Team:Minnesota/Project

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We also examined 4 mutations in the Tet operator site for each construct.
We also examined 4 mutations in the Tet operator site for each construct.
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<h2>Methods</h2>

Revision as of 17:47, 7 August 2009

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Contents

The Project

AND Gates

How it works

ANDgatetable.jpg
AND Gates are logic circuits that produce an output if and only if two inputs are present. For example, our system produces GFP only if two small molecules named aTc and IPTG are present. If one of them is not present, no GFP will be produced. Ideally an AND gate will obey the truth table shown to the right, where 1 means the input is present, and 0 means that it is not.

In order to get this behavior, we take advantage of the structure of promoter sequences in prokaryotes. When RNA polymerase(more specifically the sigma factor) binds to the promoter, it binds to region at -35 and -10 relative to the transcriptional start site. Since the entire sequence is not used by the sigma factor, there are three regions of DNA that can be changed to provide AND gate behavior. These are the upstream of -35, between -35 and -10, and downstream of -10. In each of these sites, we can place a DNA sequence that is preferentially bound by proteins in the cell. For this case, the two proteins are TetR and LacI, both of which are constitutively produced by our strain of E. coli(DH5-alpha-pro). In each of the three sites, either a lac or tet operon can replace the normal DNA. In order to create an AND gate, at least one of these sites must be a tet operon and one must be a lac operon.

ANDgate.jpg

We studied the potential of a synthetic, single promoter AND gate. This helps us understand at a fundamental level what is happening in a network with multiple regulators and opens further research paths. The device consists of parts of the Tet (tetracycline) and Lac (lactose) and responds to commonly used inducers IPTG (Isopropyl β-D-1-thiogalactopyranoside) and aTc. Three or fewer of these operators can be combined to form a promoter and the order and quantity of each operator site result in different constructs, that is, given three blank spots for operators on a promoter and the choice of either -, T or L for each spot, there are a variety of possible promoter designs. We chose to focus on four constructs:

  • T T L
  • T T -
  • T - -
  • - T -

We also examined 4 mutations in the Tet operator site for each construct.

Methods

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