Nephila clavipes is a kind of golden silk spider which is typical with most spiders.

It lives in mny warmer zones of America. It can be distinguished by large size and bright colour of the species.The male is much more smaller than the female. The mature female can make web that reach in one- meter width. The silk of N. clavipes has been used recently for promoting to help regeneration of mammalian neuronal. The N.clavipes silk is both high tensile and prominently elasticity. Therefore, it can be suggested that using this source of material for industrial and medical objectives is very helpful. The nature of the silk protein has been attempted several times for designing synthetic genes. The silk protein has been expressed into microorganisms. However, there is a real difficulty in bacteria production because of recombination from highly repetitive genes which encode the repetitively composed spider silk protein (spidorin) is genetic instability. As a result, tobacco and potato have been chosen for the production of spidorins because these plants have been successful for the production of genetic transformation products in several cases. In this approach, they carried out to design the synthetic spider silk genes to match the N.clavipes MaSp1 as an DNA complementary, i.e .we did assembly all synthetic spider silk genes. They build up a set of spidorins gene which code for various size proteins. Subsequently, these proteins were essential to determine as well as check the plant system expression and provide a panel for the optimization of downstream processing and purification. The fiborin fragment which constructed of gene encode the fusion protein FA2 is a synthetic homolog of a segment of the gene comes from fiborine heavy chains of Bomby mori, a kind of silkworm moth. After that, the synthetic spider silk genes were done by cloning into an vector which allow for expression of genetic transformation plants through cauliflower mosaic virus(CaMV) 35S promoter performance .In addition, Western blotting was also used to detect transgenic products. Then, the expression cassettles were cloned into vectors for transformation. Next, Agrobacterium tumefaciens was required to use for plasmids transformation. After transforming to tobacco and potato leaves, kanamycin-resistant plans were collected completely. They need to be tested newly expressed traits by transferring to soil. We determine the synthetic silk collection in potato and tobacco leaves. Normal growth and morphology were shown by all the first and second generation

transgenic plants. The general feasibility was proven via these outcomes. Using the Western blotting ,we compare tagged recombinant silk protein bands to standards of recombinant antibody molecules within the same intervals. Therefore, the protein amount was evaluated. The accumulation level is independent on size. By analyzing the WESTERN blotting, as indicated within the clear-cut bands, the protein tends to be more stable in leaves and tubers as well. The results show that there are the different cops could plants considered as cheap bioreactors within a large-scale spider silk protein product. Eventually, it should be done by purifying strategy for the synthetic spidorins from the tobacco and potato leaves. In other worlds, researchers had to test the solubility and heat stability of the synthetic spiroids after making purification. We have figured out that by utilizing the staining procedure, the recombinant silk proteins are in difficulty of detecting in crude extraction. Only a little amount could be recognized after the heating up process. By a great number of methods such as heat treatment, precipitating salt as well as acidification are in association with together,we would be able to obtain a considerable amount of spider silk proteins. Biological materials from naturally renewable sources may be responsible for alternatives petrol-based basic materials. Spider silk proteins can be the raw ingredients for fibers, foils manufacture and helpful for both technology and medicine. For instance, some of products such as textiles, and some future application would be applied like system for wound closure and tissue engineering scaffolds. Bioreactors of employing plant materials should boost up the level of production of spidorins. To provide the higher amount of protein, the researchers should suggest, introduce and extend expression to other crops as well as other plant organs. Another either difficulty or limitation of this approach to such material is the lack of suitable strategies to turn the raw material into produceable intermediate products. The spidroids in large-scale level in plants would create new chances to face with such this source in daily products. With respect of discussion, in addition to employ the N.clavipes, many researcher found out in addtion to use trangenic plants to produce the spider silk proteins, it can be suggested that the spiroids can also be produced via lactation(Frederick T. W, Norman E. V).The mammaliant cell cultured are low yield and high cost. On the other hand,production of spider silks within transgenic animals would provide a cost-effective way for their production.The similarity between mammary gland

and spider silk gland are more interesting at the cellular level.The approach was relevant for introducing genes of spider silk into a mouse genome and express these gene under the conditions of promoter.Transgenic mices generated by pronuclear microinjection into the pronucleid of eggs.They carried out to mate the founder mice together with non-trangeni c mice in oder to genetrate and then,the subsequent generations are generated.By using the basic molecular biotech,the gene sequence was proven that these were stabllity.These results demonstrated that the silk genes could be maintained stably in the genome within at least 3 generations.The products from non-trangenic and transgenic mouse milk were isolated completely under the specific conditions and compared to culture of cell from silk protein by Western blotting with the particular antibodies.Two key proteins were determined.Additionally,these results also demonstrated the feasibility of spider silk expression in the milk of trangenic plants.By employing the recombinant spider silk protein ,it would bring a great number of favorable conditions. In the first place,the production of spider silk is promised limitedless.They are industrial level of dairy industries.The economic value and production degree are fully known in the case of whether produced in milk goats or cows.The milk based on silk manufacture also speed up dowstream the silk process.For transgenic animals,the recombinant silk protein is still in milk solution.With a method of two steps, the silk protein sufficiency is obtained to get spinning.The first step is relevant to the removal of all milk fat.Subsequently,the silk protein need to be treated by methods of chromatographic. Moreover, it should be paid attention to The modular natureof spider silk proteins has led to several attempts to design synthetic genes (Gosiline et al,.1999) and Fanestock S.R(1996) suggested that experiments with high molecules weigh silk gene Escherichia coli and Pichia pastoris resulted also in the synthesis of truncated protein like a restricting element for bioengineering of spider silk. High solubility of recombinant silk is a popular feature because its spine dope is much more concentrated in preparation,or put another way,more protein and less water. In a similar recent publication, Lazaris et al,.(2002) claimed the successful one in culturing the mammalian cell of spiroids in size from 60 to 140 kDa. Lewis(1992) also reported that it had to be put a lot of efforts to express the cloning of natural spider silk proteins in microorganism but the production of amount of sufficient for structure of protein has not been reported. One aim of this and the same

research is to contribute experimental matters with the goal at having much deeper profound knowledge and understanding the structural language of protein polymers. In conclusion, the development of employing the transgenic plants such as potato and tobacco has been successful and brought many best achievements for the biotechnological field. A solid knowledge for this field would aid for us to use effectively this spider silk protein as high quality-evaluated product. Rerences 1. Rohit, S.G., Prateek,K. (2000) Spiders silk: Investigation of spinning process, web material and its properties. Biological Science and Bioengineering 2. Janaki Krishna, P.S. (2006) Transgenic Plants for Spider Silk-like Protein Production. ISP New Report. 3. Saravanan, D.(2006) Spider silk-structure, properties and spinning. Journal of textile and apparel technology and management ,Volume 5,1-20 4.Lutz,S. and Uwe,T.B.(2009) Protein Engineering Handbook 1 5. Rashid, A.(2009) Introduction to genetic engineering of crops in plan 6.Alberth, G.A.,Charles, H.M.,Chitaranjan,K.,Timothy, C.H.(2010) Transgenic crop plants :Utilization and Biosafety ,Volume 2 7.Frederik, T.W.,Normal,W.(2004) Natural fibers,Plastic and Composites. 8.Dennis, W.R.(2002) Introduction to molecular protein, 3RD Edition 9.Michael,B.,Mathilde, B.(2011) Governing risk in GM agriculture.Cambridge University Press, pp 30-35

Molecular Pharming (C123P3)

NAME: NGUYEN LE QUYEN

ID: 007754

TITLE: CRITIQUE FOR PRODUCTION OF SPIDER SILK PROTEIN IN TOBACCO AND POTATO

SUBMITTED: 13TH DECEMBER

TO: MS SANDY LOH