Minimal cells, synthetic cells, rewritten genomes The importance of the chassis.
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Transcript of Minimal cells, synthetic cells, rewritten genomes The importance of the chassis.
Minimal cells, synthetic cells, rewritten
genomes
The importance of the chassis
Chassis In synthetic biology the chassis is the cell.
When engineering a car, we need to match the engine to the chassis. Would a Corolla engine move a Hummer?
In synbio we need to make sure our devices will work in the cell.
Machines need to be…
Reliable
Reproducible
Not error prone
Not evolving
Programmable
No cross-talk among systems
Are cells like that?
If we pay attention to the chassis, we may prevent…
Prevent errors
Prevent evolution
Prevent cross-talk by introducing orthogonal systems
Increase ease of programming
Increase reproducibility
Increase reliability
Ways to engineer chassis
Minimal cells
Synthesized cells
Rewritten genomes
Perhaps completely artificial cells?
A computer analogy -- the genome of a cell is the operating system & the cytoplasm is the
hardware
The cytoplasm is the hardware that runs the operating system.
The chromosome is the operating system.
•The cytoplasm contains all of parts (proteins, ribosomes, etc.) necessary to express the information in the genome.
•The genome contains all information necessary to produce the cytoplasm and cell envelope and to replicate itself.
•Each is valueless without the other.
From Giovannoni et al. 2005
Mfl214, Mfl187
Mfl516, Mfl527, Mfl187
Mfl500 Mfl669Mfl009, Mfl033,Mfl318, Mfl312
Mfl666, Mfl667, Mfl668
Mfl023, Mfl024,Mfl025, Mfl026
ribose ABC transporter
glucosesucrose trehalo
sexylose
unknownfructose
sn-glycerol-3-phosphate ABC transporter
Mfl254, Mfl180, Mfl514, Mfl174, Mfl644, Mfl200, Mfl504, Mfl578, Mfl577, Mfl502, Mfl120, Mfl468, Mfl175, Mfl259Mfl039, Mfl040, Mfl041, Mfl042, Mfl043, Mfl044, Mfl596, Mfl281
Glycolysis
Mfl497 Mfl515, Mfl526 Mfl499 Mfl317?, Mfl313? ?
Mfl181
beta-glucoside
Mfl009, Mfl011, Mfl012, Mfl425, Mfl615, Mfl034, Mfl617, Mfl430, Mfl313?
PTS II SystemMfl519, Mfl565
chitin degradation
Mfl223, Mfl640, Mfl642, Mfl105, Mfl349
Pentose-Phosphate Pathwayglyceraldehyde-3-phosphate
Mfl619, Mfl431, Mfl426
Mfl074, Mfl075, Mfl276, Mfl665, Mfl463, Mfl144, Mfl342, Mfl343, Mfl170, Mfl195, Mfl372
Mfl419, Mfl676, Mfl635, Mfl119, Mfl107, Mfl679, Mfl306, Mfl648,Mfl143, Mfl466, Mfl198, Mfl556, Mfl385
Mfl076, Mfl121, Mfl639, Mfl528, Mfl530, Mfl529, Mfl547, Mfl375
Purine/Pyrimidine Salvage
glucose-6-phosphate
ribose-5-phosphate
Mfl413, Mfl658
xanthine/uracilpermease
DNA RNAMfl027, Mfl369
competence/DNA transport
DNA Polymerase
degradation
RNA Polymerase
Mfl047, Mfl048, Mfl475
Mfl237
protein translocation complex (Sec)
protein secretion (ftsY)
srpRNA, Mfl479
Signal Recognition Particle (SRP) Ribosome
Export
Mfl182, Mfl183, Mfl184
Mfl509, Mfl510, Mfl511
Mfl652Mfl557
Mfl605Mfl019
Mfl094, Mfl095, Mfl096, Mfl097,
Mfl098
Mfl015
spermidine/putrescineABC transporter
unknown amino acidABC transporterglutamine
ABC transporter
oligopeptide ABC transporter
arginine/ornithineantiporter lysine
APC transporteralanine/Na+ symporter
glutamate/Na+symporter
Mfl016, Mfl664
putrescine/ornithineAPC transporter
23sRNA, 16sRNA, 5sRNA,
Mfl122, Mfl149, Mfl624, Mfl148, Mfl136, Mfl284, Mfl542, Mfl132, Mfl082,Mfl127, Mfl561, Mfl368.1, Mfl362.1, Mfl129, Mfl586, Mfl140, Mfl080,
Mfl623, Mfl137, Mfl492, Mfl406
Mfl608, Mfl602, Mfl609, Mfl493, Mfl133, Mfl141, Mfl130, Mfl151, Mfl139, Mfl539, Mfl126, Mfl190, Mfl441, Mfl128, Mfl125, Mfl134, Mfl439, Mfl227,
Mfl131, Mfl123, Mfl638, Mfl396, Mfl089, Mfl380, Mfl682.1, Mfl189, Mfl147, Mfl124, Mfl135, Mfl138, Mfl601, Mfl083, Mfl294, Mfl440?
proteins
degradation
Mfl418, Mfl404, Mfl241, Mfl287, Mfl659, Mfl263, Mfl402, Mfl484, Mfl494, Mfl210, tmRNA
tRNA aminoacylation
ribosomal RNA transfer RNA
messenger RNA
Mfl029, Mfl412, Mfl540, Mfl014, Mfl196,Mfl156, Mfl282, Mfl387, Mfl682, Mfl673, Mfl077, rnpRNA
Mfl563, Mfl548, Mfl088, Mfl258, Mfl329, Mfl374, Mfl541, Mfl005, Mfl647, Mfl231, Mfl209
Mfl613, Mfl554, Mfl480, Mfl087, Mfl651, Mfl268, Mfl366, Mfl389, Mfl490, Mfl030, Mfl036, Mfl399, Mfl398, Mfl589,
Mfl017, Mfl476, Mfl177, Mfl192, Mfl587, Mfl355
Mfl086, Mfl162, Mfl163, Mfl161
amino acids
Amino Acid Transport
intraconversion?
Mfl590, Mfl591
Lipid SynthesisMfl230, Mfl382, Mfl286, Mfl663, Mfl465, Mfl626
fatty acid/lipid transporter
Identified Metabolic Pathways in
Mesoplasma florum
Mfl384, Mfl593,Mfl046, Mfl052
L-lactate,acetate
Mfl099, Mfl474,Mfl315, Mfl325,Mfl482
cardiolipin/phospholipids
membrane synthesis
x22
Mfl444, Mfl446, Mfl451
variable surface lipoproteins
hypotheticallipoproteins
phospholipid membrane
Mfl063, Mfl065, Mfl038,Mfl388
Mfl186 formate/nitratetransporter
Mfl060, Mfl167, Mfl383, Mfl250
Formyl-THF Synthesis
THF?
x57hypothetical transmembrane proteins
met-tRNA formylationMfl409, Mfl569
Mfl152, Mfl153, Mfl154
Mfl233, Mfl234, Mfl235
Mfl571, Mfl572
Mfl356, Mfl496, Mfl217
Mfl064, Mfl178Nfl289, Mfl037, Mfl653, Mfl193
Mfl109, Mfl110, Mfl111, Mfl112, Mfl113, Mfl114,
Mfl115, Mfl116
ATP Synthase Complex
ATP ADP
phosphate ABC transporter
phosphonate ABC transporter
metal ion transporter
Mfl583, Mfl288, Mfl002, Mfl678, Mfl675, Mfl582,
Mfl055, Mfl328
Mfl150, Mfl598, Mfl597, Mfl270, Mfl649
acetyl-CoA
cobalt ABC transporter
Mfl165, Mfl166
K+, Na+transporter
Mfl378
malate transporter?
Mfl340, Mfl373, Mfl521, Mfl588
Pyridine Nucleotide Cycling
NAD+
Electron Carrier Pathways
NADHNADPH
NADP
Flavin Synthesis
riboflavin?
FMN, FADMfl283, Mfl334
Mfl193
Mfl057, Mfl068, Mfl142,Mfl090,
Mfl275
Mfl347, Mfl558
G. Fournier02/23/04
x13+
unknown substrate transporters
PRPP
niacin?
We consider a bacterial cell to be minimal if it contains only the genes that are necessary and sufficient to ensure continuous growth under ideal laboratory conditions.
What do we mean by “minimal bacterial cell”?
Why make a minimal cell• To define a minimal set of genetic functions
essential for life under ideal laboratory conditions.
• To discover the set of genes of currently unknown function that are essential and to determine their functions.
• To have a simple system for whole cell modeling.
• To modularize the genes for each process in the cell (translation, replication, energy production, etc.) and to design a cell from those modules.
• To build more complex cells by adding new functional modules.
Why make a minimal cell• To define a minimal set of genetic functions
essential for life under ideal laboratory conditions.
• To discover the set of genes of currently unknown function that are essential and to determine their functions.
• To have a simple system for whole cell modeling.
• To modularize the genes for each process in the cell (translation, replication, energy production, etc.) and to design a cell from those modules.
• To build more complex cells by adding new functional modules.
There are 2 ways to minimize
TOP DOWN: Start with the full size viable M. mycoides JCVI syn1.0 synthetic genome. Remove genes and clusters of genes one (or a few) at a time. At each step re-test for viability. Only proceed to the next step if the preceding construction is viable and the doubling time is approximately normal.
BOTTOM UP: Make our best guess as to the genetic and functional composition of a minimal genome and then synthesize it. Craig Venter calls this the Hail Mary genome.
Our starting point for minimization is the synthetic genome M. mycoides JCVI-syn1.0
We chose to minimize Mycoplasma mycoides JCVI-syn1.0 the synthetic version of Mycoplasma mycoides because:
• It has a small genome (1.08 MB).
• It can be readily grown in the laboratory.
• We can routinely chemically synthesize its genome and clone it in yeast as a yeast plasmid.
• We can isolate the synthetic genome out yeast as naked DNA and bring it to life by transplanting it into a recipient mycoplasma cell.
• We have developed a suite of tools to genetically engineer its genome.
What bacterial cell will we minimize?
KEEP DELETEAmino acid biosynthesis 0 4Biosynthesis of cofactors 9 2Cell envelope 28 92Cellular processes 3 8Central intermediary metabolism 7 8DNA metabolism 32 32Energy metabolism 28 35Fatty acid and phospholipid metabolism 7 6Hypothetical proteins 59 110Mobile and extrachromosomal element fcns 0 14NULL (tRNAs, rRNAs, RNAs) 49 0Protein fate 22 23Protein synthesis 107 8Purines 19 7Regulatory functions 9 8Signal transduction 3 14Transcription 14 4Transport and binding proteins 35 33Unknown function 21 47Yeast vector and markers 4 0___________________________________________________________TOTAL 457 455
Hail Mary Genes by functional category
Moving life into the digital world and back
Our capacity to build microbes capable of solving human problems is limited only by our imagination
Self-Replicating Machine
For our purposes, we
define a synthetic cell as one that operates off of a chemically synthesized
genome
Assemble cassettes by homologous recombination
Assemble overlapping synthetic DNA oligonucleotides(~60 mers)
Completely assembled synthetic genome
Approach used to synthesize a bacterial cell
Cassettes (~1 kb)
Recipient cell Synthetic cell
GenomeTransplantation
Genome Synthesis
Mycoplasma capricolum
Mycoplasma mycoides
RECIPIENT CELLSgDNA DONOR
Science August 2007
Science February 2008
1/25 1/8 1/4 Whole
42 43 44 45
6kb 72kb 144kb 580kb24kb
50-77B50-77A
Yeast Vector
yeast
yeastE. coliChemicalSynthesis
RECIPIENT CELLSgDNA DONOR
Yeast
Mycoplasma capricolum
Mycoplasma mycoides
Science August 2009
Science May 2010
Whole Genome Synthesis
Approach Writing DNA
Itaya Nature Biotechnology : (2010) 28: 687–689
2 polished M. mycoides genomesCP001621 CP001668 (aka
YCpMmyc1.1)
4 “watermark” sequences
Also wrote in TetR and LacZ
Approach Writing DNA
Assembling DNA
Itaya Nature Biotechnology : (2010) 28: 687–689
Chemical synthesis of ~1kb sequences
Cloning + recombination for
10, 100 kb and 1Mb fragments
Approach Writing DNA
Assembling DNA
Transplanting DNA to M. capricolum
Itaya Nature Biotechnology : (2010) 28: 687–689
In vitro methylation and deprotonation
inactivated restriction enzyme gene (MCAP0050)
Agarose plug isolation of DNA
Technical Achievement (1): Assembly
Figure 1Science (2010) 329: 52
Technical Achievement (2): Transplantation
1.0
WT
Figure 4 and 5 Science (2010) 329: 52
PCR for watermarksDigests of genome plugs
Conclusions According to JCVI:
“The synthetic cell is called Mycoplasma mycoides JCVI-syn1.0 and is the proof of principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.”
Conclusions According to Venter on CNN:
“We built it from four bottles of chemicals.”
“So it's the first living self-replicating cell that we have on the planet whose DNA was made chemically and designed in the computer.”
“So it has no genetic ancestors. Its parent is a computer.”
http://www.cnn.com/2010/HEALTH/05/21/venter.qa/index.html
Conclusions According to Jim Collins (BU):
“This is an important advance in our ability to re-engineer organisms, not make new life from scratch…Although some of us in synthetic biology have delusions of grandeur, our goals are much more modest."
http://www.nature.com/news/2010/100520/full/news.2010.255.html
Precise manipulation of chromosomes in vivo enables genome-wide codon replacementFarren J. Isaacs, Peter A. Carr, Harris H. Wang,…JM Jacobson, GM Church - Science, 2011
rE.coliEngineering The First Organisms with Novel
Genetic Codes
http://www2.le.ac.uk/departments/genetics/vgec/education/post18/topics/dna-genes-chromosomes
Expanding the Genetic Code
Nonnatural amino acids
Mehl, Schultz et al. JACS (2003)
Nonnatural DNA bases
Geyer, Battersby, and BennerStructure (2003)
Anderson, Schultz et al. PNAS (2003)
4-base codons
Why reengineer the genome?
Designs: Design new DNA nucleotides
Design new amino acids
Design new proteins
Prevent viral infection
Prevent engineered organisms from cross breeding with wild types
Programming cells by multiplex genome engineering and accelerated evolutionHarris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest & George M. Church Nature 460, 894-898(13 August 2009)
http://profiles.umassmed.edu/profiles/ProfileDetails.aspx?From=SE&Person=240
Bacterial Conjugation
http://en.wikipedia.org/wiki/File:Conjugation.svg
http://www.flickr.com/photos/ajc1/1103490291/
32 cell lines total, target~10 modifications per cell line
E. ColiMG16554.6 MB
rE.coli - Recoding E.coli
oligo shotgun:parallel cycles
32
16
8
4
2
1
Precise manipulation of chromosomes in vivo enables genome-wide codon replacementSJ Hwang, MC Jewett, JM Jacobson, GM Church - Science, 2011
Conjugative Assembly Genome Engineering (CAGE)