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Team:EPF-Lausanne/Our Project/T7 promoter variants
From 2011.igem.org
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One major challenge in designing new regulatory parts is to determine which combinations of transcription factors and binding sequences match. | One major challenge in designing new regulatory parts is to determine which combinations of transcription factors and binding sequences match. | ||
From previous research and our own MITOMI experiments, we know which DNA sequences TetR binds to, and which residues of tetR participate in binding, but we do not know how changing these residues will affect either binding affinity or specificity. | From previous research and our own MITOMI experiments, we know which DNA sequences TetR binds to, and which residues of tetR participate in binding, but we do not know how changing these residues will affect either binding affinity or specificity. | ||
| - | Molecular dynamics simulations and other theoretical approaches have not | + | Molecular dynamics simulations and other theoretical approaches have not come any closer to answering these questions. |
In short, we know too little about protein-DNA interaction to intelligently design transcription factors. | In short, we know too little about protein-DNA interaction to intelligently design transcription factors. | ||
| - | To make up for this, we present an experimental system to select valid binding pairs from many random tetR and pTet mutants, based on an inducible lysis gene. | + | To make up for this lack of knowledge, we present an experimental system to select valid binding pairs from many random tetR and pTet mutants, based on an inducible lysis gene. |
| - | The system | + | The system -- in a way a "survival of the weakest" -- is related to directed evolution. |
A lysis system based on the K112808 lysis device is indirectly activated by tetR. | A lysis system based on the K112808 lysis device is indirectly activated by tetR. | ||
Therefore, if in a given cell the tetR variant present can bind to the tetR promoter, the cell lyses and releases its DNA into the culture media. | Therefore, if in a given cell the tetR variant present can bind to the tetR promoter, the cell lyses and releases its DNA into the culture media. | ||
From there, DNA can be recovered and amplified, tranformed, or directly sequenced. | From there, DNA can be recovered and amplified, tranformed, or directly sequenced. | ||
| - | By design, this DNA codes for a combination of TF and promoter with high affinity | + | By design, this DNA codes for a combination of TF and promoter with high mutual affinity, and therefore almost directly yields a valid regulatory part. |
In this light, it is a useful component of our transcription factor development pipeline. | In this light, it is a useful component of our transcription factor development pipeline. | ||
| - | This is a direct and practical way of solving the problem of selecting high affinity pairs among the millions of combinations of transcription factors and promoters. | + | This is a direct and practical way of solving the problem of selecting high affinity pairs among the millions of possible combinations of transcription factors and promoters. |
It can be seen as a form of DNA-based information processing, and is therefore also a neat example of a problem more efficiently solved by non-conventional computation. | It can be seen as a form of DNA-based information processing, and is therefore also a neat example of a problem more efficiently solved by non-conventional computation. | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Revision as of 23:56, 21 September 2011
Lysis Selection System
Lysis selection system Main | Lysis Characterization | DNA Recovery | DNA Selection | T7 Promoter VariantsOne major challenge in designing new regulatory parts is to determine which combinations of transcription factors and binding sequences match. From previous research and our own MITOMI experiments, we know which DNA sequences TetR binds to, and which residues of tetR participate in binding, but we do not know how changing these residues will affect either binding affinity or specificity. Molecular dynamics simulations and other theoretical approaches have not come any closer to answering these questions. In short, we know too little about protein-DNA interaction to intelligently design transcription factors. To make up for this lack of knowledge, we present an experimental system to select valid binding pairs from many random tetR and pTet mutants, based on an inducible lysis gene.
The system -- in a way a "survival of the weakest" -- is related to directed evolution. A lysis system based on the K112808 lysis device is indirectly activated by tetR. Therefore, if in a given cell the tetR variant present can bind to the tetR promoter, the cell lyses and releases its DNA into the culture media. From there, DNA can be recovered and amplified, tranformed, or directly sequenced. By design, this DNA codes for a combination of TF and promoter with high mutual affinity, and therefore almost directly yields a valid regulatory part. In this light, it is a useful component of our transcription factor development pipeline.
This is a direct and practical way of solving the problem of selecting high affinity pairs among the millions of possible combinations of transcription factors and promoters. It can be seen as a form of DNA-based information processing, and is therefore also a neat example of a problem more efficiently solved by non-conventional computation.
