Download presentation
Presentation is loading. Please wait.
1
Minimal cells, synthetic cells, rewritten genomes
The importance of the chassis
2
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.
3
Machines need to be… Reliable Reproducible Not error prone
Not evolving Programmable No cross-talk among systems Are cells like that?
4
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
5
Ways to engineer chassis
Minimal cells Synthesized cells Rewritten genomes Perhaps completely artificial cells?
6
A computer analogy -- the genome of a cell is the operating system & the cytoplasm is the hardware
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. The cytoplasm is the hardware that runs the operating system. The chromosome is the operating system.
9
From Giovannoni et al. 2005
10
Identified Metabolic Pathways in Mesoplasma florum
G. Fournier 02/23/04 PTS II System Mfl519, Mfl565 sucrose trehalose xylose beta-glucoside glucose unknown ribose ABC transporter Mfl516, Mfl527, Mfl187 Mfl500 Mfl669 Mfl318, Mfl312 Mfl009, Mfl033, fructose Mfl214, Mfl187 Mfl619, Mfl431, Mfl426 Mfl181 ATP Synthase Complex Mfl497 Mfl515, Mfl526 Mfl499 Mfl317?, Mfl313? Mfl617, Mfl430, Mfl313? Mfl425, Mfl615, Mfl034, Mfl009, Mfl011, Mfl012, ? Mfl112, Mfl113, Mfl114, Mfl109, Mfl110, Mfl111, Mfl115, Mfl116 Mfl666, Mfl667, Mfl668 glucose-6-phosphate chitin degradation Mfl558 Mfl347, ATP ADP sn-glycerol-3-phosphate ABC transporter Pentose-Phosphate Pathway Glycolysis Mfl025, Mfl026 Mfl023, Mfl024, L-lactate, acetate Mfl223, Mfl640, Mfl642, Mfl105, Mfl349 glyceraldehyde-3-phosphate Mfl254, Mfl180, Mfl514, Mfl174, Mfl644, Mfl200, Mfl504, Mfl578, Mfl577, Mfl502, Mfl120, Mfl468, Mfl175, Mfl259 Mfl039, Mfl040, Mfl041, Mfl042, Mfl043, Mfl044, Mfl596, Mfl281 Lipid Synthesis unknown substrate transporters Mfl046, Mfl052 Mfl384, Mfl593, fatty acid/lipid transporter ribose-5-phosphate acetyl-CoA Mfl230, Mfl382, Mfl286, Mfl663, Mfl465, Mfl626 Mfl590, Mfl591 Mfl325,Mfl482 Mfl099, Mfl474,Mfl315, x13+ PRPP cardiolipin/ phospholipids membrane synthesis Purine/Pyrimidine Salvage phospholipid membrane Identified Metabolic Pathways in Mesoplasma florum Mfl074, Mfl075, Mfl276, Mfl665, Mfl463, Mfl144, Mfl342, Mfl343, Mfl419, Mfl676, Mfl635, Mfl119, Mfl107, Mfl679, Mfl306, Mfl648, Mfl170, Mfl195, Mfl372 Mfl076, Mfl121, Mfl639, Mfl528, Mfl530, Mfl529, Mfl547, Mfl375 Mfl143, Mfl466, Mfl198, Mfl556, Mfl385 niacin? Mfl038, Mfl065, Mfl063, Mfl388 xanthine/uracil permease Pyridine Nucleotide Cycling variable surface lipoproteins Mfl413, Mfl658 Mfl444, Mfl446, Mfl451 Mfl340, Mfl373, Mfl521, Mfl588 Mfl583, Mfl288, Mfl002, Mfl678, Mfl675, Mfl582, Mfl055, Mfl328 Mfl150, Mfl598, Mfl597, Mfl270, Mfl649 hypothetical lipoproteins DNA Polymerase RNA Polymerase x22 competence/ DNA transport Mfl047, Mfl048, Mfl475 Electron Carrier Pathways DNA RNA K+, Na+ transporter Mfl027, Mfl369 Flavin Synthesis Nfl289, Mfl037, Mfl653, Mfl193 Mfl064, Mfl178 NAD+ Mfl165, Mfl166 ribosomal RNA transfer RNA degradation FMN, FAD Mfl193 Mfl541, Mfl005, Mfl647, Mfl258, Mfl329, Mfl374, Mfl563, Mfl548, Mfl088, Mfl231, Mfl209 Mfl282, Mfl387, Mfl682, Mfl014, Mfl196,Mfl156, Mfl029, Mfl412, Mfl540, Mfl673, Mfl077, rnpRNA Mfl283, Mfl334 malate transporter? hypothetical transmembrane proteins NADP Mfl378 x57 Ribosome metal ion transporter Signal Recognition Particle (SRP) riboflavin? Mfl356, Mfl496, Mfl217 messenger RNA tRNA aminoacylation NADPH NADH protein secretion (ftsY) srpRNA, Mfl479 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? cobalt ABC transporter Mfl237 Mfl152, Mfl153, Mfl154 proteins Formyl-THF Synthesis Export Mfl613, Mfl554, Mfl480, Mfl087, Mfl651, Mfl268, Mfl366, Mfl389, Mfl490, Mfl030, Mfl036, Mfl399, Mfl398, Mfl589, Mfl017, Mfl476, Mfl177, Mfl192, Mfl587, Mfl355 Mfl086, Mfl162, Mfl163, Mfl161 phosphonate ABC transporter met-tRNA formylation Mfl571, Mfl572 Mfl060, Mfl167, Mfl383, Mfl250 protein translocation complex (Sec) Mfl057, Mfl068, Mfl142,Mfl090, Mfl275 Mfl409, Mfl569 phosphate ABC transporter Mfl233, Mfl234, Mfl235 degradation THF? Mfl186 formate/nitrate transporter amino acids intraconversion? Mfl094, Mfl095, Mfl096, Mfl097, Mfl098 Mfl418, Mfl404, Mfl241, Mfl287, Mfl659, Mfl263, Mfl402, Mfl484, Mfl494, Mfl210, tmRNA Mfl509, Mfl510, Mfl511 spermidine/putrescine ABC transporter oligopeptide ABC transporter Mfl016, Mfl664 putrescine/ornithine APC transporter Mfl015 Mfl182, Mfl183, Mfl184 Mfl019 Mfl605 arginine/ornithine antiporter Mfl557 Mfl652 unknown amino acid ABC transporter glutamine ABC transporter lysine APC transporter alanine/Na+ symporter glutamate/Na+ symporter Amino Acid Transport
11
What do we mean by “minimal bacterial cell”?
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.
12
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.
13
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.
14
There are 2 ways to minimize
Our starting point for minimization is the synthetic genome M. mycoides JCVI-syn1.0 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.
15
What bacterial cell will we minimize?
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.
16
Hail Mary Genes by functional category
KEEP DELETE Amino acid biosynthesis Biosynthesis of cofactors Cell envelope Cellular processes Central intermediary metabolism DNA metabolism Energy metabolism Fatty acid and phospholipid metabolism Hypothetical proteins Mobile and extrachromosomal element fcns 0 14 NULL (tRNAs, rRNAs, RNAs) Protein fate Protein synthesis Purines Regulatory functions Signal transduction Transcription Transport and binding proteins Unknown function Yeast vector and markers ___________________________________________________________ TOTAL
17
Moving life into the digital world and back
Our capacity to build microbes capable of solving human problems is limited only by our imagination
18
Self-Replicating Machine
19
For our purposes, we define a synthetic cell as one that operates off of a chemically synthesized genome
21
Approach used to synthesize a bacterial cell
Assemble overlapping synthetic DNA oligonucleotides (~60 mers) Recipient cell Synthetic cell Cassettes (~1 kb) Assemble cassettes by homologous recombination Genome Transplantation Completely assembled synthetic genome Genome Synthesis
22
Mycoplasma capricolum
Science August 2007 Mycoplasma mycoides Mycoplasma capricolum gDNA DONOR RECIPIENT CELLS
23
Science February 2008 6kb 24kb 72kb 144kb 580kb yeast 1/25 1/8 1/4
42 43 44 45 Yeast Vector 50-77A 50-77B 1/25 1/8 1/4 Whole Chemical Synthesis E. coli yeast
24
Mycoplasma capricolum
Science August 2009 Mycoplasma capricolum Mycoplasma mycoides Yeast gDNA DONOR RECIPIENT CELLS
25
Science May 2010
26
Whole Genome Synthesis
27
Approach Writing DNA 2 polished M. mycoides genomes CP001621
CP (aka YCpMmyc1.1) 4 “watermark” sequences 95 sites at which 2 published genomes differ. Chose to correct all designed DNA to match 1668 reference. (19 “harmless” differences not corrected) Six frame stop codons (in green) and Asc I restriction site in grey. Encoded in the watermarks is a new DNA code for writing words, sentences and numbers. In addition to the new code there is a web address to send s to if you can successfully decode the new code, the names of 46 authors and other key contributors and three quotations: "TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE." - JAMES JOYCE; "SEE THINGS NOT AS THEY ARE, BUT AS THEY MIGHT BE.”-A quote from the book, “American Prometheus”; "WHAT I CANNOT BUILD, I CANNOT UNDERSTAND." - RICHARD FEYNMAN. Also wrote in TetR and LacZ Itaya Nature Biotechnology : (2010) 28: 687–689
28
Approach Writing DNA Assembling DNA
Cloning + recombination for 10, 100 kb and 1Mb fragments UGA = tryptophan not stop in mycoplasms Chemical synthesis of ~1kb sequences Itaya Nature Biotechnology : (2010) 28: 687–689
29
Approach Writing DNA Assembling DNA Transplanting DNA to M. capricolum
In vitro methylation and deprotonation Agarose plug isolation of DNA inactivated restriction enzyme gene (MCAP0050) Itaya Nature Biotechnology : (2010) 28: 687–689
30
Technical Achievement (1): Assembly
Figure 1 Science (2010) 329: 52
31
Technical Achievement (2): Transplantation
1.0 WT PCR for watermarks Digests of genome plugs Figure 4 and 5 Science (2010) 329: 52
32
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.”
33
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.”
34
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."
36
Engineering The First Organisms with Novel Genetic Codes
rE.coli Engineering The First Organisms with Novel Genetic Codes Precise manipulation of chromosomes in vivo enables genome-wide codon replacement Farren J. Isaacs, Peter A. Carr, Harris H. Wang,…JM Jacobson, GM Church - Science, 2011
37
Expanding the Genetic Code
Nonnatural amino acids Mehl, Schultz et al. JACS (2003) Nonnatural DNA bases Geyer, Battersby, and Benner Structure (2003) Anderson, Schultz et al. PNAS (2003) 4-base codons
38
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
44
Programming cells by multiplex genome engineering and accelerated evolution
Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest & George M. Church Nature 460, (13 August 2009)
48
Bacterial Conjugation
49
rE.coli - Recoding E.coli 32 cell lines total, target
MG1655 4.6 MB 32 cell lines total, target ~10 modifications per cell line oligo shotgun: parallel cycles 32 16 8 4 2 1
50
Conjugative Assembly Genome Engineering (CAGE)
Precise manipulation of chromosomes in vivo enables genome-wide codon replacement SJ Hwang, MC Jewett, JM Jacobson, GM Church - Science, 2011
Similar presentations
