Team:USTC-China/Safty

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<h1>Three Laws of Synthetic Biology</h1>
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<div id="textNot">
 
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<h1>Three Laws of  Synthesis Biology</h1>
 
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1).'''''Background''''':
 
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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|1 three laws]
 
-
 
 
-
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'
 
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1.Once reconstructed plasmd is spread into any other non-aimed bacteria ,the suicide system for the plasmd is stared.
 
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2.Once the mutation is accumulated into some degrees,the suicide system for the bacteria is stared.
 
-
 
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3.Once the bacterium is away from the set enviroment,the suicide system is started.
 
-
 
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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
 
-
 
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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)
 
-
 
-
[[File:3laws photo1.png|center]]
 
-
 
-
 
-
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.
 
-
[[File:搜狗截图 2011-07-14 23-03-48.png|center]]
 
-
 
-
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:
 
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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?
 
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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.
 
-
 
-
 
-
NEW:
 
'''1).Background:''' The Three Laws of Robotics, often shortened to The Three Laws or Three Laws, were written by science fiction author Isaac Asimov and later expanded upon. The rules were introduced in his 1942 short story "Runaround", although foreshadowed in earlier stories. This regulation goes like this: 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.  
'''1).Background:''' The Three Laws of Robotics, often shortened to The Three Laws or Three Laws, were written by science fiction author Isaac Asimov and later expanded upon. The rules were introduced in his 1942 short story "Runaround", although foreshadowed in earlier stories. This regulation goes like this: 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|1 three laws]
from [http://en.wikipedia.org/wiki/Three_Laws_of_Robotics|1 three laws]
The Three Laws are still followed by robot scientists worldwide today, which clearly foresaw the problems that might arise between advanced robots and humans.  
The Three Laws are still followed by robot scientists worldwide today, which clearly foresaw the problems that might arise between advanced robots and humans.  
Similarly, Synthetic Biology is faced with the same problem. If we were God, what rules should we follow so that we won't ruin the original perfect God-given world?  
Similarly, Synthetic Biology is faced with 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 are the problems that "Three Laws of Synthesis Biology" needs to deal with?  
+
 
 +
'''A. What are the problems that "Three Laws of Synthesis Biology" needs 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 (resistant to the glyphosate herbicide) could be transmitted by wind pollination to resident plants of different Agrostis 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 types.  
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 (resistant to the glyphosate herbicide) could be transmitted by wind pollination to resident plants of different Agrostis 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 types.  
Second, mutation is inevitable, but when mutation accumulated to a certain degree, it will generate a "Synthetic Killer Gene", that's why we need a system to inspect and then destroy the mutated ones.  
Second, mutation is inevitable, but when mutation accumulated to a certain degree, it will generate a "Synthetic Killer Gene", that's why we need a system to inspect and then destroy the mutated ones.  
Last, like the Killer Bees in South America, engineering bacteria may also cause catastrophes in nature. So we responsible researchers need to set up appropriate restrictions for engineering bacteria.  
Last, like the Killer Bees in South America, engineering bacteria may also cause catastrophes in nature. So we responsible researchers need to set up appropriate restrictions for engineering bacteria.  
Based on what have been discussed above we now give our type of Three Laws:
Based on what have been discussed above we now give our type of Three Laws:
-
B. 'Three Laws of Synthetic Biology'
+
 
 +
'''B. 'Three Laws of Synthetic Biology''''
 +
 
1. Once an altered plasmid gets into bacteria that does not aim at adopting the plasmid, the suicidal mechanism of the plasmid will be executed.
1. Once an altered plasmid gets into bacteria that does not aim at adopting the plasmid, the suicidal mechanism of the plasmid will be executed.
2. Once the mutation accumulates to a certain degree, the suicide mechanism of the bacteria will be executed.
2. Once the mutation accumulates to a certain degree, the suicide mechanism of the bacteria will be executed.
3. Once an infected bacterium is away from its regular surroundings, the suicide mechanism will be executed.
3. Once an infected bacterium is away from its regular surroundings, the suicide mechanism will be executed.
-
C. Further explanation  
+
 
 +
'''C. Further explanation'''
 +
 
1. The First Law is aimed at dealing with unexpected Gene Flow between engineering bacteria and wild types.  
1. The First Law is aimed at dealing with unexpected Gene Flow between engineering bacteria and wild types.  
2. The Second Law is aimed at unexpected mutations. Mutations, as we know, is inevitable and uncontrollable, thus we can now only reduce the uncertainty mutations bring about.  
2. The Second Law is aimed at unexpected mutations. Mutations, as we know, is inevitable and uncontrollable, thus we can now only reduce the uncertainty mutations bring about.  
3. The Third Law is aimed at the possibility of biological invasion caused by escaped bacteria. Because the bacteria with changed plasmid may have much more advantages over the wild types.  
3. The Third Law is aimed at the possibility of biological invasion caused by escaped bacteria. Because the bacteria with changed plasmid may have much more advantages over the wild types.  
4.It's easy to tell that to carry out the Three Laws we need to add more segments into the plasmid and this regulation will surely reduce the commercial value of engineering bacteria or Gene Segments, However, we believe only with great responsibility comes great power.  
4.It's easy to tell that to carry out the Three Laws we need to add more segments into the plasmid and this regulation will surely reduce the commercial value of engineering bacteria or Gene Segments, However, we believe only with great responsibility comes great power.  
-
D. Detailed Constructions. In order to achieve the Three Laws, several systems and methods need to be exploited. Furthermore, only if these principles be admitted by institutions and are set to be the basic rules of synthetic works worldwide can we achieve our goal of "balanced co-existence between God-given types and artificial types." That is, we need to construct new standards for synthetic biology, more parts are needed for a standard biobrick or an experiment so that the Three Laws will not be violated during all the process of construction. After some researches and discussions, we build systems that may fulfill our demands: 1.Double Plasmids Strategy (DPS): for all the synthetic works, scientists should use at least two plasmids for each artificial gene segment. (see figure 1)  
+
 
 +
'''D. Detailed Constructions.'''
 +
 
 +
In order to achieve the Three Laws, several systems and methods need to be exploited. Furthermore, only if these principles be admitted by institutions and are set to be the basic rules of synthetic works worldwide can we achieve our goal of "balanced co-existence between God-given types and artificial types." That is, we need to construct new standards for synthetic biology, more parts are needed for a standard biobrick or an experiment so that the Three Laws will not be violated during all the process of construction. After some researches and discussions, we build systems that may fulfill our demands: 1.Double Plasmids Strategy (DPS): for all the synthetic works, scientists should use at least two plasmids for each artificial gene segment. (see figure 1)  
[[File:3laws photo1.png|center]]
[[File:3laws photo1.png|center]]
-
a. Overview: Plasmid A,B is created together for the aimed gene segment(the blue section).Both of them have suicidal zymogen activator sequence, suicidal zymogen sequence as well as the restrain sequence for the other. Thus, as long as A and B were together, the plasmids will work regularly. Once one of the plasmids spread into the wild, the suicidal enzyme activator will start to translate and then activate the suicidal zymogen, as a result, the plasmid would be destroyed. In this way, Law 1 would be fulfilled.  
+
 
-
b. Further explanation: the reason why we use two segments to start the suicidal process is that missing expression is inevitable in any cell. In order to reduce the impact of DPS towards normal cells, we add the segment of 'activator'.
+
a. Overview:  
 +
 
 +
Plasmid A,B is created together for the aimed gene segment(the blue section).Both of them have suicidal zymogen activator sequence, suicidal zymogen sequence as well as the restrain sequence for the other. Thus, as long as A and B were together, the plasmids will work regularly. Once one of the plasmids spread into the wild, the suicidal enzyme activator will start to translate and then activate the suicidal zymogen, as a result, the plasmid would be destroyed. In this way, Law 1 would be fulfilled.  
 +
 
 +
b. Further explanation:  
 +
 
 +
the reason why we use two segments to start the suicidal process is that missing expression is inevitable in any cell. In order to reduce the impact of DPS towards normal cells, we add the segment of 'activator'.
c. Questions: 1.What if the suicidal parts mutated? 2. What if A&B were spread into one certain cell? 3. What can be used as suicidal parts? Are there some parts that can be used for a large scale of aimed genes? 4. What would be the proper ratio between plasmid A&B?  
c. Questions: 1.What if the suicidal parts mutated? 2. What if A&B were spread into one certain cell? 3. What can be used as suicidal parts? Are there some parts that can be used for a large scale of aimed genes? 4. What would be the proper ratio between plasmid A&B?  
-
2).Inserting New Section  
+
'''2).Inserting New Section'''
-
a. Backdrop: we found CRISPR/Cas System may be used as a method in DPS. CRISPR represents a series of DNA found in most archaeal (~90%) and bacterial (~40%) genomes .CRISPR, in combination with Cas proteins, forms the CRISPR/Cas system.  
+
 +
'''a. Backdrop:'''
 +
 
 +
we found CRISPR/Cas System may be used as a method in DPS. CRISPR represents a series of DNA found in most archaeal (~90%) and bacterial (~40%) genomes .CRISPR, in combination with Cas proteins, forms the CRISPR/Cas system.  
[[File:搜狗截图 2011-07-14 23-03-48.png|center]]
[[File:搜狗截图 2011-07-14 23-03-48.png|center]]
Line 127: Line 55:
(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) 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. Modeling: All the biobricks should be added with a CRISPR proto-spacer in the front. The proto-spacer is protected normally. Once the plasmid was spread and absorbed into any bacteria, the loci will be exposed by the restriction endonuclease, thus will be recognized by the CRISPR system and cleaned out. This model is then put together with the DPS. Plasmid 'A' contains reconstructed gene and will be added with a proto-spacer in the front, as suicidal segment, normally, 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 be launched and the proto-spacer would be exposed by restriction endonuclease, thus would be destroyed by the CRISPR system.
+
'''b. Modeling:'''  
-
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 mutation that allows the viruses to overcome immunity. Alternative 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 endonuclease? 3.Is there any other system that we can use? 3).Bacteria: auxotroph bacteria should be used as the expectation of Law three. 4).Mutation: no good ideas so far.
+
-
Question: Is there any available systems or parts that can construct several pairs of standard plasmids as used in iGEM? 1. The Plasmids pair should be stable and be able to be used as a 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 artificial parts.
+
All the biobricks should be added with a CRISPR proto-spacer in the front. The proto-spacer is protected normally. Once the plasmid was spread and absorbed into any bacteria, the loci will be exposed by the restriction endonuclease, thus will be recognized by the CRISPR system and cleaned out. This model is then put together with the DPS. Plasmid 'A' contains reconstructed gene and will be added with a proto-spacer in the front, as suicidal segment, normally, 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 be launched and the proto-spacer would be exposed by restriction 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 mutation that allows the viruses to overcome immunity. Alternative 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 endonuclease? 3.Is there any other system that we can use? 3).Bacteria: auxotroph bacteria should be used as the expectation of Law three. 4).Mutation: no good ideas so far.
-
</div>
+
'''''Question:''''' Is there any available systems or parts that can construct several pairs of standard plasmids as used in iGEM? 1. The Plasmids pair should be stable and be able to be used as a 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 artificial parts.

Latest revision as of 14:47, 20 September 2011

Three Laws of Synthetic Biology

1).Background: The Three Laws of Robotics, often shortened to The Three Laws or Three Laws, were written by science fiction author Isaac Asimov and later expanded upon. The rules were introduced in his 1942 short story "Runaround", although foreshadowed in earlier stories. This regulation goes like this: 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|1 three laws] The Three Laws are still followed by robot scientists worldwide today, which clearly foresaw the problems that might arise between advanced robots and humans. Similarly, Synthetic Biology is faced with 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 are the problems that "Three Laws of Synthesis Biology" needs 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 (resistant to the glyphosate herbicide) could be transmitted by wind pollination to resident plants of different Agrostis 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 types. Second, mutation is inevitable, but when mutation accumulated to a certain degree, it will generate a "Synthetic Killer Gene", that's why we need a system to inspect and then destroy the mutated ones. Last, like the Killer Bees in South America, engineering bacteria may also cause catastrophes in nature. So we responsible researchers need to set up appropriate restrictions for engineering bacteria. Based on what have been discussed above we now give our type of Three Laws:

B. 'Three Laws of Synthetic Biology'

1. Once an altered plasmid gets into bacteria that does not aim at adopting the plasmid, the suicidal mechanism of the plasmid will be executed. 2. Once the mutation accumulates to a certain degree, the suicide mechanism of the bacteria will be executed. 3. Once an infected bacterium is away from its regular surroundings, the suicide mechanism will be executed.

C. Further explanation

1. The First Law is aimed at dealing with unexpected Gene Flow between engineering bacteria and wild types. 2. The Second Law is aimed at unexpected mutations. Mutations, as we know, is inevitable and uncontrollable, thus we can now only reduce the uncertainty mutations bring about. 3. The Third Law is aimed at the possibility of biological invasion caused by escaped bacteria. Because the bacteria with changed plasmid may have much more advantages over the wild types. 4.It's easy to tell that to carry out the Three Laws we need to add more segments into the plasmid and this regulation will surely reduce the commercial value of engineering bacteria or Gene Segments, However, we believe only with great responsibility comes great power.

D. Detailed Constructions.

In order to achieve the Three Laws, several systems and methods need to be exploited. Furthermore, only if these principles be admitted by institutions and are set to be the basic rules of synthetic works worldwide can we achieve our goal of "balanced co-existence between God-given types and artificial types." That is, we need to construct new standards for synthetic biology, more parts are needed for a standard biobrick or an experiment so that the Three Laws will not be violated during all the process of construction. After some researches and discussions, we build systems that may fulfill our demands: 1.Double Plasmids Strategy (DPS): for all the synthetic works, scientists should use at least two plasmids for each artificial gene segment. (see figure 1)

3laws photo1.png

a. Overview:

Plasmid A,B is created together for the aimed gene segment(the blue section).Both of them have suicidal zymogen activator sequence, suicidal zymogen sequence as well as the restrain sequence for the other. Thus, as long as A and B were together, the plasmids will work regularly. Once one of the plasmids spread into the wild, the suicidal enzyme activator will start to translate and then activate the suicidal zymogen, as a result, the plasmid would be destroyed. In this way, Law 1 would be fulfilled.

b. Further explanation:

the reason why we use two segments to start the suicidal process is that missing expression is inevitable in any cell. In order to reduce the impact of DPS towards normal cells, we add the segment of 'activator'. c. Questions: 1.What if the suicidal parts mutated? 2. What if A&B were spread into one certain cell? 3. What can be used as suicidal parts? Are there some parts that can be used for a large scale of aimed genes? 4. What would be the proper ratio between plasmid A&B?

2).Inserting New Section

a. Backdrop:

we found CRISPR/Cas System may be used as a method in DPS. CRISPR represents a series of DNA found in most archaeal (~90%) and bacterial (~40%) genomes .CRISPR, in combination with Cas proteins, forms the CRISPR/Cas system.

搜狗截图 2011-07-14 23-03-48.png

Overview of the CRISPR/Cas mechanism (A) Immunization process: After the insertion of exogenous DNA from viruses or plasmids, a Cas complex recognizes foreign DNA and integrates a novel repeat-spacer unit at the leader end of the CRISPR locus. (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. Modeling:

All the biobricks should be added with a CRISPR proto-spacer in the front. The proto-spacer is protected normally. Once the plasmid was spread and absorbed into any bacteria, the loci will be exposed by the restriction endonuclease, thus will be recognized by the CRISPR system and cleaned out. This model is then put together with the DPS. Plasmid 'A' contains reconstructed gene and will be added with a proto-spacer in the front, as suicidal segment, normally, 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 be launched and the proto-spacer would be exposed by restriction 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 mutation that allows the viruses to overcome immunity. Alternative 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 endonuclease? 3.Is there any other system that we can use? 3).Bacteria: auxotroph bacteria should be used as the expectation of Law three. 4).Mutation: no good ideas so far.

Question: Is there any available systems or parts that can construct several pairs of standard plasmids as used in iGEM? 1. The Plasmids pair should be stable and be able to be used as a 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 artificial parts.