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Team:Warsaw/Stage1/Background
From 2010.igem.org
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<div class="note"> Gene expression control freaks</div> | <div class="note"> Gene expression control freaks</div> | ||
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During the brainstorming period of our project we came up with many interesting ideas and we tried to find a way to squeeze all those ideas into one E. coli cell. It lead us to one general dilemma. How many different genes can we insert into our Coli at one time and how precisely regulate their dosage. We decides to use different RBS sequences to control gene expression. | During the brainstorming period of our project we came up with many interesting ideas and we tried to find a way to squeeze all those ideas into one E. coli cell. It lead us to one general dilemma. How many different genes can we insert into our Coli at one time and how precisely regulate their dosage. We decides to use different RBS sequences to control gene expression. | ||
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We knew from the registry that B0033 is weak and for strong expression we should use B0034, but also we have learned that those RBSes were measured in one setup with standard promoter and GFP reporter gene. We faced two general questions:</p> | We knew from the registry that B0033 is weak and for strong expression we should use B0034, but also we have learned that those RBSes were measured in one setup with standard promoter and GFP reporter gene. We faced two general questions:</p> | ||
<div class="note">1) Are those 5 measured RBSes in the registry enough? Does it make sense to to use any other RBSes?</div> | <div class="note">1) Are those 5 measured RBSes in the registry enough? Does it make sense to to use any other RBSes?</div> | ||
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<p>We went downstairs, ordered lots of coffee, switched on the laptops and asked google scholar for help. | <p>We went downstairs, ordered lots of coffee, switched on the laptops and asked google scholar for help. | ||
We came across a Nature paper about standardization of synthetic biological parts and devices []. The parts' evolutionary reliability measurement was particularly interesting for us. Paper suggest that after 74 culture doublings the measured part mutated and wasn't functional anymore. System failure resulted from deletion between repeated DNA sequences due to recombination. In that specific system it occurred because terminator B0015 was used 2 times.</p><p> | We came across a Nature paper about standardization of synthetic biological parts and devices []. The parts' evolutionary reliability measurement was particularly interesting for us. Paper suggest that after 74 culture doublings the measured part mutated and wasn't functional anymore. System failure resulted from deletion between repeated DNA sequences due to recombination. In that specific system it occurred because terminator B0015 was used 2 times.</p><p> | ||
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<div class="note">2) RBSes were measured with GFP reporter. Are these RBS measurements universal. Would RBS behave differently if we put our favorite gene instead of GFP? </div> | <div class="note">2) RBSes were measured with GFP reporter. Are these RBS measurements universal. Would RBS behave differently if we put our favorite gene instead of GFP? </div> | ||
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<p>A first hint on how RBS strength is regulated comes from 1990 Van Dunin et al. paper. They mutated RBS of a phage coat protein gene and came to the conclusion that the stronger the RBS secondary structure is, the weaker the gene expression is. This idea is further explored in later papers(). In our eyes it crushed the idea of RBS measurement simply because the secondary structure is influenced by the downstream sequence - the beginning of the gene used. Therefore RBS measured with GFP might behave differently in other genetic setups. | <p>A first hint on how RBS strength is regulated comes from 1990 Van Dunin et al. paper. They mutated RBS of a phage coat protein gene and came to the conclusion that the stronger the RBS secondary structure is, the weaker the gene expression is. This idea is further explored in later papers(). In our eyes it crushed the idea of RBS measurement simply because the secondary structure is influenced by the downstream sequence - the beginning of the gene used. Therefore RBS measured with GFP might behave differently in other genetic setups. | ||
</p><p>We read a bit more and decided to investigate it computationally. Curious to find out if RBS parts have a chance to behave similarly with different genes see our modeling. | </p><p>We read a bit more and decided to investigate it computationally. Curious to find out if RBS parts have a chance to behave similarly with different genes see our modeling. | ||
</p> | </p> | ||
Revision as of 20:53, 25 October 2010
Why iGEM Warsaw team spends summer measuring RBS parts and how can you predict RBS strength
During the brainstorming period of our project we came up with many interesting ideas and we tried to find a way to squeeze all those ideas into one E. coli cell. It lead us to one general dilemma. How many different genes can we insert into our Coli at one time and how precisely regulate their dosage. We decides to use different RBS sequences to control gene expression.
We knew from the registry that B0033 is weak and for strong expression we should use B0034, but also we have learned that those RBSes were measured in one setup with standard promoter and GFP reporter gene. We faced two general questions:
We went downstairs, ordered lots of coffee, switched on the laptops and asked google scholar for help. We came across a Nature paper about standardization of synthetic biological parts and devices []. The parts' evolutionary reliability measurement was particularly interesting for us. Paper suggest that after 74 culture doublings the measured part mutated and wasn't functional anymore. System failure resulted from deletion between repeated DNA sequences due to recombination. In that specific system it occurred because terminator B0015 was used 2 times.
This scenario would be probably repeated if we used the same RBS parts. However the experiment was performed in the recA+ strain - in other words a strain that is capable of homologous recombination. 2009 Warsaw team used B0032 many times to obtain moderate gene expression, because Top10 (recA-) strain was used there were no recombination events.
CONCLUSION: In the light of these evidence we wanted to refine our design and avoid repeated sequences to make our system portable. This is a problem if you want to use RBSes of similar strength, because there is seldom more than one characterized.
A first hint on how RBS strength is regulated comes from 1990 Van Dunin et al. paper. They mutated RBS of a phage coat protein gene and came to the conclusion that the stronger the RBS secondary structure is, the weaker the gene expression is. This idea is further explored in later papers(). In our eyes it crushed the idea of RBS measurement simply because the secondary structure is influenced by the downstream sequence - the beginning of the gene used. Therefore RBS measured with GFP might behave differently in other genetic setups.
We read a bit more and decided to investigate it computationally. Curious to find out if RBS parts have a chance to behave similarly with different genes see our modeling.
