Minimal cells, synthetic cells, rewritten

genomesThe 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 Mfl669 Mfl009, 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

degradationMfl418, 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, Mfl328Mfl150, Mfl598, Mfl597,

Mfl270, Mfl649

acetyl-CoA

cobalt ABC transporter

Mfl165, Mfl166K+, Na+transporter

Mfl378malate 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 minimizeTOP 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 CodeNonnatural 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)