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 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.

From Giovannoni et al. 2005

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

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.

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 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.

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.

Hail Mary Genes by functional category KEEP DELETE Amino acid biosynthesis 0 4 Biosynthesis of cofactors 9 2 Cell envelope 28 92 Cellular processes 3 8 Central intermediary metabolism 7 8 DNA metabolism 32 32 Energy metabolism 28 35 Fatty acid and phospholipid metabolism 7 6 Hypothetical proteins 59 110 Mobile and extrachromosomal element fcns 0 14 NULL (tRNAs, rRNAs, RNAs) 49 0 Protein fate 22 23 Protein synthesis 107 8 Purines 19 7 Regulatory functions 9 8 Signal transduction 3 14 Transcription 14 4 Transport and binding proteins 35 33 Unknown function 21 47 Yeast vector and markers 4 0 ___________________________________________________________ TOTAL 457 455  

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

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

Mycoplasma capricolum Science August 2007 Mycoplasma mycoides Mycoplasma capricolum gDNA DONOR RECIPIENT CELLS

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

Mycoplasma capricolum Science August 2009 Mycoplasma capricolum Mycoplasma mycoides Yeast gDNA DONOR RECIPIENT CELLS

Science May 2010

Whole Genome Synthesis

Approach Writing DNA 2 polished M. mycoides genomes CP001621 CP001668 (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 emails 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

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

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

Technical Achievement (1): Assembly Figure 1 Science (2010) 329: 52

Technical Achievement (2): Transplantation 1.0 WT PCR for watermarks Digests of genome plugs Figure 4 and 5 Science (2010) 329: 52

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

Engineering The First Organisms with Novel Genetic Codes rE.coli Engineering The First Organisms with Novel Genetic Codes http://www2.le.ac.uk/departments/genetics/vgec/education/post18/topics/dna-genes-chromosomes 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

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

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 evolution Harris 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://www.flickr.com/photos/ajc1/1103490291/ http://en.wikipedia.org/wiki/File:Conjugation.svg

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

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