Team:SZU China/Design
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).
Fig.3 The UAA Orthogonal System in Synthetic Biology
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.
【Reference】
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Digital logic AND gate circuits:
As we all know, there many logic gates in digital circuits.Among them,AND gate is the most basic logic gate circuit.
It can be seen as a switch, but we need two or more keys to open it,and it's very according with our requirements.When we need multiple factors limit,AND gate is undoubtedly the best choice,because only all of our requirements are satisfied,the switch can be opened, and our system can run normally.
The AND gate of our project:
The AND gate of our system has two keys,and that is two promoters, hTERT and hUPll.The former is a cancer cell specific promoter,and the later is bladder cell specific promoter. When two keys exist at the same time and two promoters will start gene expression,the whole system can operate.Using AND gate to achieve specificity recognition,when it is compared with the traditional therapy,it is the biggest advantage of synthetic biology,and it’s also the basis of our project.
hRluc 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. When the system is verified having a function,it will produce an active hRluc.
【Reference】
[1].https://en.wikipedia.org/wiki/AND_gate[2].Mano,M.Morris and Charles R.Kime.Logic and Computer Design Fundamentals,Third Edition.Prentice Hall, (2004)
[3].Yuchen Liu,Yayue Zeng,Li Liu,Chengle Zhuang,Xing Fu,Weiren Huang& Zhiming Cai.Synthesizing AND gate genetic circuigts based on CRISPR-Cas9 for identification of bladder cancer cells.DOI:10.1038/ncomms6393(2014)
[4].Yoshida,W.&Yokobayashi,Y.Photonic Boolean logic gates based on DNA aptamers.Chem.Commun.(Camb.)14,195–197(2007)
[5].Wang,B.,Kitney,R.I.,Joly,N.&Buck,M.Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat.Commun. 2, 508 (2011).