Difference between revisions of "Part:BBa K1763012"

Latest revision as of 17:58, 18 September 2015

Major Spidroin Protein 1 (MaSp1) with Sticky Ends CA

This part contains the core sequence of MaSp1 that has been assembled with a 5'-CGTG-3' overhang on the 5' end of the sequence and 3'-TCAA-5' overhang on the 3' end of the sequence. This part was designed for use with Iterative Capped Assembly (ICA), along with the part collection [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics#ICA_Parts_Collection here] as an efficient method to assemble multiple repetitive MaSp sequences together. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site.

For details about Iterative Capped Assembly, as well as protocols for performing ICA, please visit the [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/Protocols protocols] page of the 2015 UCLA iGEM team.

Biology

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.

Sequence and Features

Usage

For detailed protocols, refer to the 2015 UCLA iGEM team protocols [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/Protocols here].

Large quantities of DNA are required for preparing ICA monomers. One stratagem for attaining sufficient DNA is as follows:

The plasmid containing this part should be transformed into E. coli. Grow a 100 mL culture of the transformed culture from a single freshly streaked colony. Harvest the bacteria and purify the DNA using kits, or otherwise. The final concentration of plasmid should be high enough to digest 5 ug in a single reaction. We aimed to have at least 120 ng/uL concentration of DNA.

Digest 5 ug of plasmid in a 50 uL reaction with 4 uL BsaI. Digest at 50 C for 2 hours and heat kill at 65 C for 20 min. The digestion conditions are different from those specified by NEB, but our optimized digestion increases enzyme activity and digestion efficiency. We have found that due to yield loss from gel purification, it may be necessary to perform this digestion in duplicate. (ie. digesting 10 ug of plasmid in 2x 50 uL reactions.)

Gel purify the digestion on 1.5% agarose gel. The product is the digested 102 bp fragment which will be directly used for ICA. An example digestion is shown below.

Fig. 1 BsaI Digestion of MaSp2 monomers AB, BC, CA. Each digestion was performed in duplicate. The expected product is 102 bp. The 102 bp bands were excised for subsequent gel extraction.

After gel purification, typical yields were ~30 ng/uL in 12 uL. This is ~75% extraction efficiency assuming complete digestion. The digested parts are ready to be used in ICA.