Team:GreatBay SZ/Results
Project
SPIDER SILK
Normal spider silk synthesized in specific silk glands in abdomen of spider is considered as an unique ability for spiders. Previous researches have proven its superior material property, spider silk is now recognized to be a new material with great potential in cloth industry, aerospace, and medical area. Spider silk includes the major ampullate (dragline) silk (MaSp)[1], minor ampullate (auxiliary spiral) silk (MiSp)[3], AcSp1[9], AcSp[11], PiSp[2] are gradually discovered and studied in the past. The property and accessibility of the major ampullate gland made itself the focus of spider silk research[5]. It is soon identified to have unrivalled strength and elasticity, specifically, a tensile strength of 200,000 psi (greater than steel), and an elasticity of 35% [1,2].
Spidroin consists of an non-repetitive N-terminal domain(NT), usually around a hundred extensive repetitive regions(Rep), and a non-repetitive C-terminal domain(CT)[Fig. 1.A][5,8]. Natural silk protein synthesis begins at the tail of the gland, secreted into the lumen in a soluble form, and it then passes down a narrow duct as the alignment of polypeptide chains take place. Eventually, at the end of the duct, insoluble protein is generated as extruded silk.[1] While spidroin passes through duct in natural spider gland, a gradual decrease in pH from 7.6 to <5.7 is identified. The alternation may triggers conformation change in spidroin’s terminal regions leading to a lock-and trigger mechanism. In neutral pH, NT is monomerically stored and its high solubility contributes to the solubility of entire protein structure. When passing through the duct, pH diminishes, NT forms dimers that lock spidroin together, while CT destabilized, unfolds, and instead forms β-sheet amyloid-like fibrils. CT also induced the β-sheet conformation in the repetitive region by a pH sensitive “salt bridge”[7,8][Fig. 1.C]. Accompanying this process is the dehydration of the spinning dope. It gives the protein a tangible shape subjected to shear force that may as well contributes to the β-sheet conformation[1].
Though with superior property, spider silk is hard to be obtained in an industrial level for collecting spider silk in the manner of collecting mulberry silk is impossible due to the relatively low production of spider and their territorial behavior, synthetic biology, which utilizing microorganisms to produce spidroin then spin it into silk, seems to be an ideal approach. However, while complete synthesis of natural spideroin cannot be reached due to its excessive number of repetitive regions and the large amount of glycine and alanine sequences that burden the cell to much, researchers developed mini spideroin with NT, CT, and a small amount of repetitive regions, usually from one to fifteen[Fig. 1.B]. In this way, the production of mini-spidroin can be achieved by engineering microorganisms, usually Escherichia.coli (E.coli) and yeasts, and purify protein generated. Silk is then obtained by silk spinning process triggers protein denaturing during spinning process by apply manners like electrospinning[5], hand-drawing spinning[6], and wet spinning into coagulation bath[7].
With great interest in this field and the hope to lay a solid foundation for future team research on related topic, GreatBay_SZ this year aims to establish a database of spiderin and silk collection for the iGEM community.
NT 2Rep CT
One major drawbacks in mini-spidroin production is due to the relative low solubility and pH sensitivity of certain terminal of spidroin. Because NT and CT from the same spidroin may hold mismatched solubility and pH sensitivity, reproduction of high soluble mini- spidroin is hard. Considering the high solubility and pH sensitivity of NT domain originates from Euprosthenops austrails, MaSp1, and correspondingly the CT domain originates from Araneus ventricosus, MiSp. A recombinant chimeric spidroin is constructed with such NT, CT, and two short repetitive regions from E. austrails MaSp1 (all connected through a GNS linker), named as NT2RepCT [Fig. 2.A].
To obtain the protein, we applied a synthetic biology approach to code the nucleotide sequence of NT2RepCT into pET28a plasmid withholds a 6×His tag for Ni-NTA purification. The plasmid is then transformed into E.coli BL21(DE3) for iptg induction and spidroin production. NT2RepCT is, in this manner, produced and purified (non-denaturing) to obtain a yield of 336±54mg/L(45.95±11.08mg/g) [Fig. 2.C]with an expected molecular mass of 33kDa [Fig. 2.B], according to SDS-page(12%).
Due to high sensitivity of NT2RepCT towards pH changing, we conducted silk spinning in buffer with pH=2,3,4,5,6,7 respectively to test its formation [Fig. 3.A]. For all conditions, spidroin altered from a soluble to insoluble state. When pH = 2 and 3, the protein was insoluble in initial conformation, yet soon melted into coagulation bath. When pH = 4 and 5, a glue-like fiber existed persistently in a insoluble state, yet there was no complete silk formation. When pH = 6 and 7, a continuous fiber is generated, yet the formation requires a long period of time, thus cannot be completely extracted. The phenomenon drives us to search for other coagulation bath available, we applied isopropanol instead to test the silk formation. In 100% of isopropanol, silk formation is continuous and uniform, we hence took isopropanol as our major coagulation bath.
To achieve automatic spinning process, we established a two-part hardware for spinning with constant speed. The machine also offered the silk a shear force that further the conformation. Eventually, we were able to obtain a continuous yet not transparent NT2RepCT silk of 20 meters long out of 200ul(10-20mg) of extracted protein [Fig. 3.D].To further explore the continuity and uniformity of our silk generated as well as obtain sophisticated data, we view the silk under microscope. Three randomly selected cross section is measured and the similarity in three sets of data helped to calculate the average diameter of 17.61um [Fig.3.E] for NT2RepCT. While trying to spin the silk with needles of varied diameter (110um and 60um) with corresponding crimp speed (387mm/min and 193mm/min respectively), silk generated does not distribute significant differences in both continuity and diameter. Therefore, we may conclude that both diameter of needle and the crimp speed cannot interfere with silk formation.
Other spidroin
To further expand our model, we utilized the discovery of certain proportional relationship between the number of repetitive region to the strength and extensibility of the silk[5]. Hoping to promote silk property and test the modularization of NT2RepCT, we doubled the amount of repetitive regions to generate NT4RepCT [Fig. 4.B]. Through similar protein purification process, we purified the protein with the expected molecular mass of 40kDa [Fig. 4.E].
However, though certainly demonstrated conformation in the coagulation bath, when going through the spinning process, NT4RepCT silk failed to form any continuous fiber-like silk in both isopropanol and pH. Unfortunately, the limitation of time stopped us to investigate further and achieve continuous silk.
After acknowledging different types of spider silk available for mini-spidroin, our team decided to further expand the realm by synthesizing NTWCT [Fig. 4.C], AcSp[11], with an increasing amount of repetitive region. We tried to construct NT2WCT, NT3WCT [Fig. 4.D], NT4WCT[3]. Only NTWCT and NT3WCT were successfully induced by iptg with expected molecular mass of 45kDa and 85kDa [Fig. 4,F] respectively. NTWCT existed in supernatant, thus can be extracted by non-denaturing protein purification, while NT3WCT existed in precipitate, it can only be extracted by 6M Gu-HCl denaturing protein purification. We concentrated both protein to 80-120mg/ml for wet spinning. We discovered that NTWCT prefers self-aggregation to form insoluble sediments, making the generation of continuous silk hard to be obtained [Fig. 5.A,B]. However, we surprised to find out that NT3WCT [Fig. 5.C,D]can form continuous, slim silk with the diameter of 7.89um.
Through all these efforts, we are able to obtain NT2RepCT, NT4RepCT, NTWCT, and NT3WCT will relatively ideal yield possible for industrial application. Also,we designed a specific bio-brick with NT-typeII Restriction enzyme cutting site -CT [Fig. 6.A,B] hoping it could be assist future teams in constructing a variety type of recombinant spider silk. In future, we hope to test all our silk’s corresponding material properties.
NATURAL PIGMENTS
Despite simply laying a complete database of spideroin, GreatBay_SZ this year also approached one major industry where spider silk held great potential: the cloth industry. Identifying the current chemical compound pollution during dying process as well as the damage brought by chemical fiber itself, we realized that the typically non-environmentally friendly material of cloth manufacture can be replaced by spider silk, as thread to weaved the cloth, and natural dyes, as pigments that granted cloth color.
Among these, we selected deoyviolacein (purple red color) and indigo (blue color), both natural dyes with admittedly strong stability and fascinating color tones [11], and shared a common precursor L-tyrptophan. Together, we hoped to weave a Spider-Man battle suits with our spider silk and dyes for demonstration.
Deoxyviolacein has longed be identified as secondary metabolities actively against pathogenic bacteria like Pseudomonas aeruginosa and Staphylococcus aureus, and leukemia, lung cancer, human uveal melanoma, and lymphoma cells[10,11]. It also served other purpose like natural pigments. The importance of violacein urged us to search for over production of both metabolities. The hidden pathway for production is encoded by the VioABCDE operon. Bio-synthesis starts from L-tryptophan, converted into protodeoxyviolaceinic acid by VioA, VioB and VioE enzymes, and then into deoxyviolacein is therefore produced with the activation of VioC gene[10] [Fig. 7.A].
Indigo, though without much significant medical usage, is the most classic ancient dye uniquely capable of producing the signature tones in blue denim. The current production of indigo not only release hazardous chemical compound, but also requires an excess, usually toxic, reducing agent to reduce the insoluble indigo into soluble leucoindigo. Replacement of reducing agent were identified in the past, yet none established as fast nor cost effective. We therefore researched an alternative indigo production pathway. With L-tryptophan, indole is formed by enzyme tnaA in E.coli. In the presence of indole and oxygen, FMO catalyzes the addition of a hydroxyl group to indole generating the intermediate indoxyl that can spontaneously oxidize to form indigo[Fig. 7.A].
To achieve over production of both pigments, we utilized the stabilized promoter BBa_K2753019 from GreatBay_China(2018), transcription-activator-like-effector (TALE) stabilised promoters that untie gene expression level from gene copy number. We designed to place TALEsp2 promoter on standard pSC101 backbones. Combining the pathway with promoter, we hence characterized and obtained pigments by constructing part TALEsp2-VioABEC [Fig. 7.B]for deoxyviolacein through BBa_K2753019, BBa_K726015, and a new basic part BBa_K3264008, a gene-transcript of VioC. Meanwhile, we also constructed a new composite part TALEsp2-tnaA-FMO[Fig. 7.C] for indigo.
Pigments were obtained by extracting pigments after 42 hours of shake-flask incubation (without iptg)[Fig. 8.A,D] using solvent ethanol for violacein, and DMSO for indigo respectively.
Through calculation based on standard indigo product and OD measurement of oh, 6h, 18h, 24h, 30h, 42h (peak of production)[Fig. 8.E,F], we are able to construct a yield versus time curve. Through that, we concluded our deoxyviolacein yields 85.81±9.09mg/L [Fig. 8.C]maximum and indigo yields 6.97±0.44mg/L [Fig. 8.F]maximum.
To sum up, both our natural pigment deoxyviolacein and indigo can be over produced with our part TALEsp2-VioABEC and TALEsp2-tnaA-FMO. We tested the dying effect of deoxyviolacein which demonstrates an ideal stability before, in, and after washed[Fig. 9.A,B,C]. However, we only obtained insoluble indigo that cannot be dyed. We therefore designed a new composite part TALEsp2-tnaA-FMO-UGT that helps in dying process, and established a deep collaboration with Tongji_China based on our indigo production. Eventually, we hoped to further expand various dying processes to make more color available for dying environmentally friendly while the two pigments, deoxyviolacein and indigo, help to establish a solid foundation.
Chromoprotein
Through metabolic engineering process, we are able to obtain deoxyviolacein and indigo. However, utilizing these natural dyes only allows for two types of color available, and experience in indigo production proved that characterization and expression of new pathways is time-consuming and, certainly, risky. For each natural dye requires different characterization pathway and dying techniques, one manner applicable for a certain type of pigment cannot be applied into a variety types of color. To solve this problem, we looked into redesigning chromoprotein themselves, hoping to design parts and dying technique that suit a variety of different colors.
We first applied the simplest approach by fusing super folder GFP(sfGFP) directly into the sequence of NT2RepCT to obtain two structures: sfGFP-NT2RepCT[Fig. 10.B] and NT2RepCT-sfGFP[Fig. 10.C]. After protein purification, we confirmed their molecular mass of 55 kDa[Fig. 10.D,E]. Silk generated through isopropanol demonstrates certain variability due to the terminal connected to chromoprotein. When connected to NT, that is, sfGFP-NT2RepCT, the silk is continuous and transparent. Under ultraviolet rays, the silk revealed florescent green as expected, and even held certain golden lustre under day light[Fig. 10.G；Figure11]. When connected to CT, however, the silk, with a larger diameter and less transparent, is fragile and discontinuous, making it hard to be formed in coagulating bath. The addition of chromoprotein is therefore hypothesized to interfere with CT’s function. We then tried to apply the same method on eforRed. Unexpectedly, we failed to induce both proteins. The experience with eforRed proved that this manner is subjected to a certain risk of protein formation failure and cannot help us to achieve a well-applicable model of colored spider silk.
To achieve our goal, instead of directly fusing chromoprotein on spidroin, we choose to mix chromoprotein with our spider protein. To obtain better dying effect, we constructed three different types of 2Rep-chromoprotein (sfGFP of florescent green with molecular mass of 34kDa, eforRed of florescent red with molecular mass of 33kDa, and amilCP of dark blue with molecular mass of 28kDa). Therefore, during spinning, the repetitive regions generate shear force in coagulating bath, allowing our chromoprotein to form hydrogen-bonding with NT2RepCT via repetitive regions, increasing stability of color by fixing chromoprotein with spidroin. Therefore, combing these new parts with NT2RepCT may lead to the formation of silk with “tagged” chromoprotein of color green, red, and blue[Fig. 12]. We also constructed three corresponding NT-2Rep-chromoprotein (with molecular mass of 45kDa, 46kDa, 45kDa respectively). Additionally, for control group, we characterized and expressed only chromoprotein sfGFP, eforRed, and amilCP (with molecular mass of 30kDa, with molecular mass of 29kDa, with molecular mass of 27kDa, respectively[Fig. 14]), mixing it with our spidroin, for further comparison.
Mixtures of all types were described and compared at a concentration of 100mg/ml[Fig. 13]. Mixture of pure sfGFP[Fig. 13.A.]and eforRed[Fig. 13.B.] demonstrates strong solubility judging by the high transparency. For amilCP[Fig. 13.C.], chromoprotein with low solubility, mixture with pure amilCP demonstrates large amount of precipitation. Mixture of 2Rep-chromoprotein establishes a emulsion like solution with a decrease in transparency. However, in the case of NT-2Rep-chromoprotein, presumably due to NT, solubility increases so that transparency is higher compared to the one with 2Rep.
We hence could generate silk through spinning of the mixture. For sfGFP[Fig. 15.], all three combinations can form silk, while the one with pure chromoprotein has a diameter of25.5um, with NT-2Rep a diameter of 21.0um, and with 2Rep-sfGFP a diameter of 9.6um, much less compared to the other two. In the case of eforRed[Fig. 16.], mixture with pure chromoprotein possesses a diameter of 31.6um, while with 2Rep a diameter of 24.7um. However, we were unable to obtain an evenly distributed fiber-like substance with NT-2Rep-eforRed. Also, for amilCP is a chromoprotein with relatively low solubility, it precipitates to blocked the continuous release of spidroin mixed with only amilCP[Fig. 17.] constantly and thus no silk is generated, while the rest can form silk.
Collectively evaluating all spinning results, it is clear that the structure of 2Rep-chromoprotein has a more stable silk formation, therefore a better model to realize colored silk for a variety of chromoprotein. Since we have obtained all three types of chromoprotein spinning dope, we mixed two or more of them to realize production of spider silk with different colors out of only three types of spinning dope[Fig. 18.].
To conclude, we utilized NT and 2Rep to construct chromoprotein capable of “tagging” themselves to common spidroin, directly rendering the silk color without complicated dying process. The structure of 2Rep-chromoprotein thus became a common architecture for colored spider silk formation. Finally, we GreatBay_SZ established a part collection based on information obtained through our chromoprotein trials. We expect this collection to help to lay a solid foundation for future spider silk development within the community of iGEM and more, and we genuinely looking forward to future teams joining in and further understanding in this blizzard field of study.
Reference
