombinantly expressed hagfish intermediate filament proteins

Working with the proteins that had been purified in 8M urea we developed a method to reconstitute the proteins and co-assemble them into filaments and threads that resemble those obtained from the natural proteins isolated from hagfish slime. Since we are interested in developing these proteins commercially as novel biomaterials we are not divulging the details of our proprietary co-assemble method.

Filament Reconstitution and Microscopy

Following isolation of the alpha and gamma slime intermediate filament proteins in urea we subjected them to our co-assemble protocol. Roughly equal amounts of alpha and gamma protein were used for co-assembly. As controls the co-assembly procedure was carried out for alpha and gamma subunits individually.

It was found that filaments and threads were formed when both the alpha and gamma subunits were present (the threads 1µm to 2µm in diameter). As expected under control conditions, in which only one subunit was present, little to no filamentous precipitate was formed. This suggests that the subunits are indeed forming heterodimers with then copolymerising into filaments and threads. The filamentous structure of the assembled structures could be imaged readily by bright field microscopy.

                   <img src="Seadna_co_assembled_networks_of_synthetic_alpha_and_gamma_subunits_of_the_hagfish_slime_intermediate_filament.png" />

Fig. 11 Co-assembled networks of synthetic alpha and gamma subunits of the hagfish slime intermediate filament. Magnification of the images is (from the left): 100x, 200x, 200x, 400x, 400x.

                   <img src="Seadna_Low_magnification_view_of_a_thread_formed_from_co-assembled_synthetic_alpha_and_gamma_subunits_of_the_hagfish_slime_intermediate_filament.png" />

Fig. 12 Low magnification view of a thread formed from co-assembled synthetic alpha and gamma subunits of the hagfish slime intermediate filament.

                   <img src="Seadna_high_magnification_view_of_a_thread_formed_from_co-assembled_synthetic_alpha_and_gamma_subunits_of_the_hagfish_slime_intermediate_filament.png" />

High magnification view of a thread formed from co-assembled synthetic alpha and gamma subunits of the hagfish slime intermediate filament. Individual strands of 1um diameter are highlighted.

Summary of filament formation

These results take us one step closer to making a polymer that is biodegradable, strong, elastic and lightweight by using synthetic biology. The future of this project involves characterising these proteins further, improving the solubility and optimizing their expression patterns. We hope that our work will open the opportunities to explore new polymer designs that will be superior to present day alternatives.

References

  • Downing, S.W., R.H. Spitzer, E.A. Koch, and W.L. Salo. 1984. The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions. J Cell Biol. 98:653-69.
  • Fudge, D.S., K.H. Gardner, V.T. Forsyth, C. Riekel, and J.M. Gosline. 2003. The mechanical properties of hydrated intermediate filaments: insights from hagfish slime threads. Biophys J. 85:2015-27.
  • Fudge, D.S., S. Hillis, N. Levy, and J.M. Gosline. 2010. Hagfish slime threads as a biomimetic model for high performance protein fibres. Bioinspir Biomim. 5:035002.
  • Fudge, D.S., N. Levy, S. Chiu, and J.M. Gosline. 2005. Composition, morphology and mechanics of hagfish slime. J Exp Biol. 208:4613-25.
  • Kim, B.S., K.E. Park, W.H. Park, and J. Lee. 2013. Fabrication of nanofibrous scaffold using a PLA and hagfish thread keratin composite; its effect on cell adherence, growth, and osteoblast differentiation. Biomed Mater. 8:045006.
  • Negishi, A., C.L. Armstrong, L. Kreplak, M.C. Rheinstadter, L.T. Lim, T.E. Gillis, and D.S. Fudge. 2012. The production of fibers and films from solubilized hagfish slime thread proteins. Biomacromolecules. 13:3475-82.
  • Puigbo, P., E. Guzman, A. Romeu, and S. Garcia-Vallve. 2007. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res. 35:W126-31.
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Contribution: HKUST Hagbric 2020

Authors: iGEM HKUST Hagbric 2020, Derek Shao Edit: Phillip Zuo

3D Structure Modelling

Our Aim and What we achieved
Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. However, the 3D protein structures of this subunit of hagfish slime intermediate filament haven’t been experimentally determined. Herein, with the I-TASSER server, we predicted the 3D structures of this subunit to facilitate our understanding about the properties of hagfish slime IFs (Figure.13). We encourage future teams to improve the modelling quality with finer tools. Our aim is to inspire future iGEMers to make good use of in-silicon tools when there is no experimentally determined 3D structure available for their target molecule.

Our Approach
Documentation of how we model the fibers are shared on the GitHub https://github.com/HagBric/iGEM_Dry_Lab

Figure 13: 3D structure of the intermediate gamma subunit


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 373
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 895


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Parameters
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