Team:UCLA/Project/Programming Spider Silk

iGEM UCLA




BACKGROUND

PROJECT GOALS

ICA

ACCOMPLISHMENTS























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

  1. Iterative capped assembly is similar to Golden Gate assembly, which uses unique sticky ends to assemble gene fragments in a specific order. Whereas Golden Gate is a one-pot reaction with all the pieces ligated simultaneously, ICA is a more “controlled” variation where pieces are assembled one at a time. ICA relies on using 3 different versions of the monomer to be assembled, each of which has different sticky ends such that the monomers must be assembled in an A-B-C fashion. This prevents monomers from self-ligating. Type IIS restriction enzymes (such as BsaI), which cleave outside of the recognition site are used to generate these.
  2. A basic biobrick for ICA consists of a gene monomer flanked by BsaI recognition and cleavage sites. While each of the core monomer is identical, the restriction sites are oriented such that digestion with BsaI yields distinct sticky ends for each of the three types of units. These pieces must be digested to reveal the sticky ends before assembly.
  3. Accessory pieces required in ICA include the initiator, streptavidin coated beads, the terminator, and the capping oligos.
    1. The initiator is a dsDNA fragment made by annealing two ssDNA oligos together. The initiator is designed such that one end is biotinylated, for conjugation to streptavidin coated beads. The other end has a sticky overhang and is designed to anneal to the forward sticky end of the ‘A-type’ monomer unit. This end is 5’-phosphorylated to enable ligation. The initiator also contains a primer binding site that can be used for PCR amplification, as well as other accessory sequences such as affinity tags and the biobrick prefix.
    2. Streptavidin coated beads serve as a solid support for the elongating DNA chain during ICA. The biotinylated end of the initiator binds to streptavidin to anchor the nascent construct. The ability to physically separate the DNA from solution is needed due to repeated wash and ligation steps used during ICA.
    3. The terminator is constructed similarly to the initiator, but lacks biotinylation. One end of the terminator is compatible to the reverse sticky end of the ‘C-type’ monomer unit. This end is 5’-phosphorylated to enable ligation. The terminator also contains a primer binding site that can be used for PCR amplification, as well as the biobrick suffix.
    4. The capping oligos are comprised of a single 5’-phosphorylated ssDNA oligo that can form a stable stem loop structure with a unique sticky end. There are three distinct caps, each of which can bind to the A, B, or C sticky ends
  4. In each extension step, the next sequential monomer (A, B, or C) is added onto the growing chain. Chains that failed to extend during the previous extension step are capped using a hair-pin oligo that prevents subsequent extension. These capped chains are still present in the mixture for the duration of ICA, but do not participate in any ligation event, and are not amplified in the final PCR. Each final construct is flanked by a biotinylated initiator oligo which allows immobilization onto streptavidin beads, and a terminator oligo. These two oligos provide primer annealing sites which can be used to amplify the sequence using conventional PCR.
  5. A generalized workflow is demonstrated below:
    1. The initiator, terminator, and capping oligos are prepared ahead of time by mixing the relevant oligos and ramping down from 95 C to form the working oligos.
    2. Monomers of each type (A, B, C) are digested from plasmid with BsaI and purified prior to ICA. These are termed working monomers.
    3. The initiator is attached to the streptavidin coated beads.
    4. An ‘A-type’ monomer is ligated to the end of the initiator. Afterwards, any unreacted fragments, as well as the ligase are washed off.
    5. Next, the ‘B-type’ monomer is ligated to the end of the growing chain. In this reaction mixture, an ‘A-type’ cap is also included, to terminate any chains that failed to extend in the previous ‘A-type’ ligation. Again, unreacted fragments are removed by washing.
    6. Next, the ‘C-type’ monomer is ligated. This reaction mixture contains the ‘B-type’ cap.
    7. Next the ‘A-type’ monomer is ligated. This time, the ‘C-type’ cap is also included in the mixture.
    8. This proceeds in a repetitive fashion until the desired construct length is reached.
    9. The final constructed is eluted off the beads. The eluate is used as a template for PCR to amplify the construct. Only complete constructs that contain the initiator and terminator are amplified. Capped constructs do not amplify.
    10. The amplified construct can now be used for downstream cloning.

Results

Using ICA, we have generated 10 silk constructs. These include constructs of pure MaSp2 ranging from 3-15 mers, pure MaSp1 of 9 and 12-mers and 12-mers of MaSp1/2 hybrids in 3 different ratios.

Future Directions

While we were able to construct many sequences of varying length and composition using ICA, we were unable to explore its maximum potential for cloning repetitive genes. We have not established an upper limit on the number of monomers able to be assemble using ICA. In addition, we have not explored the extended use of ICA to create extremely large (greater than 50 monomer units)

Achievements

  1. Successfully improved last year’s biobrick BBa_K1384000
    1. Redesigned MaSp1 and MaSp2 monomers with modified sticky ends, and cloned these into biobricks.
  2. Created a collection of parts to be used for Iterative Capped Assembly
  3. Demonstrated that ICA is adaptable to silk using our designed sticky ends.
    1. Optimized ICA for use with spider silk genes to enable fast, efficient assembly of repetitive constructs.
  4. Used ICA to assemble a variety of silk genetic constructs of different length and composition to examine their properties.
    1. Used ICA to create 10 different silk constructs ranging from 3-mers to 15-mers, and constructs with MaSp1 and MaSp2 in ratios of [1:2], [1:1] and [2:1].

List of Biobricks

  • MaSp2 AB BBa_K1763002
MaSp2 AB BBa_K1763002 MaSp2 BC BBa_K1763003 MaSp2 CA BBa_K1763004 MaSp2 SeqAB BBa_K1763009 MaSp1 AB BBa_K1763010 MaSp1 BC BBa_K1763011 MaSp1 CA BBa_K1763012 MaSp1 SeqAB2 BBa_K1763423 M2-3(1C3) BBa_K1763424 M2-3(T7) BBa_K1763425 M2-6(1C3) BBa_K1763426 M2-6(T7) BBa_K1763427 M2-9(1C3) BBa_K1763428 M2-9(T7) BBa_K1763429 M2-12(1C3) BBa_K1763430 M2-12(T7) BBa_K1763431 M2-15(1C3) BBa_K1763432 M2-15(T7) BBa_K1763433 M1-9(1C3) BBa_K1763434 M1-9(T7) BBa_K1763435 M1-12(1C3) BBa_K1763436 M1-12(T7) BBa_K1763437 M1/2[2:1]-12(1C3) BBa_K1763438 M1/2[2:1]-12(T7) BBa_K1763439 M1/2[1:1]-12(1C3) BBa_K1763440 M1/2[1:1]-12(T7) BBa_K1763441 M1/2[1:2]-12(1C3) BBa_K1763442 M1/2[1:2]-12(T7) BBa_K1763443

Fig 2: Variations of the MaSp1 amino acid sequences between different spider species [6]











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.

Fig. 3: Schematic of ICA from Briggs, et al.[5]

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.

Fig. 4: Immobilization of the initiator to magnetic beads. Figure from Briggs, et al.[5]

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.