USTC/OperatorComposition

From 2007.igem.org

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[[Image:USTC_OperatorCompostions.png|thumb|right|400px]]
[[Image:USTC_OperatorCompostions.png|thumb|right|400px]]
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Generally, lac repressors bind to their operators in the form of dimers. Almost all the  
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Generally, Lac repressor bind to its operator in the form of dimer. Almost all the according operators in nature are of approximate reverse symmetric structure, that is,  each half of the operators consists of about 10 nucleotides and receives the binding of a repressor monomer. It has been reported that the perfectly symmetric operator binds native Lac repressor most tightly. Based on this fact, an idea was proposed that the binding affinity of a non-symmetric operator may be able to be systematically reduced, exempt from the harm on the binding specificity of the repressor-operator pairs.
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according operators in nature are of approximate reverse symmetric structure, that is,  each half of the operators consists of about 10 nucleotides and receives the binding of a repressor monomer. Based on this fact, we were to construct a non-symmetric operator to systematically reduce the binding affinity which was exempt from the harm on the binding specificity of the repressor-operator pairs.
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Five promoters have been synthesized and tested:
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* O11, the perfectly symmetric operator
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* O1wt1, the native lac operator at +11 site of Plac promoter
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* O16, the right half of O11 replaced with a sequence which binds Lac repressor weakly, but has the same pattern as O11.
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* O17, the right half of O11 replaced with a random sequence
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* NUL, the positive control
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More concretely, we firstly preserved half of the original sequence of the operators, and rebuild another half of the operators with a random sequence or some sequences that were not involved in any other repressor-operator pairs in our whole system. With this idea we came to construct Operator 1-6 and 1-7. 1-6 means its left part is of Sequence 1 and its right part Sequence 6 which is reported in literature. Similarly, Operator 1-7 means its left part is of Sequence 1 while its right part Sequence 7 which is picked from a library of random sequences and which does not have anything to do with the lac repressors. 
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After a series of experiments, we came to the conclusion that the method of substituting the right half of the symmetric operator can systematically reduce the binding affinity. Ox6, which means the right half of a symmetric operator is substituted with a weak operator sequence with the same pattern, can be used as a "weak" operator. At the same time, we found that the pattern Ox7 is weaker than Ox6, and [https://2007.igem.org/USTC/FailureOfNANDv2b the affinity of Ox7 is too low for a logic gate].
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After a series of experiments, we came to the conclusion that the method of substituting the right half for Sequence 6 did achieve our goals of systematically impairing the binding affinity without influencing its binding specificity. However, at the same time, we found that the operator 1-7 we rebuilt has too weak the affinity to get bound by its repressor.
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Based on these experimental results, we hypothesized that the bending ability is essential to form repression effect. For more detailed information about Operator 1-7, please refer to [[USTC/Further_More|Further More]].
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Revision as of 09:13, 26 October 2007

USTC OperatorCompostions.png

Generally, Lac repressor bind to its operator in the form of dimer. Almost all the according operators in nature are of approximate reverse symmetric structure, that is, each half of the operators consists of about 10 nucleotides and receives the binding of a repressor monomer. It has been reported that the perfectly symmetric operator binds native Lac repressor most tightly. Based on this fact, an idea was proposed that the binding affinity of a non-symmetric operator may be able to be systematically reduced, exempt from the harm on the binding specificity of the repressor-operator pairs.

Five promoters have been synthesized and tested:

  • O11, the perfectly symmetric operator
  • O1wt1, the native lac operator at +11 site of Plac promoter
  • O16, the right half of O11 replaced with a sequence which binds Lac repressor weakly, but has the same pattern as O11.
  • O17, the right half of O11 replaced with a random sequence
  • NUL, the positive control

After a series of experiments, we came to the conclusion that the method of substituting the right half of the symmetric operator can systematically reduce the binding affinity. Ox6, which means the right half of a symmetric operator is substituted with a weak operator sequence with the same pattern, can be used as a "weak" operator. At the same time, we found that the pattern Ox7 is weaker than Ox6, and the affinity of Ox7 is too low for a logic gate.

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