journal club

Water Safety for the Developing World

Access to clean drinking water is a global problem. With this synthetic biology system, we can quickly and cheaply test water for contamination.

Saving the World with Synthetic Biology

In Cell-free biosensors for rapid detection of water contaminants, scientists teamed up to ask, Can we use synthetic biology to test the safety of drinking water? and Can we develop it so that it’s cheap and easy to use outside of a world-class laboratory?

The result is a system that can be adjusted to detect various chemical hazards, biological pathogens, and human health markers. It requires freeze-dried RNA samples so there’s no need for a fancy -80C freezer. Samples are run on a hand-held device to visualize results; diagnosis is as simple as asking, does it glow in the dark?

It’s also a great paper for introducing the field of synthetic biology. The authors define synthetic biology as capturing “the ability of cells to use molecular biosensors and genetic networks to sense and respond to changing conditions”. That’s a succinct summary capturing a broad field. Cells are really good at shuffling molecules and genes around in response to various stimuli. We can learn from them to solve real-world problems.

A Global Problem, A New Solution

Unsafe water conditions haunt 80% of the world’s population; using bioengineered cells to test the water has environmental concerns. So here, a simple RNA-based system called ROSALIND is used; it’s cell-free, and the RNA sequences just code for a fluorescent protein that can be turned on or off depending on if some chemical or pathogen is detected.

The Prerequisites

This paper uses a biosensor system called ROSALIND (RNA Output Sensors Activated by Ligand INDuction). (Fig. 1 is a nice representation of how this system works.) It consists of only three components: RNA polymerase, transcription factors, and DNA templates.

RNA polymerase is an enzyme involved in the Central Dogma (that is, DNA is transcribed into RNA, then RNA translated into protein). Now part of the beauty of this system is that it’s cell-free, and no protein translation is needed. Translation never occurs; transcription only is needed. The DNA sequence is written to code for a fluorescent RNA sequence. This sequence is an aptamer (it binds to some target molecule) that glows in the dark – a fluorescent aptamer.

In this way, translation can be skipped entirely. All you need is transcription, and the RNA is the output.

That’s what ROSALIND refers to, right? It’s an RNA Output system, and when it forms a Ligand (just a type of chemical bond) with some target, the system is Activated. The name describes exactly what it does.

There’s one more part to this system: the transcription factor (TF). A TF will control whether RNA polymerase can actually bind to the DNA sequence and make RNA from it. In this system, we have an allosteric transcription factor (aTF) which acts as a switch. The aTFs were engineered to inhibit transcription under normal circumstances. If a particular molecule is present, however, it binds to the aTF. That forces the aTF to detatch from the DNA sequence, and RNA polymerase can take over.

Once that enzyme gets to work, it creates a glow-in-the-dark RNA sequence.

The Details

The researchers tested various sequence-molecule combinations to show the versatility of their tool. For example, on the DNA sequence, they put random spacer sequences in between the fluorescent gene and the binding site for the aTF (Fig. 2e). In Fig. 2f, we see that short or no spacer sequences optimizes how well the switch works (micromolar equivalent fluorescein, MEF, measures how much the output glows in the dark. So MEF on the y-axis measures how strong the switch is. In Fig. 2f, TetR is the aTF for detecting the molecule, here the antibiotic tetracycline. If tetR is gone, there is no aTF to inhibit gene activation, and the samples always glow. If, as in the purple samples, tetR is present, it inhibits gene activation – and thus stops the glowing down to 0 MEF – when there are short spacers. TetR loses this off-switching ability as the spacer size increases along the x-axis.)

The real versatility of this system is showcased in Fig. 3. Target molecule-aTF pairs were made for four classes of molecules: tetracyclines, macrolides, small molecules, and metals. Molecules like tetracyclines and macrolides are antibiotics that are increasing concerns for drinking water contamination; small molecules like naringenin or uric acid act on the opposite end, serving as biomarkers for general human health. And metals are major pollutants in areas exposed to industrial waste and mining pollution.

So here, aTFs were designed so that if their target molecule (naringenin, tetracycline, or some other contaminant/biomarker) is present, the two bind. If the target isn’t there, the aTF binds to the DNA sequence, and RNA polymerase is blocked from transcription. In Fig. 3, cases where there is no target molecule is denoted by a black line.

If the target is there and binds to the aTF, the aTF releases from the DNA sequence, and RNA polymerase is free to move along the DNA sequence. This case is denoted by colored lines in Fig. 3, and as you can see, they yield much higher MEF measurements than their controls (the black lines). This means the switch has been flipped on, and the samples are glowing with fluorescent RNA.

For the most part, switches were specific enough so that each aTF responded only to its target molecule, and not to any other. However, there was cross-talk in some samples. We’ve looked at yeast cells handling cross-talk in their communication pathways before; now, we must consider how to solve the problem without a cell.

Fig. 4 shows where the cross-talk is occurring: the CsoR transcription factor, which should only respond to copper, also switches on when there’s zinc pollution in the sample. To fix this, they redesigned their DNA. If copper is in the sample, gene transcription occurs and the sample glows, as it should. But if zinc is present, two genes are transcribed. There’s the glow-in-the-dark RNA output, as before. But there’s now also a second output that stops the RNA from glowing! This reduced cross-talk and made their system specific to copper pollution only. It’s a promising strategy should other sensors be designed this way, if cross-talk is observed for them as well.

They also demonstrated methods to increase sensitivity of their switches.

Conclusions

In this paper, we’ve learned:

  1. How ROSALIND works
  2. How to apply a Bio 101 concept like The Central Dogma to real-world problems
  3. How to design cell-free RNA systems as biosensors

These resulting biosensors can be modified for target molecules of interest. Maybe we want to check a human health marker like naringenin in the water. Or maybe we want to test for specific pollutants.

Either way, this system offers a path forward. What’s better, its technology is low-maintenance as far as lab protocols go.

Once you’ve designed and mixed your DNA sequence, corresponding aTF, and RNA polymerase, you can freeze dry the mix and then store it at room temperature. That makes it really easy to transport and use in the developing world, outside of fancy laboratories! Furthermore, we have handheld devices capable of reading fluorescence. These small, cheap, quick-acting tools make the method all the more accessible in areas of the world that are actually at risk from unclean drinking water.

References

Jung, J. K., Alam, K. K., Verosloff, M. S., Capdevila, D. A., Desmau, M., Clauer, P. R., Lee, J. W., Nguyen, P. Q., Pastén, P. A., Matiasek, S. J., Gaillard, J. F., Giedroc, D. P., Collins, J. J., & Lucks, J. B. (2020). Cell-free biosensors for rapid detection of water contaminants. Nature Biotechnology. https://doi.org/10.1038/s41587-020-0571-7

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