Latest revision as of 18:33, 20 October 2009

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The Project

Although, we will only be judged for the software tools we have developed, we have also experimentally constructed, built and tested BioBricks that function as logical AND gates. The most important aspect of our work is that we combined simulations and experiments.

SynBioSS Designer (https://2009.igem.org/Team:Minnesota/Designer)

AND Gates

How they work

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 lacO or tetO2 operator can replace the normal DNA. In order to create an AND gate, at least one of these sites must be a tetO2 operator and one must be a lacO operator.

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) operons and responds to commonly used inducers IPTG (Isopropyl β-D-1-thiogalactopyranoside) and aTc (anhydortetracycline). 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. "-" represents a region of non-coding DNA from λ phage functions as a "null" operator site; no proteins in our E. coli strain should specifically bind it. "T" represents the tetO2 operator site, and "L" represents the lacO operator site. A number of these constructs were built in the Kaznessis lab using wild-type operator sites prior to this summer's iGEM team project. To tune and more thoroughly understand the function of these constructs, we replaced the wild-type tetO2 operator sites with several mutants which we found in the literature. We chose to focus on three constructs:

T T L
T T -
T - -

tetO2 mutants

The tetO2 operator site is a 19bp palindromic DNA sequence which is tightly bound by the dimer TetR, repressing the expression of downstream genes in the absence of an inducer. Helbl and Hillen have documented several mutations of tetO2 and the binding affinities of wild-type and mutant TetR for them. Since TetR is a dimer which binds both sides of the palindrome in the same way, each of these mutations were also made at the same location on both sides of the palindrome. File:TetO2.tiff

@@ Line 1: / Line 1: @@
-{|align="justify"
+{|align="center" cellpadding="25"
-|You can write a background of your team here.  Give us a background of your team, the members, etc.  Or tell us more about something of your choosing.
+|[[Image:mnlogo.jpg|300px|center|frame]]
-|[[Image:Example_logo.png|200px|right]]
-|-
-|
-''Tell us more about your project.  Give us background.  Use this is the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''
-|[[Image:Team.png|right]]
-|-
-|
-|align="center"|[[Team:Minnesota | Team Example 2]]
 |}
-<!--- The Mission, Experiments --->
+{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"
+{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"
-{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
+!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]
-!align="center"|[[Team:Minnesota|Home]]
+!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]
-!align="center"|[[Team:Minnesota/Team|The Team]]
+!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]
-!align="center"|[[Team:Minnesota/Project|The Project]]
+!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]
-!align="center"|[[Team:Minnesota/Parts|Parts Submitted to the Registry]]
+!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]
-!align="center"|[[Team:Minnesota/Modeling|Modeling]]
+!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]
-!align="center"|[[Team:Minnesota/Notebook|Notebook]]
+!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]
 |}
-(''Or you can choose different headings.  But you must have a team page, a project page, and a notebook page.'')
-== '''Overall project''' ==
-Your abstract
-== Computational Tools ==
-Both Designer and Wiki can be found via the SynBioSS [http://synbioss.sourceforge.net home page].
-=== SynBioSS Designer ===
-SynBioSS Designer is an application for the automatic generation of sets of biomolecular reactions. This software allows a user to input the molecular parts involved in gene expression and regulation (e.g. promoters, transcription factors, ribosomes, etc.) The software then generates complete networks of reactions that represent transcription, translation, regulation, induction and degradation of those parts. To facilitate the creation of detailed kinetic models of synthetic gene networks composed of BioBricks, we have adapted SynBioSS Designer to automatically generate a kinetic model from a construct composed entirely of BioBricks. A NetCDF or SBML file is generated for simulations.
-=== SynBioSS Wiki ===
-The inaccessibility of requisite kinetic data complicates the generation of detailed mechanistic models. We address this barrier by creating a web accessible database curated by users in a “Wiki” format. SynBioSS Wiki is a significant
-extension of the open-source Mediawiki software. The Wiki stores reaction kinetic data in a formatted and searchable
-scheme with references to the relevant literature. This framework allows for the input of reactions whose rates are described either by elementary first & second order rate equations or any arbitrarily complex rate equation defined using MathML (e.g. Hill type reactions). Reactions may be searched via participating molecules which may be proteins, DNA sequences, small molecules, etc. Once located, reactions of interest (along with their associated kinetic data) may be collected. The completed model may be exported in SBML format for additional editing or simulation.
-It is also through the SynBioSS Wiki databases that SynBioSS Designer can access and proliferate kinetic information related to the simulation of BioBricks, thus extending the utility of the database for the benefit of the greater modeling community. To jumpstart the process, we have entered the known biomolecular interactions in the expression and regulation of well-studied operons, such as the lactose, the tetracycline and the arabinose operons.
+<h1>The Project</h1>
+Although, we will only be judged for the software tools we have developed, we have also experimentally constructed, built and tested BioBricks that function as logical AND gates. The most important aspect of our work is that we combined simulations and experiments.
+<h2> SynBioSS Designer (https://2009.igem.org/Team:Minnesota/Designer) </h2>
+<h2>AND Gates</h2>
+<h3> How they work</h3>
+[[Image:ANDgatetable.jpg|300px|right]]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 lacO or tetO2 operator can replace the normal DNA. In order to create an AND gate, at least one of these sites must be a tetO2 operator and one must be a lacO operator.
+[[Image:ANDgate.jpg|400px|left]]
+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) operons and responds to commonly used inducers IPTG (Isopropyl β-D-1-thiogalactopyranoside) and aTc (anhydortetracycline). 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. "-" represents a region of non-coding DNA from λ phage functions as a "null" operator site; no proteins in our E. coli strain should specifically bind it. "T" represents the tetO2 operator site, and "L" represents the lacO operator site. A number of these constructs were built in the Kaznessis lab using wild-type operator sites prior to this summer's iGEM team project. To tune and more thoroughly understand the function of these constructs, we replaced the wild-type tetO2 operator sites with several mutants which we found in the literature. We chose to focus on three constructs:
+<ul>
+<li>T T L</li>
+<li>T T -</li>
+<li>T - -</li>
+</ul>
-=== The Experiments ===
-=== Part 3 ===
-== Results ==
+<h2> tetO2 mutants </h2>
+The tetO2 operator site is a 19bp palindromic DNA sequence which is tightly bound by the dimer TetR, repressing the expression of downstream genes in the absence of an inducer. Helbl and Hillen have documented several mutations of tetO2 and the binding affinities of wild-type and mutant TetR for them. Since TetR is a dimer which binds both sides of the palindrome in the same way, each of these mutations were also made at the same location on both sides of the palindrome.
+[[Image:TetO2.tiff|frame]]

Team:Minnesota/Project

From 2009.igem.org