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Silkworm biotechnology
- 2. SILKWORM Larva or caterpillar or imago of the domestic silkmoth It is an economically important insect (primary producer of silk) Preferred food white mulberry leaves, other mulberry species and osage orange Wild silkmoths are different from their domestic cousins: as they have not been selectively bred they are not as commercially viable in the production of silk Bombyx mori (Latin: "silkworm of the mulberry tree")
- 3. TYPES OF MULBERRY SILKWORM Mulberr y silkwor m Univoltine PolyvoltineBivoltine
- 4. SILKWORM1. UNIVOLTINE: Linked with geographic area within greater Europe eggs hibernate during winter cross-fertilize only by spring generates silk only once annually 2. BIVOLTINE: It is the second type and is normally found in China, Japan, and Korea. The breeding process: twice annually, resulting two life cycles. 3. POLYVOLTINE: Found in the tropics The eggs are laid by female moths and hatch within nine to 12 days, so the resulting type can have up to eight separate life cycles throughout the year TYPES OF MULBERRY SILKOWORM
- 5. GENOME Draft sequences- published in 2004 The full genome- published in 2008 by the International Silkworm Genome Consortium Genome size around 432 megabase pairs
- 7. SERICULTURE/SILKFARMING Sericulture, the practice of breeding silkworms for the production of raw silk, has been under way for at least 5,000 years in China, from where it spread to Korea and Japan, India and later the West. The silkworm was domesticated from the wild silkmoth, Bombyx mandarina Amongst species of silkworms, Bombyx mori is the most widely used. Silk is known as the “queen of textile” and “biosteel” because of its strength.
- 8. SERICULTURE
- 10. Solid and tough shell of silk nano-fibrils that surround and protect a centre of liquid cargo. can protect sensitive molecular materials tiny capsules (invisible to naked eyes) prove a significant technology in areas including food science, biotechnology and medicine. Specially-developed by microengineering process - combines the power of microfluidic manufacturing with the value of natural silk. The same technology could also be used in pharmaceuticals to treat a wide range of severe and debilitating illnesses. Increase the stability and lifetime of an antibody that acts on a protein implicated in neurodegenerative disease Tested the micrococoons with an antibody that has been developed to act on alpha- synuclein, the protein that is thought to malfunction at the start of the molecular process leading to Parkinson’s Disease. MICROCOCOONS
- 11. Silk micrococcons for protein stabilisation and molecular encapsulation Bright field microscopy images of Scale bar, 5 μm structures placed between crossed polarizers Scale bar, 10 μm 3D reconstructions of confocal images
- 12. Baculovirus Baculovirus are present in invertebrates primarily insect species. They are not infectious for vertebrates & plants. Genome: • is covalently closed circular double stranded (134 kbp) • due to its small size it can accommodate large fragments of foreign DNA They are divided into two groups on the basis of their structure as - Nucleopolyhedroviruses (NPV) Granuloviruses
- 13. Baculovirus expression system Recombinant baculovirus have become widely used as vectors to express heterologous genes in cultured insect cells and insects larvae. Baculovirus genome contains: Gene- encoding polyhedrin (viral protein) This accumulates inside the insect cell and major constituent of protein matrix The promoter of this gene is very strong, and this gene is of no use in the viral reproduction cycle. This gene or other non essential genes is replaced by with heterologous gene- Strategy used in Baculovirus expression system
- 14. Strong polyhedrin (polh) promoter results in high-level expression of target genes during the very late phase of infection AcNPV ORF 603 and ORF1629 regions flank the polh promoter and MCS for homologous recombination with baculoviral DNA and generation of recombinant baculovirus pUC origin of replication and β-lactamase gene (ampr for propagation and selection in bacterial cells
- 16. Utilizing a natural viral infection for silkworms (baculovirus), we leverage every cell in the silkworm’s body (excluding the head and abdominal cavity) to rapidly produce structurally complex and biologically active protein. Silkworm- baculovirus expression system: Universally recognized as a powerful and versatile system alternative to protein expressed in E. coli, yeast or other baculovirus expression systems. Production of proteins and used as a bioreactor Advantages of baculovirus expression system over E. coli improved solubility incorporate post- translational modifications higher yields for secreted proteins do not require aseptic conditions highly-dense disposable bioreactors
- 17. Production of proteins and used as a bioreactor
- 19. Protein production using transgenic silkworms Human type III procollagen and feline interferon were produced in cocoons using transgenic silkworms. Human μ-opioid receptor was expressed in the silk glands and fat bodies of transgenic silkworms.
- 20. Production of recombinant proteins using silkworm larvae and pupae and the improvement of silkworm expression systems by the development of the BmNPV bacmid
- 21. Baculovirus vector expression system (BCES) technology
- 22. Uninfected expresSF+ cells AcNPV infected expresSF+ cells
- 23. Prophylactic vaccines - for the prevention of infectious diseases •Influenza (seasonal and pandemic), Human papillomavirus, Emerging pathogens (SARS), Biodefense (Ebola) •Therapeutic vaccines Oncology (prostate and breast cancer), Diabetes •Gene therapy - using rAAV (recombinant adeno-associated virus) Muscular dystrophy, Genetically-based lipid disorders, CNS disorders (Alzheimer's and Parkinson's diseases) •Biologics Wound healing, Tissue repair, Antivirals (hepatitis C), Anti-inflammatory diseases/arthritis •Three BEVS-generated products have progressed through Phase III clinical development: Flublok - seasonal influenza vaccine; Protein Sciences Corporation Diamyd - therapeutic vaccine for type I diabetes; Diamyd Medical AB Glybera - gene therapy for lipoprotein lipase deficiency; uniQure APPLICATIONS
- 24. SERICIN It is a protein created by Bombyx mori in the production of silk FIBROIN: Structural centre of the silk SERICIN: Gum coating the fibres and allowing them to stick to each other Application of sericin (a silk protein) on alkali modified polyester using a crosslinking agent has been developed at IIT, New Delhi. Improved • Smoothness • Moisture retention • Wicking Made the fabric more suitable for medical and sport garments.
- 26. Silk based gels Silk based supermacroporous cryogel matrices, synthesised- at IIT, Kanpur Exploring their potential in cell-material interactions. Composite silk based biomaterials- applied for soft and hard tissue engineering applications. Nano-silk sericin based hydrogels prepared from silk industry waste sanitary napkins and baby diapers The scaffold of nano-fibroin were prepared as an ideal biopolymer for enzyme immobilisation. The dressing membrane made up of fibroin has been found to heal wound much faster.
- 27. Silk structure and material platforms. A) Silk fibroin is extracted from B. mori cocoons through an alkaline process. Silk fibers consist of repetitive β-sheet structures (hydrophobic domains) alternated with amorphous chains (hydrophilic regions). Aqueous silk solution can then be processed in a wide range of forms as hydrogels, fibers, and sponges. Sheet crystals, hydrophobic Amorphous chains, hydrophilic
- 28. Self-assembled nanoparticles prepared from Tasar Antherea mylitta Silk sericin Silks are fibrous proteins spun by a variety of species including silkworms and spiders. Sericin is removed before textile application and usually considered as a waste (Bombyx mori). This study expected that the wild silkworm sericin could be a new candidate polymer in biomedical application. Bioactive molecules can be attached to sericin by covalent bonding = bioconjugate. Sericin nanoparticles will be prepared by the attachment of polyethylene glycol (PEG). The sericin nanoparticles will be applied to cosmetic application for protecting active ingredients like retinol. Novel approach of using silk sericin as drug carrier to specific regions in the body where we can avoid immunogenic problems like in the GI tract.
Editor's Notes
- pecies that produce Tussar silk, a kind of wild silk, whaereas bombyx mori is domesticated silkworm Larvae of the Assam silkmoth: MUga
- In the study, the researchers successfully showed that silk micrococoons. , which has a range from northern India to northern China, Korea, Japan, and the far eastern regions of Russia. The domesticated silkworm derives from Chinese rather than Japanese or Korean stock. Silkworm micrococoons could be used in biotechnology and medicine. The process mimics on the microscale the way in which Bombyx mori silkworms spin the cocoons from which natural silk is harvested. Made by researchers from at the University of Cambridge with help from engineers from the University of Sheffield:
- The z-stack central cut images are shown in the inserts. Scale bar, 10 μm. NSF micrococoons:
- Heterologous genes placed under the transcriptional control of the strong polyhedrin promoter of the Autographa californica polyhedrosis virus (AcNPV) Based on site specific transposition of an expression cassette (pfast Bac with gene of interest) into a baculovirus shuttle vector (bacmid) Pol1,vCtah are the non essential genes
- for production of high quality proteins. And since the baculovirus does not infect humans, another advantage is there are no biohazards associated with its use for laboratory workers. Likewise, since silkworms are domesticated and the moths cannot fly, the biosafety risk is very low for the environment. Expression levels are 10- to 100-fold higher than using Bombix mori (silkworm) cell line or conventional insect cell lines (e.g., Sf cells)2. And only a small amount of recombinant baculovirus is needed Production of human α-interferon in silkworm larvae, using recombinant B. mori nucleopolyhedrovirus (BmNPV), the production of many proteins has been achieved.
- , which has a range from northern India to northern China, Korea, Japan, and the far eastern regions of Russia. The domesticated silkworm derives from Chinese rather than Japanese or Korean stock. Silkworm micrococoons could be used in biotechnology and medicine. The process mimics on the microscale the way in which Bombyx mori silkworms spin the cocoons from which natural silk is harvested. Made by researchers from at the University of Cambridge with help from engineers from the University of Sheffield: Step2 Co-transfectionThe recombinant transfer vector (recombinant plasmid) is co-transfected with the baculovirus DNA into the BmN cells. Recombinant viruses appear after six days in the media. Step3 Production of the Recombinant ProteinProCube™ uses a sterile syringe to manually inject a larva or pupa. ProCube™ harvests the hemolymph (larva) or tissue (pupa) after six days incubation. Then we filter and centrifuge the extract in order to obtain soluble fractions. After separation we purify our customer’s target protein using a variety of standard chromatography columns.
- Transgenic silkworms have also been used for recombinant protein production. To establish transgenic silkworms, two different systems, involving the use of an attenuated recombinant baculovirus or a piggy Bac transposon-derived vector, were adopted. A method combining the two systems was also established.
- re-engineer the baculovirus such that it programs the infected insect cells to generate large quantities of desired recombinant protein(s). Spodoptera frugiperda insect cell line, expresSF+ cells, to optimize protein production using our BEVS technology. These cells have been extensively characterized
- Our BEVS platform has been successfully used for the development of a wide range of vaccines and therapeutics:
- The synthesised materials were thoroughly characterised by scanning electron microscopy, microcomputer tomography, TF-IR, mechanical properties, in vitro and in vivo biocompatibility. at RV College of Engineering, Bangalore.
- Silk protein has two types of protein i.e fibroin and sericin. Our research goal is characterization of the wild silkworm sericin and further application of this biopolymer in the bio-medical fields.
- The Institute of Materials Research and Engineering (IMRE) in Singapore has developed a way to replace the traditional dying process necessary to make colored silk. A simple dietary change for the silkworm larva and they are able to produce silk in a variety of colors, with the color directly integrated into the fibers. The process designed by the researchers involves feeding a diet of mulberries treated with fluorescent dye to the silkworms in the last four days of the larva stage. Once the silkworms ingest the dye they then turn the color of the dye they were fed. The silkworms then spin their cocoon with the resulting color of the silk matching the dye they ingeste (Rhodamine B )