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

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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 B. Cereus. In Figure 3 we have displayed the crystal structure of the aptamer domain of this riboswitch. We tested 2 mutations 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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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.  
 
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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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We modified the previously developed chloramphenicol acetyltransferase operon (CHOP) by the 2017 East Chapel Hill iGem and nicknamed it “CHOPv2”. We used Gibson overhangs with homology to pSB1C3 so we may clone the operon into the pSB1C3 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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<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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Revision as of 20:35, 12 October 2018

East Chapel Hill
Introduction Solution Our Design References

Description

Introduction

The Impacts of Excess Fluoride:

Granite and volcanic rocks are extremely high in fluoride, due to large amounts of fluoride-rich minerals, including: biotite, fluorite, amphibole, apatite. These high-fluoride deposits rise through faults and hot springs into groundwater. Prolonged exposure to these high levels of fluoride has been correlated to diseases such as dental and skeletal fluorosis. These diseases have most severe impacts in young children, whose enamel is still developing at the time of fluoride exposure. 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. Please see our interview with Tewodros Godebo to understand more about how many are attempting to solve the issue of high-fluoride water. 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. However, one issue we encountered when developing this operon was its low affinity to fluoride in water. As a result, this operon was most effective in detecting concentrations of fluoride 75uM and above.

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 may detect concentrations of fluoride as low as ?uM.

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 domain and expressional domain. The aptamer domain primarily serves as a receptor for specific ligands to bind to. Meanwhile, the expressional domain 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 domain 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 domain of this riboswitch. We tested 2 mutations 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 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

hol up
Figure 4: Crystal structure of a fluoride channel
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

Our Design

We modified the previously developed chloramphenicol acetyltransferase operon (CHOP) by the 2017 East Chapel Hill iGem and nicknamed it “CHOPv2”. We used Gibson overhangs with homology to pSB1C3 so we may clone the operon into the pSB1C3 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.
Figure 5: Schematic of the fluoride riboswitch regulated chloramphenicol acetyltransferase operon (CHOP)

References