Programming animal physiology and behaviors through engineered bacteria

Introduction

Controlling the behaviour of animals using genetic tools is a technique commonly used by scientists, especially in model systems such as Drosophila [1,2] and C. elegans [3]. However, the existing tools are limited in the extent of control they offer and the types of behaviours that can be controlled. In most cases, the behavioural alterations arise due to dysfunction or mutation of a gene and the degree of alteration cannot be controlled.

In order to thoroughly understand or manipulate behaviours, it is necessary to have fine control over animal behaviour in a laboratory setting. This preprint addresses this issue by using C. elegans as a model system and bacteria to control behaviour.

C. elegans and bacteria share a very close relationship, as do most hosts and their resident microbes [4]. Studies abound on the myriad ways in which gut bacteria influence the metabolism and behaviours of their hosts [5,6]. Following this body of literature, this preprint provides a means by which one can exploit this relationship to genetically engineer the bacteria and thereby, control host behaviour.

Key Results

The authors used RNAi for targeted gene silencing in C. elegans. They used an elegant technique, by which RNAi constructs expressed by ingested bacteria would silence specific genes in the host. These genes in C. elegans were related to either GFP expression, twitching behaviour or fat storage.

In order to control the expression of RNAi using chemical agents, the RNAi construct was made in two ways. In the first, it was cloned as an AND gate in conjunction with a split T7 RNA polymerase. The T7 polymerase is a monomer in its native state. By splitting the protein and expressing the two halves under two different inducible promoters, the researchers ensured that the polymerase would only be expressed if both inducers were added to the medium.

In the second method, the RNAi construct was cloned as an OR gate, using the same split T7 polymerase. In this case, both inducible promoters were placed in tandem before the split polymerase, allowing the activation of either one to activate expression of both polymerase halves.

Depending on the output, the RNAi construct was made to target GFP, unc22 (for twitching behaviour), or sbp-1 (for fat storage).

The scientists were able to induce expression of the RNAi in the bacteria by adding the respective inducers to the media. They were able to demonstrate that the AND gate behaved as expected, with silencing of the target genes in C. elegans occurring only when both inducers were present. With the OR gate, the addition of either inducer was sufficient to silence the target genes.

Thus, by controlling the expression of RNAi in bacteria, the authors were able to control corresponding gene expression in host C. elegans and thereby, behaviours.

What I Like About this Preprint

The scientists use pre-existing methods and knowledge in a novel way to control behaviour in C. elegans. The interactions between C. elegans and its microbiome is well-documented, as are the influences of the microbiome on animal behaviour. The molecular tools used to control RNAi expression have also been employed by previous studies [7].

It is the combination of techniques that gives rise to a new method to control behavioural outputs. The authors also use simple solutions when troubleshooting, such as varying the length of the RNAi construct when faced with problems of leaky expression, as opposed to tuning the promoter. This would allow use of the similar construct length across different promoters, instead limiting future endeavours to promoters of specific strengths.

Questions

• Is there a time lag between ingestion of the bacteria and observed changes in behaviour? Would this depend on the gene being silenced?
• Can the RNAi expression, and hence behavioural control, be titred using different concentrations of inducer?
• Once induced, how long do the behavioural changes last?
• Can the RNAi expression be terminated at will using repressors?

References

1. Martin, F., & Alcorta, E. (2017). Novel genetic approaches to behavior in Drosophila. Journal of neurogenetics, 31(4), 288-299.
2. Luu, P., Zaki, S. A., Tran, D. H., & French, R. L. (2016). A novel gene controlling the timing of courtship initiation in Drosophila melanogaster. Genetics, 202(3), 1043-1053.
3. Melo, J. A., & Ruvkun, G. (2012). Inactivation of conserved C. elegans genes engages pathogen-and xenobiotic-associated defenses. Cell, 149(2), 452-466.
4. Khan, F., Jain, S., & Oloketuyi, S. F. (2018). Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans. Microbiological research, 215, 102-113.
5. O’Donnell, M. P., Fox, B. W., Chao, P. H., Schroeder, F. C., & Sengupta, P. (2020). A neurotransmitter produced by gut bacteria modulates host sensory behaviour. Nature, 583(7816), 415-420.
6. Cabreiro, F., & Gems, D. (2013). Worms need microbes too: microbiota, health and aging in Caenorhabditis elegans. EMBO molecular medicine, 5(9), 1300-1310.
7. Shis, D. L., & Bennett, M. R. (2013). Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants. Proceedings of the National Academy of Sciences, 110(13), 5028-5033.

Tags: behavior, genetics, microbiome, rnai, worm

doi: https://doi.org/10.1242/prelights.24345

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