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

Line 122: Line 122:
  
 
<p2 style="font-size:18px;">
 
<p2 style="font-size:18px;">
−
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.  
+
The previously developed Chloramphenicol Acetyltransferase Operon (CHOP) by the 2017 East Chapel Hill iGem team 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.  
 +
<br>
 +
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.
 +
 
 
</p2>
 
</p2>
  
−
<h2 style="text-align: left;"> What is a Riboswitch? </h2>
+
<h2 style="text-align: left;"> About the Riboswitch</h2>
  
 
<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.  
 
<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.  

Revision as of 16:38, 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 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 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 (Figure 2), 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.
Figure 2: Schematic of a transcriptional riboswitch
2015 Exeter iGEM Team, RNA Riboswitches

In our project, we will use the fluoride riboswitch from B. Cereus because it was characterized. In Figure 3 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 (Figure 3).
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

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 (Figure 4). In E. coli the gene crcB encodes a fluoride efflux channel that removes excess fluoride from the cell so that it is no longer toxic. In E. coli 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 E. coli strain so that fluoride could accumulate intracellularly.
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 constructed an operon that would enable us to regulate the expression of the gene chloramphenicol acetyltransferase with the fluoride riboswitch, called CHOP (Figure 5). 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 E. coli 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 BBa_KK2990000 for the correct overhangs for Gibson.
Figure 5: Schematic of the fluoride riboswitch regulated chloramphenicol acetyltransferase operon (CHOP)

How CHOP works:

  • Using the ΔcrcB E. coli strain, which can accumulate fluoride intracellularly
  • The Riboswitch detects fluoride
  • Fluoride activates the chloramphenicol acetyltransferase enzyme
  • Which allows for the growth of bacteria on agar plates with the antibiotic chloramphenicol

References