Difference between revisions of "Team:East Chapel Hill/Description"

 
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   <a href="#about">Introduction</a>
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   <a href="#services">Solution</a>
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   <a href="#Introduction">Introduction</a>  
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   <a href="#clients">Our Design</a>
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<h1>Description</h1>
 
<h1>Description</h1>
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<h1>Introduction</h1>
 
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<!-- <h2 style="text-align: left;"> The Impacts of Excess Fluoride:</h2> -->
 
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<h2> The Impacts of Excess Fluoride:</h2>
 
  
  
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<p2 style="font-size:18px;"> Fluoride is present in all bodies of water. Within the oceans, rivers, lakes, and groundwater, the mineral is existent and the extent to which fluoride is present depends on the amount of sediments or volcanic rocks being eroded in the area. It is when fluoride concentrations are at the toxic level when health concerns can arise. The FDA recommends that fluoride concentrations in water do not exceed 0.7mg/L, while the World Health Organization (WHO) limit is 1.5mg/L, and the Environmental Protection Agency designation for contaminated water is 4 mg/L. Fluoride concentrations at or above 1 mg/kg of body weight are deemed poisonous. Ingesting this amount in one sitting requires immediate medical attention. While constantly being exposed to 10 mg/L to 6 mg of fluoride everyday can lead to dental and skeletal fluorosis, in which the teeth and bones decay and deform. More severely, doses above 4.5 mg/kg body weight can cause developmental and reproductive concerns. Therefore fluoride concentrations can affect the growth and the IQ of people. In countries like China, India, and Sri Lanka, water sources are decentralized and residents in some areas experience concentrations of fluoride as high as 30 mg/L (<b>Figure 1</b>).
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<p2 style="font-size:18px;"> Granite and volcanic rocks are extremely high in fluoride due to large amounts of fluoride-rich minerals including biotite, fluorite, amphibole, and apatite. These high-fluoride rock deposits rise through faults and hot springs into groundwater. Prolonged exposure to high levels of fluoride correlates to diseases such as dental and skeletal fluorosis. These diseases can severely impact young children, whose enamel is still developing. Please see our interview with <a href="https://2018.igem.org/Team:East_Chapel_Hill/MaikoSuzuki">Maiko Suzuki</a> to learn specifically how fluorosis manifests in the teeth.  
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Unfortunately, mitigating fluoride problems has proven to be very expensive and challenging. One of the issues we are attempting to address with our project is diligently tracking fluoride concentrations after treatment attempts. In rural communities, even once there has been treatment to high-fluoride water, it is difficult to monitor fluoride concentrations after the treatment.
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<figcaption>Figure 1: Map of documented occurrences of high-fluoride groundwater <br>
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    <font size="2">Source: http://www.bgs.ac.uk/research/groundwater/health/fluoride.html</font></figcaption>
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We hope that the operon we have developed may assist the monitoring of fluoride concentrations in small, low-technology villages after treatment of the water has been administered.
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<h1>Solution</h1>
 
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In order to combat excess fluoridation of water in third world countries, we envision solutions that utilize the recently discovered fluoride riboswitch, a structured piece of RNA that kind interact with fluoride and regulate the expression of a downstream gene. We envision technologies utilizing fluoride riboswitches that can be used to sequester, bioremediate, or detect fluoride in water. We think these strategies can be used in cell-free and cell based systems. However, before we can work on developing these technologies we first needed to better characterize the responsiveness of fluoride riboswitches and develop a way to select for riboswitches with a higher responsiveness to fluoride.  
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The previously developed Chloramphenicol Acetyltransferase Operon (CHOP) by the 2017 East Chapel Hill iGem team was our first attempt in creating an accessible device that may serve as a visual indicator of fluoride in water. This year, we tested a series of promoters and riboswitch constructs to determine which are conducive to an operon with highest binding ability to fluoride. We were successful in being able to alter the previous CHOP operon so that it could detect concentrations of fluoride as low as 100uM.  
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<h2 style="text-align: left;"> What is a Riboswitch? </h2>
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<h2 style="text-align: left;"> About the Riboswitch</h2>
  
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<p2 style="font-size:18px;">A riboswitch is a piece of mRNA that regulates gene expression. There are primarily two types of riboswitches: translational and transcriptional riboswitches. The fluoride riboswitch is a transcriptional riboswitch (<b>Figure 2</b>), which means that a terminator is formed when the riboswitch is transcribed that limits the processivity of the RNA polymerase transcribing downstream genes. When the aptamer (ligand-binding) region of the fluoride riboswitch interacts with fluoride, the terminator is not formed allowing the RNA polymerase to proceed and transcribe the downstream gene.  
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<p2 style="font-size:18px;">A riboswitch is a segment of messenger RNA that is able to control gene expression by selectively binding to certain ligands. Riboswitches have 2 main domains: the aptamer and expression. The aptamer primarily serves as a receptor for specific ligands to bind to. Meanwhile, the expression may switch between 2 secondary structures, controlling gene expression. 
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<br><br>
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Riboswitches may be translational or transcriptional. A transcriptional riboswitch has a “switching sequence” in the aptamer that directs the formation of a transcriptional terminator, which signals to RNA polymerase to stop transcription. One may think of this process as an “on” or “off” switch, with “on” allowing for transcription of a gene.  
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When the aptamer (ligand-binding) region of the fluoride riboswitch interacts with fluoride, the terminator is not formed allowing the RNA polymerase to proceed and transcribe the downstream gene.  
 
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In our project, we will use the <b>fluoride riboswitch</b> from <i>B. Cereus</i> because it was characterized. In <b>Figure 3</b> you can see a crystal structure of the aptamer domain of the fluoride riboswitch. How can a negatively charged piece of RNA bind to a negatively charged fluoride ion? The fluoride riboswitch encapsulated three Mg2+ ions that can bind to the fluoride ion (<b>Figure 3</b>).  
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The riboswitch we used is from the previously characterized <i>B. cereus</i>. In Figure 3 we have displayed the crystal structure of the aptamer of this riboswitch. We tested two variations of this riboswitch, which we labeled FRS1 and FRS2. Figure 4a shows the predicted folding structure of FRS1, and Figure 4b shows the predicted folding structure of FRS2. We were interested in determining how the predicted folding structure may influence the binding ability of this riboswitch to fluoride.
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<br><br>
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In nature, this riboswitch regulates the expression of genes that are able to pump high levels of fluoride out of the cell. The <i>crcB</i> gene in <i>E.coli</i> bacteria encodes the fluoride efflux channel, which is capable of pumping fluoride out of the cell so that it is no longer toxic. In our experiments, we used a modified <i>crcB</i> <i>E.coli</i> strain so that fluoride may accumulate in the cell.  
 
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  <img src="https://static.igem.org/mediawiki/2018/3/37/T--East_Chapel_Hill--FRS1.png" style="width:75%;height:auto;">
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  <figcaption> <I> Figure 4a: Predicted folding structure of fluoride riboswitch "FRS1" </I>
  
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  <figcaption>Figure 3: Crystal structure of a fluoride riboswitch <br>
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    <font size="2">Aiming Ren, Kanagalaghatta R. Rajashankar, Dinshaw J. Patel “Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch” 2012 Nature 486, 85–89
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In nature, the riboswitch regulates the expression of genes that help the organism deal with high levels of fluoride. These genes are often pumps that allow fluoride to be exported out of the cell (<b>Figure 4</b>). In <i>E. coli</i> the gene crcB encodes a fluoride efflux channel that removes excess fluoride from the cell so that it is no longer toxic. In <i>E. coli</i> when the crcB gene is genetically deleted (ΔcrcB), the phenotype is increased sensitivity to fluoride and concentrations above 500μM are lethal. In our experiments we needed to utilize the ΔcrcB <i>E. coli</i> strain so that fluoride could accumulate intracellularly.
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   <img src="https://static.igem.org/mediawiki/2018/7/7c/T--East_Chapel_Hill--FRS2.png" style="width:75%;height:auto;">
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   <figcaption> Figure 4: Crystal structure of a fluoride channel<br>
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   <figcaption> <I>Figure 4b: Predicted folding structure of fluoride riboswitch "FRS2" </I>
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  <font size="2">Randy B. Stockbridge, Ludmila Kolmakova-Partensky, Tania Shane, Akiko Koide, Shohei Koide, Christopher Miller & Simon Newstead "Crystal structures of a double-barrelled fluoride ion channel." 2015 Nature 525, 548-51</font>
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<h1>Our Design</h1>
 
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We constructed an operon that would enable us to regulate the expression of the gene chloramphenicol acetyltransferase with the fluoride riboswitch, called CHOP (<b>Figure 5</b>). We ordered the synthetic operon from IDT DNA with overhangs that have homology to the pSB1A3 vector so we could clone our operon in with Gibson. We used the pSB1A3 vector because we are regulating the chloramphenicol acetyltransferase gene and we need to use the ΔcrcB <i>E. coli</i> strain, that is kanamycin resistant. We constructed the operon so that it is easy for future users to use Gibson cloning to add a new “promoter riboswitch segment” by cutting with HindIII or a new gene by cutting with XhoI. Check out our part <a href="http://parts.igem.org/Part:BBa_K2290000">BBa_KK2990000</a> for the correct overhangs for Gibson.
 
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   <img src="https://static.igem.org/mediawiki/2017/e/e5/ T--East_Chapel_Hill--design-f3.png" style="width:100%;height:auto;">
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   <figcaption>Figure 5: Schematic of the fluoride riboswitch regulated chloramphenicol acetyltransferase operon (CHOP)
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<a name="Our Design"></a>  
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   <figcaption>Figure 3: Crystal structure of a fluoride riboswitch <br>
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    <font size="2">Aiming Ren, Kanagalaghatta R. Rajashankar, Dinshaw J. Patel “Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch” 2012 Nature 486, 85–89
  
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<h2 style="text-align: left;"> How CHOP works:<h2>
 
  
  
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<li>Using the ΔcrcB <i>E. coli</i> strain, which can accumulate fluoride intracellularly</li>
 
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<li>The Riboswitch detects fluoride</li>
 
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<li>Fluoride activates the chloramphenicol acetyltransferase enzyme </li>
 
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<li>Which allows for the growth of bacteria on agar plates with the antibiotic chloramphenicol</li>
 
  
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<h1>References</h1>
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<h1> Our Design </h1>
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We modified the previously developed chloramphenicol acetyltransferase operon (CHOP) by the 2017 East Chapel Hill iGem. We used Gibson overhangs with homology to pSB1A3 so we could clone the operon into the pSB1A3 vector. This operon was designed so that future users may easily test a library of promoters and riboswitches simply by cutting with restriction enzyme HindIII. One may even test the expression of a new gene by using the XhoI restriction enzyme.
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  <img src="https://static.igem.org/mediawiki/2018/d/d1/T--East_Chapel_Hill--OPERON.png" style="width:75%;height:auto;">
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The fluoride binding mutant has two point mutations that prevent the antiterminator loop from forming. Therefore, fluoride can’t bond and there should be no growth. This acts as a control to verify that bacterial growth is directly a result of fluoride concentrations.
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<img src="https://static.igem.org/mediawiki/2018/c/c1/T--East_Chapel_Hill--LOWFBM.png" style="width:75%;height:auto;"> </img>
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<figcaption> <I>The figure above shows the conserved fluoride riboswitch</I>
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<img src="https://static.igem.org/mediawiki/2018/a/aa/T--East_Chapel_Hill--fbmhighfluoride.png" style="width:75%;height:auto;">
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<figcaption> <I>The figure above shows the 2 point mutation of the fluoride riboswitch, creating the fluoride binding mutant</I>
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Latest revision as of 03:38, 18 October 2018

East Chapel Hill
Introduction Solution Our Design

Description

Introduction

Granite and volcanic rocks are extremely high in fluoride due to large amounts of fluoride-rich minerals including biotite, fluorite, amphibole, and apatite. These high-fluoride rock deposits rise through faults and hot springs into groundwater. Prolonged exposure to high levels of fluoride correlates to diseases such as dental and skeletal fluorosis. These diseases can severely impact young children, whose enamel is still developing. Please see our interview with Maiko Suzuki to learn specifically how fluorosis manifests in the teeth.

Unfortunately, mitigating fluoride problems has proven to be very expensive and challenging. One of the issues we are attempting to address with our project is diligently tracking fluoride concentrations after treatment attempts. In rural communities, even once there has been treatment to high-fluoride water, it is difficult to monitor fluoride concentrations after the treatment.

We hope that the operon we have developed may assist the monitoring of fluoride concentrations in small, low-technology villages after treatment of the water has been administered.

Solution

The previously developed Chloramphenicol Acetyltransferase Operon (CHOP) by the 2017 East Chapel Hill iGem team was our first attempt in creating an accessible device that may serve as a visual indicator of fluoride in water. This year, we tested a series of promoters and riboswitch constructs to determine which are conducive to an operon with highest binding ability to fluoride. We were successful in being able to alter the previous CHOP operon so that it could detect concentrations of fluoride as low as 100uM.

About the Riboswitch

A riboswitch is a segment of messenger RNA that is able to control gene expression by selectively binding to certain ligands. Riboswitches have 2 main domains: the aptamer and expression. The aptamer primarily serves as a receptor for specific ligands to bind to. Meanwhile, the expression may switch between 2 secondary structures, controlling gene expression.

Riboswitches may be translational or transcriptional. A transcriptional riboswitch has a “switching sequence” in the aptamer that directs the formation of a transcriptional terminator, which signals to RNA polymerase to stop transcription. One may think of this process as an “on” or “off” switch, with “on” allowing for transcription of a gene. When the aptamer (ligand-binding) region of the fluoride riboswitch interacts with fluoride, the terminator is not formed allowing the RNA polymerase to proceed and transcribe the downstream gene.
Figure 2: Schematic of a transcriptional riboswitch
2015 Exeter iGEM Team, RNA Riboswitches

The riboswitch we used is from the previously characterized B. cereus. In Figure 3 we have displayed the crystal structure of the aptamer of this riboswitch. We tested two variations of this riboswitch, which we labeled FRS1 and FRS2. Figure 4a shows the predicted folding structure of FRS1, and Figure 4b shows the predicted folding structure of FRS2. We were interested in determining how the predicted folding structure may influence the binding ability of this riboswitch to fluoride.

In nature, this riboswitch regulates the expression of genes that are able to pump high levels of fluoride out of the cell. The crcB gene in E.coli bacteria encodes the fluoride efflux channel, which is capable of pumping fluoride out of the cell so that it is no longer toxic. In our experiments, we used a modified crcB E.coli strain so that fluoride may accumulate in the cell.
Figure 4a: Predicted folding structure of fluoride riboswitch "FRS1"


Figure 4b: Predicted folding structure of fluoride riboswitch "FRS2"
Figure 3: Crystal structure of a fluoride riboswitch
Aiming Ren, Kanagalaghatta R. Rajashankar, Dinshaw J. Patel “Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch” 2012 Nature 486, 85–89

Our Design

We modified the previously developed chloramphenicol acetyltransferase operon (CHOP) by the 2017 East Chapel Hill iGem. We used Gibson overhangs with homology to pSB1A3 so we could clone the operon into the pSB1A3 vector. This operon was designed so that future users may easily test a library of promoters and riboswitches simply by cutting with restriction enzyme HindIII. One may even test the expression of a new gene by using the XhoI restriction enzyme.

Schematic of operon BBa_K2843000
The fluoride binding mutant has two point mutations that prevent the antiterminator loop from forming. Therefore, fluoride can’t bond and there should be no growth. This acts as a control to verify that bacterial growth is directly a result of fluoride concentrations.


The figure above shows the conserved fluoride riboswitch


The figure above shows the 2 point mutation of the fluoride riboswitch, creating the fluoride binding mutant