Synthetic minimal cells -toolbox for biosensing and monitoring biodiversity Kate Adamala MIT Media Lab, MIT Department of Biological Engineering.

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Synthetic minimal cells-toolbox for biosensing and monitoring biodiversity

Kate Adamala

MIT Media Lab, MIT Department of Biological Engineering

Kate Adamala

Readout of biology where?End-pointRemove samples, analyze in the lab

ContinuousAnalytes are continuously monitored in the natural context.

ContinuousEnd-pointKate Adamala2Kate Adamala

Readout of biology in situ how?Electronic sensors+ Fast, reliable, remote readout + More sensitive and versatile, at the current state of technology- Electronic waste in the environment (if portable assay is available)- Needs a biolab (if no portable assay)

Biosensors+ Fast, in situ readout+/- Protein sensors, genetically encoded parts- More difficult to develop for each analyte- More sensitive to environmental stressors

Kate Adamala3Kate Adamala

BiosensorsModified bacteria+ very cheap to propagate, stable under wide array of conditions- They breed!

Liposomal biosensors (synthetic minimal cells)+ completely bio-orthogonal- More difficult to prepare, less stable

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Biosensors =/= invasive speciesInvasive species a cautionary taleRabbits, foxes in AustraliaGray squirrels in Europe

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Biosensors =/= invasive speciesNot in the Amazon!(at least not more from us)

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BiosensorsModified bacteria+ very cheap to propagate, stable under wide array of conditions- They breed!

Liposomal biosensors (synthetic minimal cells)+ completely bio-orthogonal- More difficult to prepare, less stable

Kate Adamala7semi-permeable, selective membraneKate Adamala

Synthetic minimal cells

protocella.k.asynthetic minimal cellgenetic materialenzymesKate Adamala8semi-permeable, selective membraneKate Adamala

Synthetic minimal cells

protocella.k.asynthetic minimal cellgenetic materialenzymes

2-way talk- enhancing sensing abilities of natural cells

- controlling cells by releasing signals from protocells

- reading out cells by detecting excreted chemicals

Kate Adamalatalk = communication9

cell Synthetic minimal cellsKate Adamala

cell Synthetic minimal cellsKate Adamala

cell

Synthetic minimal cellsKate Adamala

Protocells: the origin of life

Boundaries of life

Biosensorssynthetic cell

Liposome bioreactors

Cellular models

Living technologies

Actuators with natural cells

Synthetic minimal cells - applicationsKate Adamala

The anatomy of the synthetic cellThe compartmentThe encapsulated machinery

Kate AdamalaThe anatomy of the synthetic cellThe compartmentThe encapsulated machinery

Isolate from the environment while allowing the metabolites and waste transport. - semi-permeable lipid bilayer

Encapsulated enzymes and reagents.Content depends on the function.- gene expression: cell-free Tx/Tl systems.- small molecule payloads.

Kate AdamalaThe anatomy of the synthetic cell compartment

PhospholipidsCreate stable, rigid, impermeable membranes.

Polar headgroupsHydrophobic tails

Lipid bilayer membranes

CholesterolUsed as an additive. Modulates membrane fluidity.Kate AdamalaThe anatomy of the synthetic cell compartmentAlternative: encapsulation in emulsions.

wateroilwater/oil droplets

Video courtesy of Rebecca Turk MacLeod, University of Glasgow Kate AdamalaThe anatomy of the synthetic cellThe compartmentThe encapsulated machinery

Isolate from the environment while allowing the metabolites and waste transport. - semi-permeable lipid bilayer

Encapsulated enzymes and reagents.Content depends on the function.- gene expression: cell-free Tx/Tl systems.- small molecule payloads.

Kate AdamalaCell-free Tx/TlCell free transcription and translation (Tx/Tl)- No transfection, cell culture, protein purificationKate AdamalaCell-free Tx/TlCell free transcription and translation (Tx/Tl)- No transfection, cell culture, protein purification

Rosenblum, G. & Cooperman, B.S., 2014. FEBS Letters, 588(2), pp.261268. In vivoCell-freeKate AdamalaCell-free Tx/TlCell free transcription and translation (Tx/Tl)- No transfection, cell culture, protein purification- Easier to add control factors, chaperones, inhibitors, binding and folding partners- Easier to remove proteases, nucleases- Rapid, small scale production of isotope labeled proteins (for NMR and kinetics studies) and selenomethionine labeled proteins (for X-ray crystallography) - Possible to make toxic proteins- Avoid inclusion bodies

Kate AdamalaCell-free Tx/TlCell free Tx/Tl system components:- Amino acids- Nucleotides- Salts- Energy- Enzymes (ribosomes, polymerases, acetyltransferases)- tRNAs

- Your gene(s)

Hong, S.H., Kwon, Y.-C. & Jewett, Frontiers in chemistry, 2(34)Kate AdamalaCell-free Tx/TlSystemAdvantagesDisadvantagesE. coliVery high protein yieldRelatively tolerant of additivesMany eukaryotic proteins insoluble upon expressionEukaryotic co- and post-translational modifications not possibleDifferent codon usage than eukaryotesWheat germTranslation of large proteins possibleDevoid of off-target endogenous mammalian proteinsHigh protein yieldMammalian co- and post-translational modifications are not possiblePremature termination of productsInsectTranslation of large proteins possibleNo endogenous mammalian proteinsCertain forms of protein glycosylation possibleMammalian co- and post-translational modifications are not possibleRabbit ReticulocyteMammalian systemCap independent translationProtein glycosylation not possibleCo-expression of off-target proteinsHumanCo- and post-translational modifications are possibleSynthesis of functional proteinsLower yieldsNew systemThermoFisher Scientific 2015, modifiedKate Adamalasemi-permeable, selective membraneKate Adamala

Synthetic minimal cells

protocella.k.asynthetic minimal cellgenetic materialenzymes

2-way talk- enhancing sensing abilities of natural cells

- controlling cells by releasing signals from protocells

- reading out cells by detecting excreted chemicals

Kate Adamalatalk = communication24Biosensing

Vamvakaki, V. & Chaniotakis, N. a. Pesticide detection with a liposome-based nano-biosensor. Biosens. Bioelectron. 22, 28482853 (2007).Acetylcholinesterase encapsulated within liposome, porins facilitate entry of the analyte (organophosphates).Response readout: pH sensitive dye (detect acetic acid). Tunable by adding enzyme inhibitor.Kate AdamalaBiosensingLentini et.al, NATURE COMMUNICATIONS 5:4012 DOI: 10.1038/ncomms5012Expanding the sensing capacity of bacteria

Kate AdamalaBiosensingActivating ribozymesAdamala, Engelhart and Szostak J. Am. Chem. Sci. 2014

Kate AdamalaBiodiversity population dynamicsAdamala, K. & Szostak, J.W., 2013. Nature chemistry, 5(6), pp.495501.

Kate Adamala

Miller, D. & Gulbis, J. Life 5, 10191053 (2015).Metabolic engineering1D protein-nucleic acid scaffold2D and 3D nucleic acid scaffoldAssembly of metabolic pathways.Kate AdamalaRNA imagingvan Nies, P. et al. ChemBioChem 14, 19631966 (2013).

membrane dye, red, synthesized YFP (green) mRNA (Spinach, cyan).Kate AdamalaThank you!Thanks to Beno Juarez @ FabLab Lima!

Thanks to all members of Boyden Lab, especially Ed Boyden, Daniel Martin-Alarcon, Kiryl Piatkevich and Daniel SchmidtThanks to all members of Szostak Lab, especially Jack Szostak, Aaron Engelhart, Neha Kamat and Anders BjorkbomThanks to Pierluigi Luisi and Pasquale Stano,and everyone in Luisi Lab

and big thanks to everyone else for comments and support!

Funding from NSF CBET, Howard Hughes Medical Institute and NASA Exobiology

Kate Adamala31Kate Adamala