This part is the coding sequence for the silk like polypeptide. This sequence was assembled from a consensus of the MaSp1 from spiders. This protein, in spiders, allows resistance and elasticity used for spinning the webs. The synthetic gene encodes a synthetic protein "silk-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.
The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for the assembling.
major ampullate spidroin 1 (MaSp1) gene from spiders
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN2) and a Strep-tag behind it. SubunitN2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS. The Strep-tag, fused at the C terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin.
The DNA sequence was codon-optimized for Bacillus Subtilis. Moreover the nucleotide sequence was optimized in order to accomplish the production standard of IDT. A couple of optimized codons were affected by this.
The amino acids sequence of the silk protein was obtained from MaSp2. The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
Spider Silk 1x Subunit "F" (Fewest tRNA codon optimized)
Spider silk (1x) 2E subunit optimized to use the fewest number of tRNA codons; contains two elasticity domains and one strength domain.
Artificially designed based on BBa_K844002 and DNA was synthesized.
lac/IPTG inducible Spider Silk 1x "F" Subunit fused to GFP
This part is a spider silk 1x "F" subunit with GFP fused to its C-terminus. This generator is lactose/ITPG inducible.
Assembly standard #23 assembly of basic parts
This part contains the core sequence of MaSp2 that has been assembled with a 5'-AGTT-3' overhang on the 5' end of the sequence and 3'-ACAG-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp2 sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site. Sequence and Features
The gene sequence for MaSp2 is particularly GC-rich, which may complicate PCR.
This part is derived from the MaSp2 gene found in the spider N. Clavipes.
Spider Silk 1x 1E Subunit "U" (native sequence)
Spider silk subunit (1x) native sequence; contains one elasticity domain and one strength domain.
Based on native 1E sequence from Brooks, et al. (2008) and DNA was artificially synthesized
MaSp2 12 mer in pSB1C3. Does not contain regulatory elements
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE1) with Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitE1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites. The amino acid sequence is based on the adapted MaSp2 sequences discribed in the paper of Brooks et. al. 2008.
The amino acids sequence of the silk protein was obtained from the paper by Brooks et al. (2008). The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
Antimicrobial spider silk generator with C-terminus 10x His-Tag
Human (Homo sapiens) and Spider (Argiope aurantia)
Spider Silk Generator - 10x "F" Silk Construct with His Tag
Created with standard assembly (standard #23)
Major Spidroin Protein 2 (MaSp2) with sticky ends BC
This part contains the core sequence of MaSp2 that has been assembled with a 5'-TGTC-3' overhang on the 5' end of the sequence and 3'-GCAC-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp2 sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site.
RBS Start-codon EstA Strep-tag SpiderSilkSubunitN1 SpiderSilkSubunitN2
MotB Strep-tag SilkSubunitN1 SilkSubunitN2
RBS Start-codon EstA Strep-tag SpiderSilkSubunitN1
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN1) with a Bacillus Subtilis ribosome binding site (RBS), a coding sequence for the signal protein EstA and a Strep-tag infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. The EstA codes for a signal protein that that is there to fasilitate secretion of the protein. The Strep-tag, fused at the N terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin. SubunitN1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites.
RBS Start-codon SpiderSilkSubunitE1 SpiderSilkSubunitE2 SilkTail Strep-tag stop-codon
RBS Start-codon Strep-tag SpiderSilkSubunitE1
The part contains the coding sequence for part of the spider silk protein together with Bacillus Subtilis ribosome binding site (RBS) and the Strep-tag.
This part is a fusion of the Apis mellifera (Western Honey Bee) silk protein coding region and the superfolder GFP coding region. A T7 promoter regulatory element has been added upstream, along with a ribosomal binding site. (in progress)
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN1) with Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitN1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites.
RBS Start-codon EstA Strep-tag SpiderSilkSubunitE1
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE1) with a Bacillus Subtilis ribosome binding site (RBS), a coding sequence for the signal protein EstA and a Strep-tag infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. The EstA codes for a signal protein that that is there to fasilitate secretion of the protein. The Strep-tag, fused at the N terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin. SubunitE1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites. The amino acid sequence is based on the adapted MaSp2 sequences discribed in the paper of Brooks et. al. 2008.
The amino acid sequence of the silk protein was obtained from the paper by Brooks et al. (2008). The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE1) with Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitE1 codes for a spider silk protein that is optimized to prevent the forming of secundary RNA structures in close proximity to the RBS. This is given priority over reducing the number of restriction sites. However, the primary goal of the algorithm was still to optimize the codon selection based on their availbility scores.
Spider Silk Generator - 4x "B" Silk Construct with His Tag
Protein generator part for Spider Silk. Protein is a 4x fusion of spider silk "B" subunits, which were codon optimized to use a reduced set of tRNA codons (1 to 2 codons per amino acid). Contains a lactose/IPTG inducible , RBS, and a 10x-His tag for purification of protein. IMPORTANT NOTE: This part uses Assembly Standard #23 (Silver Fusion) scar sites, which the sequence data below will not reflect (it shows Assembly Standard #10 scars).
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN2). SubunitN2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS.
RBS Start-codon Strep-tag SpiderSilkSubunitE1 SpiderSilkSubunitE2
This BioBrick contains the coding sequence for two parts of the spider silk proteins (SubunitE1 and SubunitE2) with a Bacillus Subtilis ribosome binding site (RBS) and a Strep-tag infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. The Strep-tag, fused at the N terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin. SubunitE1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites. SubunitE2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS.
This part is a fusion between the coding region of the honeybee silk protein and SpyCatcher protein which has a high affinity for SpyTag
This BioBrick contains the coding sequence for silk tail and ends with a stop codon. The Tail ....
The amino acid sequence of the tail was obtained from MaSp2. The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE2) and a Strep-tag behind it. SubunitE2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS. The amino acid sequence is based on the adapted MaSp2 sequences discribed in the paper of Brooks et. al. 2008. The Strep-tag, fused at the C terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin.
Spider Silk 1x Subunit "B" (Balanced tRNA codon optimized) with Met (ATG) added
Spider silk subunit optimized to use a reduced set of tRNA codons (1 or 2 codons per amino acid); contains two elasticity domains and one strength domain, also contains 5??? Met (ATG) codon.
Created using mutagenesis PCR to add upstream Met (ATG) to part BBa_K844004
Studies investigating the potential for co-spinning, a process in which the terminal domains of the functional B. mori silk protein heavy chain are fused with genes encoding functional binding domains, and subsequently are combined with native silk proteins to form crystalline fibers, have shown some success (Sponner et al., 2005). In vivo co-spinning simulations have previously been conducted using the transgene in silkworms, producing functionally fluorescent N-terminal ??? GFP- C-terminal fusions that successfully incorporate into endogenous Bombyx mori silkworm silk monomers (Kojima et. al., 2007). These N-Terminal and C-terminal regions are not only easier to clone due to their non-repetitive structures, but additionally allows for storage and assembly of endogenous ampullate spidroins (Askarieh et al., 2010). Composite silkworm-spider silk chimeras have shown improved mechanical properties, and may increase the ease in high-yield production due to the easier manipulation of silkworm silk monomers (Teule, et. al. 2012). Yet, this process still relies on the use of transgenic silkworms, which have longer developmental cycles, require larger storage and nutrient conditions, and difficulty to harvest relative to bacterial counterparts (Murphy et al., 2009). An ex situ co-spinning process has been developed for the production of composite N. clavipes ??? B. mori silk fibers with functional protein fusions in bacterial chassis (namely, E. coli). This process results in the construction of a transgene that can be modularized using restriction enzymes to incorporate a wide range of functional peptide binding sites or other proteins, including avidin-biotin binding domains, RGD-cadherin motifs, albumin binding domains, or antibody affinity domains (Jansson et al., 2014). Specifically, this composite structure focuses on the ability to express functional sfGFP in silk fibers as a marker for successful co-spinning, and verifies proper NC-sfGFP expression using SDS-PAGE analysis. A Standard Registry part encoding sfGFP (BBa_K515005) flanked by conserved B. mori N- and C- silk fibroin heavy chain terminal domains (NC-sfGFP) has been produced. This part is ligated in the standard registry pSB1C3 vector backbone, and transformed into electrocompetent Escherichia coli cell cultures. Expression of the fusion protein is induced using IPTG, and extracts are purified using Immobilized Metal Affinity Chromatography (IMAC) using a Ni-NTA Sepharose resin bed designed to pull down polyhistidine tagged protein constructs.
We decided to use a LacI promoter to regulate expression of the large protein construct in E. coli K12 laboratory strains, in lieu of utilizing a T7 promoter or constitutive Anderson family promoters. This is to maximize the protein yield of the co-spinning module without causing a tradeoff between regular cell growth and housekeeping protein synthesis. Additionally, we decided to use sfGFP as the reporter gene, instead of a standard mut3b GFP, due to the ability of sfGFP to function even when fused to large bulky protein domains (in this case, FibNT and FibCT). Lastly, we designed BsaI TypeIIS restriction sites flanking the reporter gene that when cut, generates sticky ends compatible with EcoRI and SpeI sites. This was created to allow iGEM teams to take protein domains of interest from the Standard Registry of Parts, and fuse them into the co-spinning module by a simple Digest and Ligate assembly to swap the sfGFP construct. However, the protein domain to be added must have the Silver Fusion prefix and suffix, to allow for in-frame cloning of the protein domain. Without this in-frame cloning, the scar sites of the tradition prefix and suffix will cause a frameshift mutation that mutate the translation product, and renders it non-functional.
The LacI promoter (BBa_R0010), RBS (BBa_B0034), and super folder GFP (BBa_I746916) were derived from sequences found in the Standard Registry of Parts. The Bombyx mori silk fibroin heavy chain N-terminal (FibNT) and C-terminal (FibCT) domains were derived from the sequenced consensus genome of Bombyx mori silkworm species, and were synthesized using IDT gBlocks gene fragments.
BsaI Restriction Site (EcoRI Compatible)