Transcript Biosensors and biodiversity monitoring | Kate Adamala (pptx, 8.4 MB)

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-point
Remove samples, analyze in the lab
Continuous
Analytes are continuously monitored in the natural context.
End-point
Continuous
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 Adamala
Biosensors
Modified 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 Adamala
Biosensors =/= invasive species
Invasive species – a cautionary tale
Rabbits, foxes in Australia
Gray squirrels in Europe
Kate Adamala
Biosensors =/= invasive species
Not in the Amazon!
(at least not more from us…)
Kate Adamala
Biosensors
Modified 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 Adamala
Kate Adamala
Synthetic minimal cells
genetic
material
enzymes
semi-permeable,
selective
membrane
protocell
a.k.a
synthetic minimal cell
Kate Adamala
Synthetic minimal cells
genetic
material
enzymes
semi-permeable,
selective
membrane
protocell
a.k.a
synthetic minimal cell
2-way talk
- enhancing sensing
abilities of natural cells
- controlling cells by
releasing signals from
protocells
- reading out cells by
detecting excreted chemicals
Kate Adamala
Synthetic minimal cells
cell
Kate Adamala
Synthetic minimal cells
cell
Kate Adamala
Synthetic minimal cells
cell
Kate Adamala
Synthetic minimal cells - applications
Biosensors
Cellular models
Living technologies
Liposome bioreactors
Actuators with natural cells
synthetic cell
Protocells:
the origin of life
Boundaries of life
The anatomy of the synthetic cell
The compartment
Kate Adamala
The encapsulated machinery
The anatomy of the synthetic cell
The compartment
Isolate from the environment while
allowing the metabolites and waste
transport.
- semi-permeable lipid bilayer
Kate Adamala
The encapsulated machinery
Encapsulated enzymes and
reagents.
Content depends on the function.
- gene expression: cell-free Tx/Tl
systems.
- small molecule payloads.
The anatomy of the synthetic cell – compartment Kate Adamala
Lipid bilayer membranes
Polar headgroups
Hydrophobic tails
Phospholipids
Create stable, rigid, impermeable
membranes.
Cholesterol
Used as an additive.
Modulates membrane
fluidity.
The anatomy of the synthetic cell – compartment Kate Adamala
Alternative: encapsulation in emulsions.
water
oil
water/oil
droplets
Video courtesy of Rebecca Turk
MacLeod, University of Glasgow
The anatomy of the synthetic cell
The compartment
Isolate from the environment while
allowing the metabolites and waste
transport.
- semi-permeable lipid bilayer
Kate Adamala
The encapsulated machinery
Encapsulated enzymes and
reagents.
Content depends on the function.
- gene expression: cell-free Tx/Tl
systems.
- small molecule payloads.
Cell-free Tx/Tl
Cell free transcription and translation (Tx/Tl)
- No transfection, cell culture, protein purification
Kate Adamala
Cell-free Tx/Tl
Kate Adamala
Cell free transcription and translation (Tx/Tl)
- No transfection, cell culture, protein purification
In vivo
Cell-free
Rosenblum, G. & Cooperman, B.S., 2014. FEBS Letters, 588(2), pp.261–268.
Cell-free Tx/Tl
Kate Adamala
Cell 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
Cell-free Tx/Tl
Cell free Tx/Tl system
components:
- Amino acids
- Nucleotides
- Salts
- Energy
- Enzymes (ribosomes,
polymerases,
acetyltransferases)
- tRNA’s
- Your gene(s)
Hong, S.H., Kwon, Y.-C. & Jewett, Frontiers in chemistry, 2(34)
Kate Adamala
Cell-free Tx/Tl
Kate Adamala
System
Advantages
Disadvantages
E. coli
•Very high protein yield
•Relatively tolerant of additives
•Many eukaryotic proteins insoluble upon
expression
•Eukaryotic co- and post-translational
modifications not possible
•Different codon usage than eukaryotes
Wheat germ •Translation of large proteins possible
•Mammalian co- and post-translational
•Devoid of off-target endogenous mammalian modifications are not possible
proteins
•Premature termination of products
•High protein yield
Insect
•Translation of large proteins possible
•No endogenous mammalian proteins
•Certain forms of protein glycosylation
possible
Rabbit
•Mammalian system
Reticulocyte •Cap independent translation
Human
•Co- and post-translational modifications are
possible
•Synthesis of functional proteins
•Mammalian co- and post-translational
modifications are not possible
•Protein glycosylation not possible
•Co-expression of off-target proteins
•Lower yields
•New system
ThermoFisher Scientific 2015, modified
Kate Adamala
Synthetic minimal cells
genetic
material
enzymes
semi-permeable,
selective
membrane
protocell
a.k.a
synthetic minimal cell
2-way talk
- enhancing sensing
abilities of natural cells
- controlling cells by
releasing signals from
protocells
- reading out cells by
detecting excreted chemicals
Biosensing
Acetylcholinesterase encapsulated within liposome,
porins facilitate entry of the analyte (organophosphates).
Response readout: pH sensitive dye (detect acetic acid).
Tunable by adding enzyme inhibitor.
Vamvakaki, V. & Chaniotakis, N. a. Pesticide detection with a liposome-based
nano-biosensor. Biosens. Bioelectron. 22, 2848–2853 (2007).
Kate Adamala
Biosensing
Expanding the sensing capacity of bacteria
Lentini et.al, NATURE COMMUNICATIONS 5:4012 DOI: 10.1038/ncomms5012
Kate Adamala
Biosensing
Kate Adamala
Activating ribozymes
Adamala, Engelhart and Szostak J. Am. Chem. Sci.
2014
Biodiversity – population dynamics
Kate Adamala
Adamala, K. & Szostak, J.W., 2013. Nature chemistry, 5(6), pp.495–501.
Metabolic engineering
Kate Adamala
Assembly of metabolic pathways.
1D protein-nucleic acid scaffold
2D and 3D nucleic acid scaffold
Miller, D. & Gulbis, J. Life 5, 1019–1053 (2015).
RNA imaging
Kate Adamala
membrane dye, red,
synthesized YFP (green)
mRNA (Spinach, cyan).
van Nies, P. et al. ChemBioChem 14,
1963–1966 (2013).
Kate Adamala
Thank you!
Thanks to Beno Juarez @ FabLab Lima!
Thanks to all members of Boyden Lab, especially Ed
Boyden, Daniel Martin-Alarcon, Kiryl Piatkevich and Daniel
Schmidt
Thanks to all members of Szostak Lab, especially Jack
Szostak, Aaron Engelhart, Neha Kamat and Anders Bjorkbom
Thanks 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 Adamala