Team:UCLA/Project/Programming Spider Silk
Programming Spider Silk
Background
Abstract
A major obstacle in creating recombinant spider silk is the highly repetitive nature of the genes that encode it. Silk genes are comprised of a repetitive core region containing ~100 repeats of a spidroin gene which precludes the use of traditional cloning techniques due to non-specificity in primer binding. While other techniques such as head-to-tail assembly or concatemerization have been developed to facilitate spider silk cloning, none of these techniques can assemble silk genes in a quick and directed manner. We have adapted the use of Iterative Capped Assembly (ICA) as a technique to construct silk genes in a rapid and sequence specific manner. By creating a number of different constructs this summer, we show that ICA can greatly facilitate the engineering of recombinant spider-silk.
Introduction
Recombinant spider-silks with customizable properties present an appealing biomaterial that can be used in textiles, tissue scaffolds, and other unique applications. Spider silk is a proteinaceous fiber whose proteins consist of non-repetitive N and C terminal domains, and a highly repetitive central core that consists of up to 100 repeats of Spidroin1 (MaSp1) or Spidroin 2 (MaSp2). These spidroin repeats are directly responsible for the final properties and behavior of the spider silk. MaSp1 contributes strength to the fiber, while MaSp2 contributes elasticity. Importantly, the relative content of MaSp1 and MaSp2 monomers in silk proteins can dictate the fiber properties when spun. Engineering recombinant spider silk genes of varying lengths and spidroin content is nearly impossible using traditional cloning methods, due to the repetitive core. These repeats essentially forbid the use of primers to amplify said genes due to the possibility of non-specific priming. These obstacles have made engineering recombinant spider silk a difficult process.
Existing techniques seek to remove reliance of cloning on primer annealing (Tokareva et al, 2013). Generally, these techniques break the silk genetic into monomeric sequences, then assemble the monomers into the final construct. Head-to-tail cloning assembles gene constructs by ligating two plasmid halves together. Each half carries one of the silk monomers, and the resulting complete plasmid has been doubled. This technique can be used recursively to assemble increasingly large silk genes in a specified manner. Directional recursive ligation uses a similar tactic, where individual monomers are ligated one at a time into a receiving plasmid. Concatemerization is another technique where a pool of monomers are ligated in a single reaction, then cloned into plasmids. This particular technique is useful for creating a library of sizes and compositions.
These existing techniques are not ideal for engineering recombinant silks, because they require repeated and extensive cloning for large constructs, as in the case of head-to-tail assembly and directional recursive ligation, or do not offer any control over the length or genetic composition, as is the case for concatemerization. As it currently stands, there is no one technique that offers rapid and controllable assembly of recombinant spider silk genes.
Iterative Capped Assembly (ICA) is a cloning method that is used to sequentially assemble long, repetitive DNA sequences. This technique was developed by Briggs et al. in 2012 as a method to assemble Transcription Activator-Like Effector Nucleases (TALENs) which are sequence specific DNA binding proteins that consist of multiple repetitive monomers. Each repeat monomer is responsible for binding to a specific nucleotide in the target sequence. Due to the repetitive nature of TALE genes, conventional PCR is unable to reliably amplify these sequences due to non-specific primer binding.
Although ICA was developed using TALE construction as a model problem, this technique can be used to construct long, repetitive DNA constructs in a directly controllable fashion. ICA assembles repetitive sequences one monomer unit at a time, while preventing the elongation of incomplete nucleotide chains. The full length sequence is flanked by unique primer annealing sites, which allows the PCR amplification of the final product. This entire process is performed using a solid substrate, which greatly facilitates the construction of long sequences.
This summer, we adapt the use of Iterative Capped Assembly (ICA) as a technique for cloning recombinant spider silks in a time-efficient and specific manner that is unparalleled by existing methods. Our experience shows that ICA can be used as a method for the quick assembly of spider silk genes.
Methodology
A major obstacle in creating recombinant spider silk is the highly repetitive nature of the genes that encode it. Silk genes are comprised of a repetitive core region containing ~100 repeats of a spidroin gene which precludes the use of traditional cloning techniques due to non-specificity in primer binding. While other techniques such as head-to-tail assembly or concatemerization have been developed to facilitate spider silk cloning, none of these techniques can assemble silk genes in a quick and directed manner. We have adapted the use of Iterative Capped Assembly (ICA) as a technique to construct silk genes in a rapid and sequence specific manner. By creating a number of different constructs this summer, we show that ICA can greatly facilitate the engineering of recombinant spider-silk.
Iterative Capped Assembly
Assembling spider silk monomers such as MaSp1 and MaSp2 together can prove to be rather difficult. Due to the repetitiveness of our silk monomers, common techniques such as Gibson assembly and direct synthesis of a full-length gene product could be ineffective and less efficient in building silk sequences. Therefore, a technique called iterative capped assembly (ICA), which allows rapid assembly of repeating monomers, would be a better option in working with our highly repetitive sequences. This technique has previously been applied in order to assemble other repetitive modules. For example, ICA has been demonstrated to efficiently assemble very repetitive transcription activator-like effector nucleases (TALENs) up to 21 monomers long[5].
ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.
Silk monomers for ICA were built using PCR to attach BsaI recognition site onto the MaSp sequences, as well as different end extensions in the form of 4bp overhang that is essential for ligation. BsaI is a type IIs endonuclease that cleaves outside the recognition site and therefore generates overhangs that are still part of the native silk sequence. This leaves our digested products, or building blocks, free of any remaining recognition site, which is usually formed with other types of restriction enzymes.
We have designed 3 types of overhangs that the Bsa1 enzyme can generate. These are the A overhang, (AGCA), the B overhang (TGCA) and the C overhang (TGCT).
The key idea here is that monomers with the same overhang are complementary and can be ligated together. Each silk monomer was modified via pcr to have one of these overhangs at the 5’ end of the sequence and another at the 3’ end of the sequence. One version of our silk monomer had an A overhang at the 5’ end and a B overhang at the 3’ end, which we called (5’)AB(3’). Another version was (5’)BC(3’), and the final version was (5’)CA(3’). We therefore end up with 3 versions of the same silk monomer. Making these modifications for all of our different types of silk monomers potentially gives us the ability to assemble a hybrid silk gene composed of different monomers in a matter of hours. Following restriction digestion of all our monomers with BsaI to create the sticky ends, each silk monomer would be able to ligate to the preceding piece due to the complementing 4bp on their ends. For example, if we wished to ligate a MaSp 2 monomer to a (5’)BC(3’)MaSp1 monomer, we would simply add a (5’)CA(3’) MaSp2. With the 3 subsets of MaSP1 and MaSP2, we could eventually program gene sequence of desired physical properties with various ratios and orders of each monomer type.
A key aspect of ICA is that the gene to be assembled is fixed to a solid support as it is being ligated together. Streptavidin beads act as the solid support in this case. Conjugating our gene to the beads therefore necessitates an “initiator oligo”, a biotynilated sequence that contains both the biobrick prefix as well as one of the A,B, or C overhangs at its 3’ end. An advantage of fixing our growing sequence to the beads is that it allows us to remove all traces of the previous ligation in a “wash” step before adding the next silk monomer to the growing gene.
When dealing with repetitive sequences, regular assembly techniques would give products of various lengths due to the uncontrolled ligation among pieces. Another powerful aspect of ICA is that it increases the frequency of producing a full-length sequence that we intend to build. This is achieved by adding capping oligos that ligate to a chain where a previous monomer piece fail to attach. Any incomplete or incorrect sequence due to unsuccessful ligations would be blocked from further extending, and thus monomers that are added later would ligate to the right sequences at a higher frequency.
Once the full gene sequence has been generated, the terminator oligo containing the biobrick suffix is added to complete the assembly. The gene can then be released from the streptavidin beads by heating and disrupting the Streptavidin biotin interaction[5].
ICA is a proficient technique to standardize assembly of any custom gene sequence. Not only does it provide flexibility in producing custom gene sequence, it is also compatible with the iGEM biobrick system. The initiator contains the prefix sequence of a biobrick and the terminator contains the suffix sequence. These contain primer sites allow PCR amplification of the full-length constructs after the release from the beads. In addition, restriction sites within the initiator and terminator enable the insertion of the complete, assembled product into a biobrick backbone easily using Golden-Gate cloning.
What We've Done
Over the course of the summer, we modified the nucleotide sequence of MaSp1 and MaSp2 so that it is both compatible with the iGEM standards as well as with our ICA assembly scheme. We generated these sequences by ordering single stranded oligos and doing multiple rounds of pcr to assemble the full construct. These sequences contain the biobrick prefix and suffix as well as the required Bsa1 restriction sites for the ICA/Golden Gate reaction. Furthermore, we designed and assembled the other nucleotide sequences necessary for ICA from primers that we ordered. These sequences include the biotinylated initiator oligo, the ICA terminator oligo, as well as the capping oligos (see above for description of each).
We ran some trial runs of ICA by trying to assemble only a few monomers together. We only were able to run a few trials, and unfortunately, were not able to create the full silk construct. We were able to ligate the initiator oligo to the terminator oligo, showing that the initiator oligo was successfully conjugating to the streptavidin beads and that the ligation reaction reaction worked. Hopefully with some optimizations and troubleshooting, we can use this protocol to assemble a full length silk construct in the near future.
References
[1] Lewis, Randolph V. "Spider silk: ancient ideas for new biomaterials." Chemical reviews 106.9 (2006): 3762-3774.
[2] Gatesy, John, et al. "Extreme diversity, conservation, and convergence of spider silk fibroin sequences." Science 291.5513 (2001): 2603-2605.
[3] Xia, Xiao-Xia, et al. "Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber." Proceedings of the National Academy of Sciences 107.32 (2010): 14059-14063.
[4] Huemmerich, Daniel, et al. "Novel assembly properties of recombinant spider dragline silk proteins." Current Biology;14.22 (2004): 2070-2074.
[5] Briggs, Adrian W., et al. "Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers." Nucleic acids research(2012): gks624.
[6] Teulé, Florence, et al. "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." Nature protocols 4.3 (2009): 341-355.