Description

Introduction

In 2017, the Orange Water and Sewer Authority (OWASA) was shut down for a week due to human error, which caused an overfeed of fluoride into the water used by Chapel Hill and Carrboro residents. Both Chapel Hill and Carrboro were placed under states of emergency, and many residents had to go without water for more than 24 hours. Fluoride levels were measured to be 5.90 mg/L, which exceeded the federal and state drinking water standards.

In a privileged environment like Chapel Hill, one day without running water can shut down businesses, schools, and homes. However, in rural areas of developing countries, residents must live their entire lives dependent on overly fluoridated water. Prolonged exposure to increased levels of fluoride has been shown to cause dental and skeletal fluorosis, birth defects, reproductive toxicity, and mutagenicity.

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.

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

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 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.

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

Modifying Characterization Technique

Last year, we encountered reliability issues that arose from using a plating assay to characterize our operon. We decided to transition to another measurement technique. We decided to use a growth-based viability assay to allow for high throughput characterization of our operon. Bacteria were first exposed to our inhibitory condition, which included chloramphenicol, and then diluted into a fresh medium. A growth curve was then used as quantitative data for the viability of the bacterial cells. This approach allowed us to obtain more conclusive and quantitative characterization of the CHOP operon.

The figure below illustrates our procedure for the growth based viability assay.