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PowerPoint Presentation
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
Kate Adamala4Kate Adamala
Biosensors =/= invasive speciesInvasive species a cautionary taleRabbits, foxes in AustraliaGray squirrels in Europe
Kate Adamala5Kate Adamala
Biosensors =/= invasive speciesNot in the Amazon!(at least not more from us)
Kate Adamala6Kate 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
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