MAXED OOT Maxicells: a novel chassis maximised for use OOTside the lab

Why Synthetic Biology needs MaxED OOT Maxicells

Time and time again great feats of genetic engineering, that have the potential to solve innumerable global and local problems, must remain simply conceptual. It is often the risk associated with the environmental release of transgenic and gene-edited prokaryotes that prevents their practical deployment, and limits the potential of synthetic biology as a whole.

Maxed OOT maxicells are a chassis that is safe for environmental release. Maxicells are achromosomal, non-replicating cells and therefore cannot accumulate mutations over successive generations. Further to their natural properties, we have enhanced their biosafety by putting a block on dangerous horizontal gene transfer - attenuating both of the main risks associated with the environmental release of a transgenic/ gene-edited prokaryote.

For synthetic biology to make the world a better place, synthetic biology itself must first become better. Our novel chassis will breath new life into old iGEM and synthetic biology projects.

Our Project

The overall aim of our project was to optimise maxicells as a chassis that is suitable for environmental release. Our project was therefore split into two sides: optimising maxicell biosafety, and optimising their usefulness as a chassis.

Biosafety

Naturally, maxicells are unable to reproduce and therefore already come with a degree of safety not found with any prokaryotic chassis. However, they may still allow the horizontal gene transfer of advantageous genes to environment-native cells. In order the make our maxicells fully safe for environmental release, we have put a block on horizontal gene transfer using our triple lock system.

Figure 1.^[1] DNA uptake (HGT) in Vibrio cholerae

Practicality

Maxicells would be no use as a chassis if they were diffeicult to produce and if they could not carry out the functions we require from a chassis. So to demonstrate how easy they are to produce, use, and their huge potential as a chassis we have:

• Evaluated 3 different maxicell production methods for their ease and efficiency
• Quantified their active metabolic time frame
• Demonstrated their function as a biosensor

Applications of Maxed OOT Maxicells

We believe maxicells have huge potential for use in synthetic biology. For any future iGEMers or synthetic biologists reading this, here are some applications that we think maxicells are particularly suited for...

Biosensors

Maxed OOT maxicells have the unique ability to house organisationally and structurally sophisticated mechanisms whilst preventing HGT, this can be used to create biosensors for use outside the lab. They provide new options in designing mechanisms to sense and report pollutants in drinking or groundwater or particular pathogens in food, patient samples or the environment ^[2,3].

Bioremediation

Because of the complexity of activity afforded by maxicells a system could be created to remove arsenic from a body of water with cysteine rich proteins ^[4] then the maxicells would produce gas vesicles to float to surface of water ^[5] to be skimmed off the top.

Another possibility would be for Maxed OOT maxicells to house the enzymes and/or metabolites needed for a reaction which turns a harmful chemical into a harmless one. These enzymes and metabolites can be protected from inhibitory conditions by being in the maxicell.

Agriculture

Maxicells could be used to create a signal to recruit and help set up an appropriate niche for a healthy rhizosphere around crop roots. They may have use as a spray to house pesticide mechanisms, the new and wider options granted by maxed OOT maxicells over cell free systems ^[6] may allow design of a safer/more effective pesticide.

Drug Delivery

Specific adherence proteins and surface markers on the maxicells may be used to target a specific cell type/body tissue and release the maxicells drug payload upon arrival. ^[7]This would decrease severity of side effects such as toxicity and allow a far lesser dosage of the drug to be administered.

References

1. Ellison C, Dalia T, Vidal Ceballos A, Wang J, Biais N, Brun Y et al. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nature Microbiology. 2018;3(7):773-780.
2. Ahmed, A., Rushworth, J. V., Hirst, N. A., & Millner, P. A. (2014). Biosensors for Whole-Cell Bacterial Detection. Clinical Microbiology Reviews, 27(3), 631–646. http://doi.org/10.1128/CMR.00120-13
3. Hardeep Kaur, Rabindra Kumar, J. Nagendra Babu, SunilMittal (2015) Advances in arsenic biosensor development – A comprehensive review Biosensors and Bioelectronics, 63, 533-545. https://doi.org/10.1016/j.bios.2014.08.003
4. Hai-nan Zhang, Lina Yang, Jian-ya Ling, Daniel M. Czajkowsky, Jing-Fang Wang, Xiao-Wei Zhang, Yi-Ming Zhou, Feng Ge, Ming-kun Yang, Qian Xiong, Shu-Juan Guo, Huang-Ying Le, Song-Fang Wu, Wei Yan, Bingya Liu, Heng Zhu, Zhu Chen, and Sheng-ce Tao (2015).Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic National Academy of Sciences, vol. 112 no. 49 15084-15089. https://doi.org/10.1073/pnas.1521316112
5. Tashiro, Y., Monson, R. E., Ramsay, J. P., & Salmond, G. P. C. (2016).Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria. Environmental Microbiology, 18(4), 1264–1276. http://doi.org/10.1111/1462-2920.13203
6. Zemella, A., Thoring, L., Hoffmeister, C., & Kubick, S. (2015). Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. Chembiochem, 16(17), 2420–2431. http://doi.org/10.1002/cbic.201500340
7. MacDiarmid J, Mugridge N, Weiss J, Phillips L, Burn A, Paulin R et al. Bacterially Derived 400 nm Particles for Encapsulation and Cancer Cell Targeting of Chemotherapeutics. Cancer Cell. 2007;11(5):431-445.

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