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Revision as of 11:13, 18 September 2015
Orthogonal system:
Parallel and independent orthogonal system is one of the foundations of synthetic biology. This system has no or little cross with the natural biological system which is composed of unnatural base pair,shift code,unnatural amino acid,orthogonal aaRS(aminoacyl tRNA synthetase), orthogonal RNA synthetase,promoter and the orthogonal ribosome.These components work corporately or separately in the organism, programming cells with new functions, and also providing new techniques for researchers.
UAA Orthogonal genectical system：
1.What is UAA?
UAA is short for Unnatural Amino Acids.In most organisms,there are 20 kinds of amino acids in DNA encoding,which form the vast majority of proteins in the present life.Part of the ancient bacteria (Archaea) and (eubacteria) can encoding selenium (selenocysteine) or the pyrrolysine.The amino acids which do not exist in the natural protein sequence are called UAA.The unnatural amino acids used in the system must be stable, and can be stable in the cell.If the unnatural amino acids were inserted into the proteins expressed by the genetic system of encoding,the new physical and chemical properties of the proteins will expand the space of protein operation.
2.The UAA using in our system:Acetyllysine(ACK)
Acetyllysine(ACK) is one of unnatural amino acid and only corresponds to a special aminoacyl-tRNA(tRNA). It can be added to the protein by Semantic coding of termination codon(UAG).
3.Use UAA as a switch to start the translation of proteins:
To transmit the genetic information of mRNAs to proteins,after aa-tRNAs were synthesized by natural amino acids and tRNA via aaRS,they entered, and then entered the protein or peptide.To expand the genetic code and to lead new amino acids into the biosynthetic system,we need to identify the unnatural orthogonal codon,the tRNA with blank codon,the aaRS which can load UAA onto the tRNA specifically,the orthogonal mRNA,which can identify and highly efficient orthogonal translation,appropriate ribosome-binding site (RBS). These components need to satisfy the following conditions: (1) the only code must be recognized by the new tRNA, and has a link-coupled aaRS; (2) the aaRS/tRNA combination must be highly specific for unnatural amino acids in the host cells; (3) aaRs/tRNA pairs must be orthogonal differentiating endogenous aaRS/tRNA pairs(That is not cross in using).
About our Bladder Cancer Sniper：
1.Methodology
2.How the sniper was formed?
(1)The construction of Orthogonal system:
In our project,when AND gate operates the tRNA as well as AckRS are desperately synthesized.The certain tRNA can identify and combine terminations codon UAG. AckRS can achieve the attachment between unnatural amino acid Ack and the tRNA. Then the tRNA charged with unnatural amino acid Ack is able to specifically promote a certain protein translation. Eventually the certain mRNA can be translate successfully and output some effectors to regulate tumors growth, motility and apoptosis. The benefits of our UAA orthogonal system is that it can only working in our bladder while unable to interfere our regular genetic system.
Bladder cell specific promoters hUPII and hTERT are the receptors that respond to the endogenous transcription signals sent by bladder cancer cells. When two promoters are activated at the same time , the expression of downstream effector genes will start. HUPII and hTERT promoters only work in bladder cancer cells rather than normal cells or other tumor cells with its capability to identify and intervent bladder cancer cells selectively.The effector genes was mutated in amber mutation, where a kind of unnatural amino acid Acetyllysine(Ack) is added. This process enable the effector genes to continue the translation of protein.
(2)The combination of AND gate and Orthogonal system:
2015 SZU-iGEM constructed SV40(with Enhancer) and Rluc with one codon being amber mutated in the same plasmid. This plasmid, together with two other plasmids, are inserted into the cell. Only when the three plasmids work simultanuously can our orthogonal system behave its function, specifically recognise bladder cancer cells and kill them.Our unnatural amino acid orthogonal system consists of three devices(plasmids).
(1)hUPll+AckRS(BBa_K1722007):
hUPll is a bladder-cell specific promoter. when it's activated, AckRS, a tRNA synthetase, will be produced.
(2)hTERT+tRNA(BBa_K1722010)/ shTERT+tRNA(BBa_K1722011):
hTERT and shTERT are cancer-cell specific promoters. tRNA can be expressed out when the promoter is activated.
(3)SV40+Rluc(BBa_K1722012):
SV40 is a widely used strong promoter. Rluc is a reporter that can produce RLUC which is a kind of luciferase.
There is an amber stop codon UAG in the sequence of Rluc. The tRNA that is produced from the second plasmid has CUA as its anticodon, which can pair with the stop codon of the mRNA chain of Rluc. AckRS can achieve the attachment of Ack, the unnatural amino acid, and the tRNA. In this way, when all the three promoters are activated inside bladder cancer cell, Ack can be charged to the specific tRNA and then the anticodon of tRNA can pair with the stop codon on the mRNA chain of Rluc. In natural condition, this Rluc gene cannot be fully expressed because of the amber stop codon. However, with our orthogonal system, it can be produced and detected.
【Source】
[1]Mcelroy D, Brettell R. Foreign gene expression in transgenic cereals[J]. Trends, Biotechnol, 1994,12(2): 62-68
[2] Jongchan W, Matthew HH, Albrecht G. Structure-function studies on the active site of the coelenterazine-dependent luciferase from Renilla, Proteinscience, 17(10): 725-735
[3] P.A.V. Anderson, J.F. Case, Electrical activity associated with luminescence and other colonial behaviour in the pennatulid Renilla kollikeri, Biol. Bull. 149(1975): 80–95.
[4] Chang C. Liu and Peter G. Schultz,Adding new chemistries to the genetic code,The Scripps Research Institute, La Jolla, California 92037
[5]GE Yongbin, HONG Jiong, WANG Dongmei Orthogonal genetic system in synthetic biology Department of Physics and Chemistry, Bozhou Normal College, Bozhou 236800, China;School of Life Science, University of Science and Technology of China, Hefei 230026, China
[6]Appella DH. Non-natural nucleic acids for synthetic bi-ology. Curr Opin Chem Biol, 2009, 13: 687-696
[7]An W, Chin JW. Synthesis of orthogonal transcrip-tion-translation networks. Proc Natl Acad Sci USA, 2009, 106: 8477-8482
Placeholder text by Designer Ipsum.
Profile images are licensed under a Creative Commons BY-NC-SA 2.0 license. The images are from Greg Peverill-Conti's 1,000 faces project.
Web Design
Scenario archetype complementary responsive script pixel sidebar sitemap keep it simple. Complementary visuals footer CSS from alan cooper delightful. Photoshop iconography simplicity user experience affordance narrative ascenders contour. Slab serif interstitial skeuomorphism illustrator design by committee simplicity alan cooper eye tracking. Typography contrast mental model typesetting affordance narrative from CSS. Retina simplicity design by committee typography oblique.
Delightful ascenders contrast prototype. Ligature jakob nielsen user measure. Ligature contrast glyph rule of thirds composition interstitial dribbble. Mental model typography urbanized balance. Resolution rounded corners IDEO constraints dribbble persona. Portfolio sketch baseline 66-character line. Usability testing mental model simplicity aspect ratio pencil type. Usable sans-serif visualization leading prägnanz baseline pencil fireworks clarity omnigraffle.
Responsive
Card-sorting dropdown constraints alan cooper prägnanz iconography. Stakeholder balsamiq italic vector composition ethnography CSS monospace hierarchy. Eye tracking serif persona focus group.
Typesetting artifact rounded corners eye tracking braindump drawer menu mockup dropdown measure. Jony ive eye tracking script monospace heuristic tabs sketches. Affordance eye tracking scenario usable ligature typesetting clarity responsive. Information architecture golden ratio drawer menu header affordance aspect ratio. Balsamiq slab serif IDEO cognitive dissonance kerning pixel pixel-perfect. Pixel rounded corners header workflow portfolio.
Scenario navigation complementary jony ive helvetica archetype interstitial serif front-end. Constraints pencil usability gestalt design language mockup usability testing affordance jakob nielsen.
Prototype
Delightful ascenders contrast prototype. Ligature jakob nielsen user measure. Ligature contrast glyph rule of thirds composition interstitial dribbble. Mental model typography urbanized balance. Resolution rounded corners IDEO constraints dribbble persona. Portfolio sketch baseline 66-character line. Usability testing mental model simplicity aspect ratio pencil type. Usable sans-serif visualization leading prägnanz baseline pencil fireworks clarity omnigraffle.
Paper prototype urbanized headroom typography splash screen pencil modal branding. Retina omnigraffle objectified descender navigation adobe ethnography. Innovate design by committee modern hero message. Contrast user-centered color theory keep it simple visuals bevel adobe descender splash screen. From focus group accessibility sans-serif archetype pixel-perfect complementary skeuomorphism. Focus group iconography figure-ground navigation user-centered omnigraffle from.
Typography
Typesetting artifact rounded corners eye tracking braindump drawer menu mockup dropdown measure. Jony ive eye tracking script monospace heuristic tabs sketches. Affordance eye tracking scenario usable ligature typesetting clarity responsive. Information architecture golden ratio drawer menu header affordance aspect ratio. Balsamiq slab serif IDEO cognitive dissonance kerning pixel pixel-perfect. Pixel rounded corners header workflow portfolio.
Fireworks mobile skeuomorphism sitemap. Workflow iconography interaction design pixel-perfect serif. Mental model monospace typeface behavior change bauhaus from usability testing. Color theory user experience paper prototype narrative palette serif gradient header. Oblique modal 66-character line sketch responsive portfolio. Comic sans fireworks prägnanz monospace gradient design language jakob nielsen. Figure-ground pixel aspect ratio sketches rounded corners jony ive constraints mental model splash screen.
Placeholder text by Designer Ipsum.
Profile images are licensed under a Creative Commons BY-NC-SA 2.0 license. The images are from Greg Peverill-Conti's 1,000 faces project.