Team:Missouri Miners/Project

From 2011.igem.org

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<h1>Project </h1><br />
<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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<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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Part 1:
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We incorporated a eYFP gene, ribosome binding site, and Omp-R promoter into E. coli as a plasmid. The plasmid consists of two main parts, the promoter and a reporter system. The promoter is an osmolarity activated promoter, we dictate this part as OmpR. The reporter system is a DNA sequence which codes for a yellow florescence protein (eYFP) gene with a ribosome binding site (RBS). These parts were prepped to be put together by digesting out the eYFP gene at the Xba1 and Pst1 restriction enzyme sites and opening up the RBS plasmid by cutting at the Spe1 and Pst1 restriction enzyme sites. Gel electrophoresis was used to purify the digested segments and then ligated together creating our intermediate part, BBa_K621000, the reporter system. This DNA was transformed into chemically competent E. coli to amplify our part. After a plasmid prep of our transformation another digest was used to cut out the reporter system, RBS/eYFP, and open up the OmpR plasmid. Another set of gel electrophoresis, purification, transformation, and plasmid prep our part, BBa_K621001 was ready to be tested.
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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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Part 2:
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<h3>Next Steps</h3>
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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. For testing to see if our part, BBa_K621001, is in the order its supposed to be the DNA was sequenced. The sequencing data gave us the order of the nucleic acid bases, then the data can be compared with the known sequence of each part. To tell if the part works glucose was added to agar plates at a 25% concentration, no florescence was seen and very little growth occurred. Next we used agar/glucose plates 0%, 1%, 5%, 10%, and 15% concentrations were tried, aging resulting in no florescence. Later it was realized our mistake of trying to use solid media plates for testing a osmolarity sensor instead of liquid media. Osmolarity is the measure of solutes in a solution that change the osmotic pressure of the solution. Making the switch to liquid cultures so the part could sense osmolarity was just what was needed to happen so that our part fluoresced. 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 - LB broth was used. This was later changed from a regular concentration of LB broth to half the normal concentration, because after some research it was seen that glucose and LB broth have very similar osmolarities and so adding one to the other did not cause a change in osmolarity. This problem was quickly fixed by going to .5X concentration LB broth.
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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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Part 3:
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The next step of the project would be to go through a mutagenesis process to try to change the sensitivity of the OmpR promoter to be able to be turned on by a smaller change in osmolarity. Mutagenesis uses a strain of E. coli, MutD, that has no proof reading capability in its genome. This increases the rate at which mutations occur by a factor of 1000. We
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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.

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