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Three Laws of Synthesis Biology

1).'''''Background''''': The Three Laws of Robotics, often shortened to The Three Laws or Three Laws, were written by science fictionauthor Isaac Asimov and later expanded upon. The rules are introduced in his 1942 short story "Runaround", although they were foreshadowed in a few earlier stories. 1.A robot may not injure a human being or, through inaction, allow a human being to come to harm. 2.A robot must obey any orders given to it by human beings, except where such orders would conflict with the First Law. 3.A robot must protect its own existence as long as such protection does not conflict with the First or Second Law. (from http://en.wikipedia.org/wiki/Three_Laws_of_Robotics) The Three Laws are still followed by robotor scientists worldwide today,and definatly pointted out the problems may arouse bewtten advanced robots and human. Similarly,Sythesis Biology is facing the same problem. If we were God,what rules should we follow so that we won't ruin the original perfect God-given world? A.what's the problems that "Three Laws of Synthesis Biology" need to deal with? First,there are reports like this "A 2004 study performed near an Oregon field trial for a genetically modified variety of creeping bentgrass (Agrostis stolonifera) revealed that the transgene and its associate trait (resistance to the glyphosate herbicide) could be transmitted by wind pollination to resident plants of differentAgrostis species, up to 14 km from the test field.So,'undesirable and uncontrolled gene flow into wild populations'is what we have to deal with to protect the wild type. Second,mutation is inavoidable,but when mutation accumulated into some grade,it may lead to a "Sythetic Killer Gene",that's why we need a system to inspect and destroy the mutation ones. Last,like the Killer Bees in South America,engineering bacteria may also cause calamitise in the nature world.So,as resposible researchers,restrictions are needed for engineering bacteria. Basied on the stiuation as above,we now give our type of 3 laws: B.'Three Laws of Synthesis Biology' 1.Once reconstructed plasmd is spread into any other non-aimed bacteria ,the suicide system for the plasmd is stared. 2.Once the mutation is accumulated into some degrees,the suicide system for the bacteria is stared. 3.Once the bacterium is away from the set enviroment,the suicide system is started. C.further explanation 1.The First Law is aimed at dealing with unexpected Gene Flow beteew engineering bacteria and the wild types. 2.The Second Law is aimed at unexpected mutations.Mutations,as we know,is avoidable and uncontrollable,thus we can now only reduce the uncertaincy which mutations bring. 3.The Thrid Law is aimed at the possibility of Biogical invasion caused by escaped bacteria.Because the bacteria with changed plasmid may have much much more advantages over the wild types. 4.It's easy to tell that 3 Laws need us to add more segment into the plasmid and will surely reduce the commercial value of the engineering bacterica or Gene Segments,however,we believe,only with D.Detail Constructions. In order to achieve the Three Laws,several systems and methods need to be used.Further more,only if these principles be admitted by labs and are set to be the basic rules of Synthetic works worldwide can we achieve our goal of "balance co-existence between God-given types and artificial types."That is,we need to construct a set a new standards for Sythetic biology,some parts more are needed for a standard biobrick or an experiment so that the Three Laws are not broken during all the process of construction. After some researches and discussions,we build systems that may fulfil as follows: 1.Double Plasmids Strategy(DPS): for all the systhetic works,scientists should use at least two plasmids for each artificial gene segement.(see figure 1) [https://static.igem.org/mediawiki/2011/6/67/3laws_photo1.png] a.overview: Plasmid A,B is created together for the Aimed Gene segment(the blue section).Both of them have suicide zymogen activator sequence,cuicide zymogen sequence as well as the restrain sequence for the other.Thus,as long as A&B were together,the plasmids can work as normal.Once one of the plasmid is spread into the wild,the suicide enzyme activator will start to translate and then activate the suicide zymogen,as the result,the plasmid would be destroyed In this way,the Law 1 would be fulfiled. b.further explanation: the reason why we use two segment to start the suicide process is that missing expression is unaviodable in any cell.In order reduce the impact of DPS towards normal cells,we add the segement of 'activitor'. c.questions: 1.what if the suicide parts mutated? 2.what if A&B was spread into one certain cell ? 3.What can be used as suicide parts?Is there some parts that can be used for a large scale of aimed gene? 4.What would be the proper ratio between plasmid A&B? 2).Inserting New Section a.backdrops: we found CRISPR/Cas System may be used as a method in DPS. CRISPR represents a family of DNA found in most archaeal (~90%) and bacterial (~40%) genomes .CRISPR,in combination with Cas proteins, forms the CRISPR/Cas systems. [https://static.igem.org/mediawiki/2011/f/f5/%E6%90%9C%E7%8B%97%E6%88%AA%E5%9B%BE_2011-07-14_23-03-48.png] Overview of the CRISPR/Cas mechanism of action. (A) Immunization process: After insertion of exogenous DNA from viruses or plasmids, a Cas complex recognizes foreign DNA and integrates a novel repeat-spacerunitattheleaderendoftheCRISPRlocus.(B) Immunity process: The CRISPR repeat-spacer array is transcribed into a pre-crRNA that is processed into mature crRNAs, which are subsequently used as a guide by a Cas complex to interfere with the corresponding invading nucleic acid. Repeats are represented as diamonds, spacers as rectangles, and the CRISPR leader is labeled L. b.model: All the biobricks should add a CRISPR proto-spacer in the front.The proto-spacer is protected normally.Once the plasmid was spread and obsorbed into any bacterium,the loci will be exporsed by the restriction endonuclease,thus will be recongnized by the CRISPR system and cleaned out. Put this model together with the DPS.Plasmid 'A' contains reconstructed gene and will be added a proto-spacer in the front,as suicide segement,normally,it will be restricted thus won't express or be recognized by the CRISPR/Cas system,however once 'A' is spread into the wild and absorbed by the bacteria,the activator will start and the proto-spacerit would be exposed by restroction endonuclease,thus would be destroyed by the CRISPR system. c.deficiency These viral particles have specifically mutated the proto-spacer (sequence within the invading nucleic acid that matches a CRISPR spacer), with a single point mutation that allows the viruses to overcome immunity. Alterniative strategies that allow viruses to escape CRISPR, such as suppressors that could interfere with crRNAs biogenesis or Cas machinery remain uncovered. d.Questions: 1.What if the proto-spacer mutates? 2.What if the CRISPR/Cas system works slower than the reconstructed plasmid and the product of the plasmid can influence the restriction endornuclease? 3.Is there any other system that we can use? 3).Bacteria:auxotraph bacteria should be used as the expectation of Law three. 4).mutation:no good ideas so far. Question: Is there any avilable systems or parts that can construct several pairs of standard plasmids as used in iGEM? 1. The Plamids pair should be stable and be able to used as biobrick. And users would only need to change the working parts according to their own work. 2.The sections should have little influence to most artifical parts.