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

Why Maxed OOT Maxicells are Useful

Time and time again a great idea for a genetic engineering project for use outside the lab has been prevented from being used to its full effect because of the risk of horizontal gene transfer (HGT). Currently in Europe and other regions release of GMOs into the environment is restricted and it is very difficult and expensive to get your GMO approved for release.

Maxed OOT maxicells are a chassis for safe GM release into the environment. Maxicells are achromosomal, non replicating cells which can’t accumulate mutations. Further to their natural properties we've enhanced their biosafety allowing them to have many potential applications in the environment potentially breathing new life into old iGEM and Synth Bio projects. We’ve shown they can be used as biosensors and to produce light.

Maxed OOT maxicells don’t run the same risk of HGT as “Regular live” cells do and they have advantages over cell free systems [1] such as being able to use membrane proteins and protecting enzymes and prosseses from outside conditions allowing a wider array of proteins to be used.

Applications of Maxed OOT Maxicells

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 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. [6] This would decrease severity of side effects such as toxixity and allow a far lesser dosage of the drug to be administered.

Why we don’t Release GMOs

Introduction of a modified or “new” organism onto an environment may have unforeseen impacts on the ecology of the environment it’s being introduced to. Also, horizontal gene transfer from GMO to wild cells has the potential grant wild cells the advantages we’ve given the GMOs which again could cause disruption to ecological balance. These 2 inherent flaws are arguably the major cause of skepticism and fear amongst the many in public and this political discussion surrounding GMOs.

What have we done?

We wanted to make maxicells into as much of a perfect chassis for environmental release as possible and to broaden its potential applications. First we explored the merits of 3 of the most promising maxicell making protocols. Next we worked to characterise maxicells metabolic properties to allow users of the chassis to understand what it can be used for and make designing applications for the chassis easier. Lastly we made maxicells safe for use in the environment by creating our triple lock system which prevents wild cells using maxicell DNA.

Proof of Concept and Fully Realised Chassis

By modeling our colicin kill switch, proving that recoding of a resistance gene is enough to stop it being used by non Maxed OOT cells and creating a plasmid with a resistance for triclosan instead of an antibiotic we have all the parts for proving the concept of out triple lock. Future work would be to assemble all these parts together to have a fully realized triple lock in maxicell making it “Maxed OOT”.

Real world Functionality

Our project was to create this chassis, to make it safe for use in the environment and to make it easy to work with and design for. It’s real potential will be unlocked when future teams take it and improve the world with it. Maxed OOT maxicells are the beginning not the end.

References

1. 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
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

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