Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Fasih Ahsan
Date created: 2015-09-14 11:00:00
Date modified: 2016-02-10 11:26:30
Bombyx mori silk co-spinning module
| Types | DnaRegion |
| Roles | Generator engineered_region |
| Sequences | BBa_K1763444_sequence (Version 1) |
Description
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.
Notes
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.Source
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.| Sequence Annotation | Location | Component / Role(s) |
| LacI (BBa_R0010) Bba prefix/suffix assembly scar site Elowitz RBS (BBa_B0034) 6x N-terminal polyhistidine tag B. mori FibNT BsaI Restriction Site (EcoRI Compatible) superfolder GFP (BBa_I746916) B. mori FibCT Ribosome Double-Stop Site | 1,200 201,208 209,220 230,247 248,712 719,724 725,1438 1451,1627 1628,1633 | feature/promoter promoter feature/scar ribosome_entry_site feature/rbs feature/tag tag feature/protein CDS sequence_feature feature/dna feature/protein CDS CDS feature/protein feature/stop stop_codon |