Team:East Chapel Hill/Design-color2

Solution

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.

Introduction

The Impacts of Excess Fluoride:

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/mL, 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 (Figure 1).

Figure 1: Map of documented occurrences of high-fluoride groundwater
Source: http://www.bgs.ac.uk/research/groundwater/health/fluoride.html

What is a 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. 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

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

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