Team:Missouri Miners/Project

From 2011.igem.org

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<div id="instructions" style="text-align: center; font-weight: regular; font-size: large; color: silver; padding: 5px;"><p><font face="Berlin Sans FB"><font size="7" face="Berlin Sans FB" color=#42C555>I</font>n the bodies of people with diabetes, the ability to recognize and respond to glucose concentrations in the blood has been compromised. As a result, glucose accumulates to dangerous levels. High blood glucose concentrations can cause irreversible damage to critical organs, impairing their functionality. With parts from the iGEM registry, our team has created a osmolarity controlled promoter linked to a yellow fluorescence production gene in E. coli. Osmolarity is the measure of solutes that contribute to the osmotic pressure of a solution, so as the glucose concentration goes up the osmolarity changes. The concentrations of glucose to which the promoter responds are initially unknown. Once the concentrations are known, the promoter can be mutated so that it will be activated by varying concentrations of glucose and be used as a glucose sensor for people with diabetes. In the future, an insulin gene could be added to this system for use in insulin pumps, where specific glucose levels trigger insulin production in E. coli.
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<div id="instructions" style="text-align: center; font-weight: regular; font-size: large; color: silver; padding: 5px;"><p><font face="Berlin Sans FB"><font size="7" face="Berlin Sans FB" color=#42C555>P</font>art 1:
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As a standard of iGEM, E.coli will be used as the bacterium for this research. We will
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incorporate the eYFP, RBS, and Omp-R genes into the DNA of the E.coli in the form of a
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plasmid. The plasmid will consist of two main parts. The first is an iGEM part: a glucose
 +
concentration activated promoter gene named OmpR. The second is a DNA sequence
 +
which codes for a yellow florescence protein (eYFP). Third is a ribosome binding site. The
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promoter will regulate the expression of the eYFP. Restriction enzymes will be
 +
used to cut the circular E.coli DNA at specific points during a digestion. Because eYFP has
 +
more base pairs than RBS (ribosome binding site) and Omp-R, we will cut the eYFP genes
 +
completely out of the plasmid so that eYFP and RBS can be combined, with eYFP as the
 +
insert and RBS as the vector. After these have been ligated together, the new
 +
eYFP/RBS plasmid will be cut and reassembled with eYFP/RBS as the insert and Omp-R as
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the vector.
 +
<div id="instructions" style="text-align: center; font-weight: regular; font-size: large; color: silver; padding: 5px;"><font face="Berlin Sans FB"><font size="7" face="Berlin Sans FB" color=#42C555>P</font>art 2
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Once the three parts are together and in the correct order OmpR/RBS/eYFP testing needed
 +
to be done to see if they were in fact in the correct order, and also what concentration of
 +
glucose it would fluoresce at. For testing to see if our part BBa_K621001 is in the order its
 +
supposed to be in the DNA will be sequenced. The sequencing data will give us the order
 +
of the bases that it reads back, then the data can be compared with the known sequence of
 +
each gene. To tell if the part works as it should glucose was added to agar plates at a 25%
 +
concentration, no florescence was seen. Next using agar/glucose plates 0%, 1%, 5%, 10%,
 +
and 15% concentrations were tried, aging resulting in no florescence. Later it was realized
 +
that our part was an osmolarity sensor which senses changes in a substance thickness and
 +
ability to move through a cell. This caused all of our testing to happen in Lb liquid cultures.
 +
Making the switch to liquid cultures so the part could sense osmolarity was just what
 +
needed to happen the part fluoresced and worked as it should. Next to get an idea of what
 +
concentrations of glucose would still let the part glow 0%, 1%, 3%, 5%, 8%, 10%, 15%,
 +
20,and 25% glucose was used.
 +
<div id="instructions" style="text-align: center; font-weight: regular; font-size: large; color: silver; padding: 5px;"><font face="Berlin Sans FB"><font size="7" face="Berlin Sans FB" color=#42C555>P</font>art 3
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The next step of the project would be once we know the lowest concentration the part will
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<h4 style="color: silver; font-size: large">Part 1:</h4>
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still glow at use cells from that culture and begin mutations. Mutate until the part works at
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As a standard of iGEM, E.coli will be used as the bacterium for this research. We will incorporate the eYFP gene, RBS, and Omp-R promoter into the DNA of the E.coli in the form of a plasmid. The plasmid will consist of two main parts. The first is an iGEM part: a glucose concentration activated promoter gene. The second is a DNA sequence which codes for a yellow florescence protein (eYFP). The promoter will regulate the expression of the eYFP by the EnvZ signal transduction pathway. Restriction enzymes will be used to cut the circular E.coli DNA at specific points. Because eYFP has more base pairs than RBS (ribosome binding site) and Omp-R, we will cut the eYFP genes completely out so that eYFP and RBS can be combined with eYFP as the insert and RBS as the vector([[Team:Missouri_Miners/Modeling|Modeling]]). After these have been ligated together, the new eYFP/RBS plasmid will be cut and reassembled with eYFP/RBS as the insert and Omp-R as the vector.
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low enough levels to sense fluctuating glucose levels in human blood
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=== The Experiments ===
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To check results and separate plasmids and genes, the team will use Gel electrophoresis. For instance, after the initial digests in which Omp-R, eYFP, and RBS have all been cut in the appropriate places, the team will run a gel first: to make sure that the digestion worked as planned, and second: to separate the various pieces from their original plasmids. We will also be useing cultures with Ampicillin to make sure that the ligations are viable and that they have the right genes. Each Digestion will be followed by a Gel and each ligation will be followed by transformation. To veiw the results of these experiments, visit the [[Team:Missouri_Miners/Notebook|Notebook]] tab.
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Revision as of 22:34, 28 September 2011


Welcome to the Missouri Miners Wiki page!



Part 1:

As a standard of iGEM, E.coli will be used as the bacterium for this research. We will incorporate the eYFP, RBS, and Omp-R genes into the DNA of the E.coli in the form of a plasmid. The plasmid will consist of two main parts. The first is an iGEM part: a glucose concentration activated promoter gene named OmpR. The second is a DNA sequence which codes for a yellow florescence protein (eYFP). Third is a ribosome binding site. The promoter will regulate the expression of the eYFP. Restriction enzymes will be used to cut the circular E.coli DNA at specific points during a digestion. Because eYFP has more base pairs than RBS (ribosome binding site) and Omp-R, we will cut the eYFP genes completely out of the plasmid so that eYFP and RBS can be combined, with eYFP as the insert and RBS as the vector. After these have been ligated together, the new eYFP/RBS plasmid will be cut and reassembled with eYFP/RBS as the insert and Omp-R as the vector.

Part 2

Once the three parts are together and in the correct order OmpR/RBS/eYFP testing needed to be done to see if they were in fact in the correct order, and also what concentration of glucose it would fluoresce at. For testing to see if our part BBa_K621001 is in the order its supposed to be in the DNA will be sequenced. The sequencing data will give us the order of the bases that it reads back, then the data can be compared with the known sequence of each gene. To tell if the part works as it should glucose was added to agar plates at a 25% concentration, no florescence was seen. Next using agar/glucose plates 0%, 1%, 5%, 10%, and 15% concentrations were tried, aging resulting in no florescence. Later it was realized that our part was an osmolarity sensor which senses changes in a substance thickness and ability to move through a cell. This caused all of our testing to happen in Lb liquid cultures. Making the switch to liquid cultures so the part could sense osmolarity was just what needed to happen the part fluoresced and worked as it should. Next to get an idea of what concentrations of glucose would still let the part glow 0%, 1%, 3%, 5%, 8%, 10%, 15%, 20,and 25% glucose was used.

Part 3

The next step of the project would be once we know the lowest concentration the part will still glow at use cells from that culture and begin mutations. Mutate until the part works at low enough levels to sense fluctuating glucose levels in human blood

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