Team:UC Chile/Bactomithril

From 2012.igem.org

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== Overview ==
== Overview ==
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Spider dragline silk is an exceptionally strong and elastic fiber and is an ideal material for numerous biomedical and industrial applications. In this secondary project we developed a novel secretion system that allows to export proteins to the extracellular medium and tried to add the first spider-silk biobrick to the Registry. This biobrick would have the synthetic silk gene ADF-3 from the orb weaving spider Araneus diadematus. Nevertheless, due to the repetitive nature of the spider silk monomer we could not finish this biobrick for de Regional Jamboree (despite of several intents and strategies). </p>
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Spider dragline silk is an exceptionally strong and elastic fiber and is an ideal material for numerous biomedical and industrial applications. In this secondary project we developed a novel secretion system that allows to export proteins to the extracellular medium and tried to add the first spider-silk biobrick to the Registry. This biobrick would have the synthetic silk gene ADF-3 from the orb weaving spider Araneus diadematus. Nevertheless, due to the repetitive nature of the spider silk monomer we could not finish this biobrick for the Regional Jamboree (despite several attempts and strategies).
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<big> Animation describing the first secretion system </big>
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https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Animacion-1-secrecion-1_v2.gif
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<html>
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<center>
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<a href="https://static.igem.org/mediawiki/2012/3/3a/UC_Chile-Animacion-1-secrecion-1_v2(2).gif">Click here to open this animation from the begining in a new tab </a>
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</center>
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</html>
== Introduction ==
== Introduction ==
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Spider dragline silk is an exceptionally strong and elastic biomaterial and one of the strongest natural fibers known. It is five times stronger by weight than steel and three times tougher than the top quality man-made fiber Kevlar [1]. That’s why is referred to as “biosteel”. Furthermore, this material is biocompatible, biodegradable and has potential for processing in aqueous solution under ambient conditions.
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Spider dragline silk is an exceptionally strong and elastic biomaterial and one of the strongest natural fibers known. It is five times stronger by weight than steel and three times tougher than the top quality man-made fiber Kevlar [1]. That is why it is referred to as “biosteel”. Furthermore, this material is biocompatible, biodegradable and has potential for processing in aqueous solution under ambient conditions.
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== Construct: a novel secretion system ==
== Construct: a novel secretion system ==
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<big>           Working Plasmid </big>
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<big> Working Plasmid ([http://partsregistry.org/Part:BBa_K743011 BBa_K43011]) </big>  
[[File:UC_Chile-OmpA+HIV.png|center]]
[[File:UC_Chile-OmpA+HIV.png|center]]
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<center>This is an intermediate plasmid that we used to build the GFP Reporter Plasmid an the Protease Producer Plasmid.</center>
<center>This is an intermediate plasmid that we used to build the GFP Reporter Plasmid an the Protease Producer Plasmid.</center>
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<big> GFP Reporter Plasmid </big>
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<big> GFP Reporter Plasmid ([http://partsregistry.org/Part:BBa_K743012 BBa_K43012])</big>
[[File:UC_Chile_OmpA+HIV+GFP.png|center]]
[[File:UC_Chile_OmpA+HIV+GFP.png|center]]
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<center>The description of the mechanism of this construct is explained in the animation below.</center>
<center>The description of the mechanism of this construct is explained in the animation below.</center>
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<big> Protease Producer Plasmid </big>
 
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[[File:UC_Chile-OmpA+HIV+Protease.png|center]]
 
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<center>This construct induces the expression of the HIV protease.</center>
 
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<big> Animation describing the first secretion system </big>
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<big> Protease Producer Plasmid ([http://partsregistry.org/Part:BBa_K743013 BBa_K43013])</big>
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https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Animacion-1-secrecion-1_v2.gif
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[[File:UC_Chile-OmpA+HIV+Protease.png|center]]
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<html>
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<center>This construct induces the expression of the HIV protease.</center>
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<center>
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<a href="https://static.igem.org/mediawiki/2012/3/3a/UC_Chile-Animacion-1-secrecion-1_v2(2).gif">Click here to open this animation from the begining in a new tab </a>
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</center>
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</html>
 
== Notebook ==
== Notebook ==
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== Results==
== Results==
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<big> Experiment 1 </big>
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You can see detailed informarion of our results in  [https://2012.igem.org/Team:UC_Chile/bactoresults this section]. Nevertheless, we can briefly summarize the following: we know that sfGFP expresses in our e. coli, but we don’t know if it is attached to the external side of the membrane, or if the HIV-1 protease works as expected. As this secretion system seems to be failing, we chose to try other alternatives.
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We transformed E. Coli with our GFP Reporter Plasmid (B2) and with the Protease Producer Plasmid (B3). We did a little experiment with those bacteria.
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== Future Prospects: The construction of the first spider silk biobrick ==
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The following samples were prepared:
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B2
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We tried with a lot of patience (in SEVERAL attempts with many different strategies) to make a biobrick with the spider silk monomer ADF-3 or the sequence of DNA corresponding to ADF-3 and tags necessary to identify and export the protein with the Type 3 Secretion System of Salmonella (SPI 2). The original sequences are described in
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B2+
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Widmaier, D. M., Tullman-Ercek, D., Mirsky, E. a, Hill, R., Govindarajan, S., Minshull, J., & Voigt, C. a. (2009). Engineering the Salmonella type III secretion system to export spider silk monomers. Molecular systems biology, 5(309), 309. doi:10.1038/msb.2009.62
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B2+ & B3
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This sequence was kindly sent by Dr. Christopher Voigt to us.
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B2+ & supB3
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A detailed description of this work is in the [https://2012.igem.org/Team:UC_Chile/Bactomithril/notepad notebook section]. After tons of hours of hard work, when we finally obtained PCR amplified bands of the correct size, the digestion tests were positive, colony PCR were succesful and Gibson Assemblies seemed to work, we sent our final construct for sequencing and the results did not match with our designs. Probably this is due to the hihgly repetitive nature of the spider silk monomers.
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== References ==
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Nomenclature
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In this section you will find references about production and modelling of recombinant spider silk proteins.
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B2-  (coli transformed with GFP Reporter Plasmid)
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<big> Recommended literature </big>
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B2+ (coli transformed with GFP Reporter Plasmid, induced with Arabinose)
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Teulé, F., Cooper, A. R., Furin, W. a, Bittencourt, D., Rech, E. L., Brooks, A., & Lewis, R. V. (2009). A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nature protocols, 4(3), 341-55. doi:10.1038/nprot.2008.250
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B3+  (coli transformed with Protease Producer Plasmid, induced with Arabinose)
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Widhe, M., Johansson, J., Hedhammar, M., & Rising, A. (2012). Invited review current progress and limitations of spider silk for biomedical applications. Biopolymers, 97(6), 468-78. doi:10.1002/bip.21715
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supB3+ (supernatant of coli transformed with Protease Producer Plasmid induced with Arabinose)
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Widmaier, D. M., Tullman-Ercek, D., Mirsky, E. a, Hill, R., Govindarajan, S., Minshull, J., & Voigt, C. a. (2009). Engineering the Salmonella type III secretion system to export spider silk monomers. Molecular systems biology, 5(309), 309. doi:10.1038/msb.2009.62
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Xia, X.-X., Qian, Z.-G., Ki, C. S., Park, Y. H., Kaplan, D. L., & Lee, S. Y. (2010). Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proceedings of the National Academy of Sciences of the United States of America, 107(32), 14059-63. doi:10.1073/pnas.1003366107
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Then we observed each sample with an epifluorescence microscope. The results were promising. B2- did not express sfGFP, but B2+ did. Additionally, in both cases (B2+ & B3) and (B2+ & supB3) the fluorescence of sfGFP disappeared almost completely.
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<big> Complementary literature </big>
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[[File:UC_Chile_7_mayo_2012.jpg|center]]
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<big> Experiment 2 </big>
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An, B., Hinman, M. B., Holland, G. P., Yarger, J. L., & Lewis, R. V. (2011). Inducing β-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching. Biomacromolecules, 12(6), 2375-81. doi:10.1021/bm200463e
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We prepared for second time samples of B2+ & B3+ to observe in the epifluorescence microscope the effect of time in the samples.
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Askarieh, G., Hedhammar, M., Nordling, K., Saenz, A., Casals, C., Rising, A., Johansson, J., et al. (2010). Self-assembly of spider silk proteins is controlled by a pH-sensitive relay. Nature, 465(7295), 236-8. doi:10.1038/nature08962
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This time B2+ & B3+ presented much more fluorescence, and it persisted over time (at least 1 hour with no significant variation).
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Ayoub, N. a, Garb, J. E., Tinghitella, R. M., Collin, M. a, & Hayashi, C. Y. (2007). Blueprint for a high-performance biomaterial: full-length spider dragline silk genes. PloS one, 2(6), e514. doi:10.1371/journal.pone.0000514
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[[File:UC_Chile-17_mayo_2012_-_B2_C_UV_1.jpg|center||962px]]
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<big> Experiment 3 </big>
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Bauer, F., & Scheibel, T. (2012). Artificial egg stalks made of a recombinantly produced lacewing silk protein. Angewandte Chemie (International ed. in English), 51(26), 6521-4. doi:10.1002/anie.201200591
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We did another experiment to investigate the interactions between our bacteria B2+ and B3, but this time with a serious protocol and recording the data for later analysis.  
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Bayley, H. (1998). Puri ® cation and characterization of recombinant spider silk expressed in Escherichia coli. Cell, 1, 31-38.
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We expected that our results were coherent with our theory. [https://static.igem.org/mediawiki/2012/3/3a/UC_Chile-Animacion-1-secrecion-1_v2(2).gif]. Both HIV protease and protease K should be able to cut the HIV cleavage site, releasing the sfGFP.
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Bogush, V. G., Sokolova, O. S., Davydova, L. I., Klinov, D. V., Sidoruk, K. V., Esipova, N. G., Neretina, T. V., et al. (2009). A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 4(1), 17-27. doi:10.1007/s11481-008-9129-z
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The samples used this time were:
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Dams-Kozlowska, H., Majer, A., Tomasiewicz, P., Lozinska, J., Kaplan, D. L., & Mackiewicz, A. (2012). Purification and cytotoxicity of tag-free bioengineered spider silk proteins. Journal of biomedical materials research. Part A, 1-9. doi:10.1002/jbm.a.34353
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B2+ :
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Das, R., & Baker, D. (2008). Macromolecular modeling with rosetta. Annual review of biochemistry, 77, 363-82. doi:10.1146/annurev.biochem.77.062906.171838
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E. coli transformed with GFP Reporter Plasmid and induced with Arabinose
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Kenney, J. M., Knight, D., Wise, M. J., & Vollrath, F. (2002). Amyloidogenic nature of spider silk. European Journal of Biochemistry, 269(16), 4159-4163. doi:10.1046/j.1432-1033.2002.03112.x
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B2+ / SN B3+ :  
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Keten, S., & Buehler, M. J. (2010). Nanostructure and molecular mechanics of spider dragline silk protein assemblies. Journal of the Royal Society, Interface / the Royal Society, 7(53), 1709-21. doi:10.1098/rsif.2010.0149
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E. coli transformed with GFP Reporter Plasmid and induced with Arabinose, and Supernatant of coli transformed with Protease Producer Plasmid induced with Arabinose
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Lazaris, A., Arcidiacono, S., Huang, Y., Zhou, J.-F., Duguay, F., Chretien, N., Welsh, E. a, et al. (2002). Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science (New York, N.Y.), 295(5554), 472-6. doi:10.1126/science.1065780
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B2+ / SN B3- :
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Moisenovich, M. M., Pustovalova, O. L., Arhipova, a Y., Vasiljeva, T. V., Sokolova, O. S., Bogush, V. G., Debabov, V. G., et al. (2011). In vitro and in vivo biocompatibility studies of a recombinant analogue of spidroin 1 scaffolds. Journal of biomedical materials research. Part A, 96(1), 125-31. doi:10.1002/jbm.a.32968
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E. coli transformed with GFP Reporter Plasmid and induced with Arabinose, and Supernatant of coli transformed with Protease Producer Plasmid not induced with Arabinose
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Rising, A., Widhe, M., Johansson, J., & Hedhammar, M. (2011). Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications. Cellular and molecular life sciences : CMLS, 68(2), 169-84. doi:10.1007/s00018-010-0462-z
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Tobar, J. a, Carreño, L. J., Bueno, S. M., González, P. a, Mora, J. E., Quezada, S. a, & Kalergis, A. M. (2006). Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells. Infection and immunity, 74(11), 6438-48. doi:10.1128/IAI.00063-06
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B2+ / protease K :
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Vollrath, F. (1999). Biology of spider silk. International journal of biological macromolecules, 24(2-3), 81-8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10342751
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E. coli transformed with GFP Reporter Plasmid and induced with Arabinose, and protease K.
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Vollrath, F., & Knight, D. P. (2001). Liquid crystalline spinning of spider silk. Nature, 410(6828), 541-8. doi:10.1038/35069000
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Winkler, S., Szela, S., Avtges, P., Valluzzi, R., Kirschner, D. a, & Kaplan, D. (1999). Designing recombinant spider silk proteins to control assembly. International journal of biological macromolecules, 24(2-3), 265-70. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10342773
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Subsequently we proceeded to observe the samples in an epifluorescence microscope. In the following figure you can see one micrograph of each sample at 100x.
 
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[[File:UC_Chile2012-13_junio_bryon_2012.jpg|center]]
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<html>
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<a href="https://2012.igem.org/Team:UC_Chile/Results/Gibson"><img src="https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Continue_button.jpg" align="right">
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Additionally, we analyzed 4 micrographs of each sample (n=4) with the software ImageJ to compare their fluorescence intensity.
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</html>
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[[File:UC_Chile-Resultados_micrografias_Bryon2.png|center]]
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All the different treatments presented significant differences, except (B2+ & protease K) and (B2+). Remarkable is the difference between (B2+ / SN B3+) and (B2+ / NS B3-). Also, the direct application of protease K over the samples decreased even more the fluorescence. However, the low fluorescence of B2+ is very weird. We will have to repeat this experiment, but at least the major part of the results makes is what we expected.
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<big> Experiment 4 </big>
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We repeated the experiment to investigate the interactions between our bacteria B2+ and B3+ with a serious protocol and recording the data for later analysis. This time we added the control B2-.
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In the following figure you can see one micrograph of each sample at 100x obtained from a fluorescence microscope.
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[[File:UC_Chile15_junio_max_2012_UV%2C_light%2C_superpuestas%2C_4xlow.jpg|center|962px]]
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In the figure each row represents one sample. The first row contains the micragraphs with UV filter. The second row  contains the micrographs seen with light. The third row contains the previous micragraphs overlayed (the light image is in grayscale, and the UV image has its black background removed). The forth image is an amplification of the third image (1:4 ratio). Click over the image to open it in a higher resolution.
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Additionally, we analyzed 4 UV micrographs of each sample (n=4) with the software ImageJ
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[[File:UC_Chile-15_junio_max_2012_UV,_light,_superpuestas,_4xlow.jpg|center|962px]]
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The results were quite different compared to the ones of our previous experiment. Remarkable is that, as expected, B2- and B2+ present the lowest and highest fluorescence intensity. Additionally, (B2+ & B3+) and (B2+ & Protease K) present very similar fluorescence. However, for second time we obtained a weird result: (B2+ & B3+) and (B2+ & B3-) present the same amount of fluorescence.
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In conclusion, we know that sfGFP expresses in our e. coli. But we don’t know if it is attached to the external side of the membrane, or if the HIV-1 protease works as expected. As this secretion system seems to be failing, we chose to try other alternatives.
+
-
 
+
-
== The construction of the first spider silk biobrick ==
+
-
 
+
-
We tried with a lot of patience (in SEVERAL intents with many different strategies) to amplify the spider silk monomer ADF-3 and the the sequence of DNA corresponding to ADF-3 and tags necessary to identify and export the protein with the Type 3 Secretion System of Salmonella (SPI 2). The original sequences correspond to the described in
+
-
 
+
-
Widmaier, D. M., Tullman-Ercek, D., Mirsky, E. a, Hill, R., Govindarajan, S., Minshull, J., & Voigt, C. a. (2009). Engineering the Salmonella type III secretion system to export spider silk monomers. Molecular systems biology, 5(309), 309. doi:10.1038/msb.2009.62
+
-
 
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This sequence was kindly sent by Dr. Christopher Voigt to us.
+
-
 
+
-
A detailed description of this work is in the [https://2012.igem.org/Team:UC_Chile/Bactomithril/notepad notebook section]. After tons of hours of hard work, when finally PCR amplified bands of the correct size, the digestion tests were positive, Colony PCR were succesful, and Gibson Assemblies seemed to work, we sent our final sequence for sequencing, but the results did not match with our designs. Probably this is due to the hihgly repetitive nature of the spider silk monomers.  
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Latest revision as of 23:50, 23 October 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012