Transcript Synthetic Life

Creating a “Synthetic” Bacterial
Cell
John Glass
The J. Craig Venter Institute,
Rockville, MD and San Diego, CA
Self-Replicating Machine
NASA Conference Publication 2255 (1982), based on the Advanced Automation for Space
Missions NASA/ASEE summer study Held at the University of Santa Clara in Santa Clara,
California, from June 23-August 29, 1980
Self-Replicating Machine
For our purposes, we
define a synthetic cell
as one that operates off of
a chemically synthesized
genome
A computer analogy -- the genome of a cell is
the operating system & the cytoplasm is the
hardware
• The cytoplasm contains
The cytoplasm is
the hardware that
runs the operating
system.
The chromosome is
the operating
system.
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.
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
Science August 2007
Mycoplasma mycoides
gDNA DONOR
Mycoplasma capricolum
RECIPIENT CELLS
Science February 2008
6kb
24kb
72kb
144kb
580kb
yeast
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Yeast Vector
50-77A
Chemical
Synthesis
1/25
1/8
E. coli
50-77B
Whole
1/4
yeast
Science August 2009
Mycoplasma capricolum
Mycoplasma mycoides
Yeast
gDNA DONOR
RECIPIENT CELLS
Science May 2010
Synthetic Organism Designer 1.0
Desig
n
Codon
Opt.
Oligo
Synthesis
Cells are complex machines with
thousands of moving parts
Tomography
Mycoplasma pneumoniae from Proteome organization in a
genome-reduced bacterium. Kuhner et al. 2009 Science 326: 1235-
Cells are 25-50% Dark Matter
Hypothetical
Proteins
Small Peptides of
unknown function
Epigenetic
Modifications
Small RNAs of
unknown
function
?
Moonlighting
Proteins
Tomography
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.
What bacterial cell will we minimize?
We chose to minimize Mycoplasma mycoides JCVIsyn1.0 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 YCp.
•
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.
Synthesis of the
Mycoplasma
mycoides
JCVI-syn1.0
Genome
Gibson et al., 2010
Science 329, 52-56
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.
For both approaches, we need to identify
genes that are non-essential and are therefore
candidates for removal. We are doing this in
three ways.
1. Identify genes with functions that are usually nonessential such as IS elements, R-M systems,
integrative and conjugative elements, etc.
2. Perform global transposon mutagenesis to identify
individual genes that can be disrupted without loss
of viability.
Tn5- puromycin
global
mutagenesis of
M. mycoides
•Illumina sequencing
yielded 10,902 unique
insertion sites
•754 genes hit, 160 not
hit.
So many genes are hit
because there is
extensive functional
redundancy.
For example, there are
2 rRNA operons and
only one is necessary.
Top down approach: Stepwise genome
reduction
Top down approach: Stepwise genome
reduction
M. mycoides wild type
1089 kb
M. mycoides JCVI-syn 1.0
1079 kb
M. mycoides JCVI-syn 1.0 – 6RM(12 genes, 17 kb) 1062 kb
M. mycoides JCVI-syn 1.0 – 6RM(12 genes, 17 kb)
– 6 IS (12 genes, 9 kb)
1051 kb
M. mycoides JC syn1.0 – 6RM(12 genes, 17 kb)
– 6 IS (12 genes, 9 kb) – ICE (44 genes, 71 kb)
M. mycoides JC syn1.0 – 6RM(12 genes, 17 kb)
– 6 IS (12 genes, 9 kb)
– ICE (44 genes, 71 kb)
– D5 deletions (52 kb)
980 kb
928 kb
We plan to continue removing the large clusters, testing for viability at
each step. After that, small clusters and individual non-essential genes
will be removed to arrive at the minimal genome.
The bottom up approach
Design and synthesis of a “Hail Mary” genome
Use the Tn5 transposon single gene
disruption by insertion map data and
our knowledge of essential functions in
the cell to make the best guess as to
which genes to include in a minimal
genome.
“Hail Mary Deletions” mapped onto M. mycoides JCVI-syn1.0
West Coast Design
“Hail Mary” Minimal Genome Construction
16,000 oligos (70 bases)
↓
370 stage-1 assemblies (1.4 kb)
↓
74 stage-2 assemblies (6.7 kb)
↓
8 stage-3 assembles (50-75 kb)
↓
483 kb Minimal Genome
1/8th molecules
can be
individually
tested for
functionality and
mixed with
subsequent
designs
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
Design of a modular genome
Can genes within individual functional categories
be clustered into modules?
Can all 30 tRNA genes of M. mycoides be organized in one module?
2
9
4
M. mycoides JCVI-syn 1.0
2
5
tRNA gene clusters are enlarged to show the
direction of transcription. JCVI-syn 1.0 has 8
single tRNA genes and 5 clusters of 2 to 9
genes, for a total of 30.
Key
promoter
tRNA gene
terminator
Moving life into the digital world and back
Our capacity to build microbes capable of solving human
problems is limited only by our imagination
Possible future uses of synthetic
& engineered species
• Increase basic understanding of life
• Increase the predictability of synthetic biological circuits
• Become a major source of energy
• Replace the petrol-chemical industry
• Enhance bioremediation
• Materials science – expand biology’s use of the periodic
table
• Drive antibiotic and vaccine discovery & production
• Gene therapy via stem cell engineering
It Takes a Village to Create a Cell
Algire, Mikkel
Alperovich, Nina
Assad-Garcia, Nacyra
Baden-Tillson, Holly
Benders, Gwyn
Chuang, Ray-Yuan
Dai, Jianli
Denisova, Evgeniya
Galande, Amit
Gibson, Daniel
Glass, John
Hutchison, Clyde
Iyer, Prabha
Jiga, Adriana
Krishnakumar, Radha
Lartigue, Carole
•Ma, Li
•Merryman, Chuck
•Montague, Michael
•Moodie, Monzia
•Moy, Jan
•Noskov, Vladimir
•Pfannkoch, Cindi
•Phang, Quan
•Qi, Zhi-Qing
•Ramon, Adi
•Saran, Dayal
•Smith, Ham
•Tagwerker, Christian
•Thomas, David
•Tran, Catherine
•Vashee, Sanjay
•Venter, J. Craig
•Young, Lee
•Zaveri, Jayshree
•Johnson, Justin
•Brownley, Anushka
•Parmar, Prashanth
•Pieper, Rembert
•Stockwell, Tim
•Sutton, Granger
•Viswanathan, Lakshmi
•Yooseph, Shibu
Funding from
DARPA Living Foundries
Synthetic Genomics Inc.
DOE GTL program
Dollars per basepair
DNA synthesis is getting easier,
faster, and cheaper
$100.00
$0.15 / bp
$10.00
$1.00
$0.10
$0.01
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