Team:Edinburgh OG/Silk

Synthetic Spider Silk

Synthetic biology has the power to engineer microorganisms for brewing valuable biomaterials such as spider silk. Spider silk is three times stronger than Kevlar fiber and five times tougher than steel [1]. Silk fabrics had been employed in the textile and fashion industry for several years. However, farming spiders is not convenient, and fermentation has a greater potential for scale than farming silkworms. At scale, synthetic silk could replace petrochemical based fabrics as nylon and polyester for a sustainable future. Synbio companies as Bolt Threads and Spiber had recently launched apparel products made by synthetic silk. The apparel market size is approximated $2 trillion USD, and synthetic silk has many other applications in the automobile, aerospace, medical and construction sectors.

Dragline silk results from the combination of silks polymers MaSp1 and MaSp2. The natural protein size is around 3,500 amino acids [3], composed of large central domains of monomer repeats. Flanking the central motif are non-repetitive N- and C- terminal domains of around 100 amino acids. These domains ensure protein stability during fiber formation in the spinning duct of the spider [4].  However, truncated versions of synthetic silk had been designed, constructed and successfully expressed in several hosts including bacteria. Since commercial gene synthesis providers struggle to manufacture repetitive genes, nowadays it is not possible to order a gene encoding a ready-to-express functional silk protein. Different multimerization strategies have been developed to build monomer repeat sequences. We decide to create monomer repeats of an MaSp1 truncated version with added methionine for improving β-sheet folding. We to use the head-to-tail multimerization strategy for building the repeats.

Design

An MaSp1 truncated version reported in the literature has been selected for multimerization used the heat-to-tail strategy. Previous MaSp1 biobricks had been submitted into the iGEM registry. However, this version contains 2 methionine and a different amino acid arrangement. The addition of the methionine intends that the polyalanines motifs fold in such a way that they leave the methionine side chains exposed to the oxidizing agent [10]. Therefore, the methionine amino acids flanking the polyalanine region is hypothesized to work as a redox trigger to control the solubility of spider silk proteins [10].

In silico genetic constructs were designed in Benchling to create truncated versions of MaSp1 silk. The head-to-tail method has been selected due to its control over repeat numbers. The multimerization construct was designed to contain Type II (BsaI) restriction sites compatible with the RFC1000 assembly while preserving a functional open reading frame. The plasmid used for multimerization and expression was the pET28a-GG-RFP-CD (provided by the Wallace Lab), which includes a His-tag region for protein purification.

For building the monomer repeats, the constructs include NheI (left) and SpeI (right) restriction sites were placed besides of the monomer repeat. This strategic allocation of restriction sites allows the creation of monomer doublings without doubling the solubilizing blocks. Monomer doubling requires two products from digestions using different enzyme combinations of the same plasmid. The first digestion SpeI and PvuI (restriction site on resistance gene region) result in a large fragment that contains the monomer repeat and the NheI site. The second reaction uses NheI and PvuI, resulting in a small fragment which includes the monomer repeat and the SpeI site. The ligation of these two fragments produces a doubling monomer leaving a small scar of two amino acids (See Figure 1). Ligations were performed as mentioned in the experiments section.

Figure 1. One-step directional approach mechanism of action. Mechanism of action of selected multimerization strategy. Black stars symbols show restriction site to cut. The first phase refers to the introduction of the whole construct into the selected plasmid (pET28a-GG-RFP-CD). The second phase involves the two parallel digestions (SpeI/NheI). The third phase shows the new plasmid with two monomers after successful ligation. Diagram adapted from [5].

As part of the In silico design, Golden Gate assembly, and monomer doublings were tested by the digestion and ligation Assembly Wizard using Benchling. This validation ensured that the constructs were inserted correctly, and the multimerization strategy worked without disruption to the open reading frame or left undesired restriction sites. For testing the multimerization strategy, gel electrophoresis was used to verify the increase of size between doubling of monomer repeats. Additionally, protein expression on E. coli BL21 was induced using IPTG and tested using SDS-PAGE visualization. On the next table are the expected band sizes and protein molecular weight for each monomer doubling.

Table 1. Expected size dimensions forecast of plasmid, multimerization constructs, and monomer-chain, plus construct molecular protein weight.

Experiments

DNA synthesis was provided by Integrated DNA Technologies (IDT). The synthesized DNA included the whole constructs containing the one-step directional restriction enzyme sites and the selected MaSp1 spider silk gene. The plasmid used for multimerization and expression through this project was the pET-28a-GG-RFP-CD provided by Ph.D. Laura Tuck from the Wallace Lab (University of Edinburgh). The plasmid pET28a is commonly used for achieving high levels of bacterial gene expression induced by IPTG and using kanamycin as bacterial resistance antibiotic. Additionally, pET28a plasmids possess a high copy level. The pET-28a-GG-RFP-CD version has been modified to facilitate Golden Gate assembly and reporting IPTG induction through the expression of Red Fluorescent Proteins (mCherry region) for red/white screening. Also, pET28a-GG-RFP-CD includes a His-tag region which facilitates protein purification and verification through binding assays.

Plasmid DNA extraction was routinely isolated from 5ml of overnight culture using the Qiagen miniprep kit according to the manufacturer instructions. For super-concentrated DNA, in the dilution step 30μl of EB buffer were added instead of 50μl that the protocol indicates. Digestion reactions were set for checking clones to purify DNA fragments of interest. For clone verification, digestions were prepared with a final volume of 20µl, containing 1x appropriate enzymatic buffer, 500-1,000 units of cutting enzymes, 100-200ng of purified plasmid DNA. For DNA extraction, the digestion reactions were prepared to 50µl final volume with 500-1000ng of plasmid DNA. Digestions were incubated for 2 hours at 37°C. Following digestions, DNA fragments were separated by gel electrophoresis. Bands of interest from the digested plasmid were excised from the gel to further isolate the target DNA using the Wizard SV Gel and PCR Clean-Up System toolkit. The procedure was performed according to manufacturer instructions (PROMEGA). The Wizard SV toolkit is suitable to extract and purify DNA fragments from 100bp to 10kb.

Ligations to create monomer repeat chains were performed using New England Biolabs T4 DNA ligase. Each reaction was prepared to 20µl final volume in 10x T4 DNA ligase buffer and 1,000 units of T4 DNA ligase enzyme at 2,000U/µl. After independent digestions using SpeI and NheI, two band of different sizes were polarized in electrophoresis gels. Assuming a 100% DNA presence after digestion, higher bands were ~75% and lower bands ~25% of the total DNA. On that basis, DNA band extraction of products for ligation were calculated to determine the DNA concentration in ng/µl from 30µl elution, leading to the final amount of product required to have a 2:1 DNA mass in a 100ng vector (NEB Ligation Calculator).

Gel electrophoresis agarose: Head-to-tail Multimerization measurement of Truncated MaSp1 Versions from 1 to 8 monomer repeats.

Plasmid DNA verification, colony PCR, and restriction enzyme digestions were routinely separated by agarose gel electrophoresis. Agarose gels were prepared using powder agarose (BIOLINE) on Tris-acetate-EDTA (TAE) buffer. DNA verification and colony PCR gels were 0.7%. For DNA extraction, gels were prepared at 1% final concentration. SYBR Safe DNA Gel Stain (Invitrogen) was supplemented at 1μgml−1 for band visualization under UV light. All samples were mixed with 1μl of 6x Purple Loading Dye DNA Buffer (NEB) per each 5μl. To determine band sizes, 0.5ng of NEB 1kb ladder was run as standard. The electrophoresis gels were run under a constant 105v voltage for 45 minutes to 1 hour. After electrophoresis, gels were visualized using the UV transilluminator device (UVP BioDoc-It Imagining System).