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Team:Warsaw/Stage1/Background
From 2010.igem.org
Why iGEM Warsaw team spends summer measuring RBS parts and how can you predict RBS strength
During the brainstorm 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 abut standardization of synthetic biological parts and devices []. The parts' evolutionary reliability measurement was particularly interesting for us. Paper suggest that after 74 culture doubling 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.
