Team:Missouri Miners/Project

From 2011.igem.org

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<h1>Project </h1><br />
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<h3>Abstract</h3>
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<p>In 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 created a glucose-controlled promoter linked to a yellow fluorescence production gene in E. coli. The concentrations of glucose to which the promoter responds can be determined. Once the concentration is 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.</p>
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<br />
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<h3>Our System Overview</h3>
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[[File:IGEM System.JPG|thumbnail|right|360px|Two Component Regulatory System Pathway]]
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<p>We submitted two biobricks to the registry: an [http://partsregistry.org/wiki/index.php?title=Part:BBa_K621000| intermediate part] with a ribosome binding site and eYFP, and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K621001|  intermediate plus the ompC operator]. The ompC operator has three binding sites for phosphorylated OmpR. OmpR and EnvZ work together in a two-component regulatory system as shown in the diagram below. </p>
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<br />
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[[File:IGEM binding.JPG|thumbnail|right|360px|When one or two OmpR bind, RNAP transcribes the gene.]]
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[[File:IGEM binding2.JPG|thumb|right|360px|When all OmpR binding sites are occupied, RNAP can't bind.]]
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<p>EnvZ is an inner membrane protein that senses osmolarity. Phosphorlyation of OmpR by EnvZ positively correlates with the osmolarity of the system. Phosphorylated OmpR (OmpR-P) can bind to the three sites on the ompC operator. When one or two of the binding sites are occupied by OmpR-P, RNA polymerase is recruited to begin downstream transcription of the reporter gene, eYFP.  However, when all three OmpR binding sites are occupied by OmpR-P, RNA polymerase cannot bind, the reporter gene can no longer be produced, and therefore the system is inhibited. In summary, as osmolarity increases from very low levels, the fluorescence produced by the system also increases until the system reaches a threshold osmolarity. Once the system reaches the threshold, the fluorescence will decrease with increasing osmolarity due to the inherent down-regulation of the system. The activity of the system can be quantified using the fluorescence of the cells because the two-component regulatory system of EnvZ and OmpR regulates transcription of the eYFP gene, dictating the level of fluorescence.</p>
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<div style="height: 795px">
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<h3>Making Our Parts</h3>
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[[File:Igemmodel3.jpg|right|400px]]
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<p>We started with three parts from the iGEM registry:
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*BBa_B0032: RBS-3, a medium-strength ribosome binding site
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*BBa_E0030: eYFP gene, codes for yellow fluorescence
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*BBa_R0082: omp-c operon, contains three binding sites for phosphorylated OmpR</p>To build our part our team performed restriction digests and ligations as indicated by the figure to the right .
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<div id="template" style="font-weight: regular; font-size: medium; color: silver; padding: 5px;">
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<br />
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<h1>Welcome to the Missouri Miners Wiki page! </h1><br />
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<h3>Testing</h3>
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<p>To measure fluorescence our team used a 96-well plate reader.  Overnight cultures of bacteria were grown in 0.5X LB media and then aliquoted into 1.5ml tubes.  Serial dilutions of the desired glucose concentrations were made. Glucose was diluted using 0.5X LB media so that the concentration of LB remained constant. The glucose was administered to the cells in a fashion that did not dilute the cell densities in the cultures. Samples of each treatment were added to a 96-well plate and were imaged using the plate reader. See [[Team:Missouri_Miners/data| data]] page for results. </p>
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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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<h3>Next Steps</h3>
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<p>In the future, our system has applications in glucose sensing devices. For our system to be sensitive to different changes in osmolarity the OmpC operon will need to be mutated. The mutagenesis process begins with transforming our system into a mutagenic strain of E. coli, in which, the cells have no proof reading capability during DNA replication. In these cells the rate of mutation is increased by 1000x. Using this process mutant  strains could be produced, sequenced, and characterized. Possible applications of a characterized system include economical glucose testing strips, a wider range of test strip availability, and precursors to advanced insulin pump systems. </p>
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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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</div>
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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
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concentration activated promoter gene named OmpR. The second is a DNA sequence
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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
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used to cut the circular E.coli DNA at specific points during a digestion. Because eYFP has
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more base pairs than RBS (ribosome binding site) and Omp-R, we will cut the eYFP genes
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completely out of the plasmid so that eYFP and RBS can be combined, with eYFP as the
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insert and RBS as the vector. After these have been ligated together, the new
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eYFP/RBS plasmid will be cut and reassembled with eYFP/RBS as the insert and Omp-R as
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the vector.
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<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
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to be done to see if they were in fact in the correct order, and also what concentration of
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glucose it would fluoresce at. For testing to see if our part BBa_K621001 is in the order its
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supposed to be in the DNA will be sequenced. The sequencing data will give us the order
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of the bases that it reads back, then the data can be compared with the known sequence of
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each gene. To tell if the part works as it should glucose was added to agar plates at a 25%
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concentration, no florescence was seen. Next using agar/glucose plates 0%, 1%, 5%, 10%,
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and 15% concentrations were tried, aging resulting in no florescence. Later it was realized
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that our part was an osmolarity sensor which senses changes in a substance thickness and
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ability to move through a cell. This caused all of our testing to happen in Lb liquid cultures.
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Making the switch to liquid cultures so the part could sense osmolarity was just what
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needed to happen the part fluoresced and worked as it should. Next to get an idea of what
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concentrations of glucose would still let the part glow 0%, 1%, 3%, 5%, 8%, 10%, 15%,
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20,and 25% glucose was used.
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<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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still glow at use cells from that culture and begin mutations. Mutate until the part works at
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low enough levels to sense fluctuating glucose levels in human blood
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Latest revision as of 03:52, 29 September 2011

Project


Abstract

In 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 created a glucose-controlled promoter linked to a yellow fluorescence production gene in E. coli. The concentrations of glucose to which the promoter responds can be determined. Once the concentration is 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.


Our System Overview

Two Component Regulatory System Pathway

We submitted two biobricks to the registry: an [http://partsregistry.org/wiki/index.php?title=Part:BBa_K621000| intermediate part] with a ribosome binding site and eYFP, and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K621001| intermediate plus the ompC operator]. The ompC operator has three binding sites for phosphorylated OmpR. OmpR and EnvZ work together in a two-component regulatory system as shown in the diagram below.


When one or two OmpR bind, RNAP transcribes the gene.
When all OmpR binding sites are occupied, RNAP can't bind.

EnvZ is an inner membrane protein that senses osmolarity. Phosphorlyation of OmpR by EnvZ positively correlates with the osmolarity of the system. Phosphorylated OmpR (OmpR-P) can bind to the three sites on the ompC operator. When one or two of the binding sites are occupied by OmpR-P, RNA polymerase is recruited to begin downstream transcription of the reporter gene, eYFP. However, when all three OmpR binding sites are occupied by OmpR-P, RNA polymerase cannot bind, the reporter gene can no longer be produced, and therefore the system is inhibited. In summary, as osmolarity increases from very low levels, the fluorescence produced by the system also increases until the system reaches a threshold osmolarity. Once the system reaches the threshold, the fluorescence will decrease with increasing osmolarity due to the inherent down-regulation of the system. The activity of the system can be quantified using the fluorescence of the cells because the two-component regulatory system of EnvZ and OmpR regulates transcription of the eYFP gene, dictating the level of fluorescence.

Making Our Parts

Igemmodel3.jpg

We started with three parts from the iGEM registry:

  • BBa_B0032: RBS-3, a medium-strength ribosome binding site
  • BBa_E0030: eYFP gene, codes for yellow fluorescence
  • BBa_R0082: omp-c operon, contains three binding sites for phosphorylated OmpR

    To build our part our team performed restriction digests and ligations as indicated by the figure to the right .


Testing

To measure fluorescence our team used a 96-well plate reader. Overnight cultures of bacteria were grown in 0.5X LB media and then aliquoted into 1.5ml tubes. Serial dilutions of the desired glucose concentrations were made. Glucose was diluted using 0.5X LB media so that the concentration of LB remained constant. The glucose was administered to the cells in a fashion that did not dilute the cell densities in the cultures. Samples of each treatment were added to a 96-well plate and were imaged using the plate reader. See data page for results.


Next Steps

In the future, our system has applications in glucose sensing devices. For our system to be sensitive to different changes in osmolarity the OmpC operon will need to be mutated. The mutagenesis process begins with transforming our system into a mutagenic strain of E. coli, in which, the cells have no proof reading capability during DNA replication. In these cells the rate of mutation is increased by 1000x. Using this process mutant strains could be produced, sequenced, and characterized. Possible applications of a characterized system include economical glucose testing strips, a wider range of test strip availability, and precursors to advanced insulin pump systems.