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 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.

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 cellSynthetic cell Genome Transplantation Genome Synthesis

Mycoplasma capricolum Mycoplasma mycoides RECIPIENT CELLS gDNA DONOR Science August 2007

Science February /251/8 1/4 Whole kb 72kb 144kb 580kb 24kb 50-77B 50-77A Yeast Vector yeast E. coli Chemical Synthesis

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

Science May 2010

Desig n Codon Opt. Oligo Synthesis Synthetic Organism Designer 1.0

Tomography Cells are complex machines with thousands of moving parts Mycoplasma pneumoniae from Proteome organization in a genome-reduced bacterium. Kuhner et al Science 326:

Tomography Cells are 25-50% Dark Matter Hypothetical Proteins Small Peptides of unknown function Small RNAs of unknown function Epigenetic Modifications Moonlighting Proteins ?

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.

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 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. What bacterial cell will we minimize?

Gibson et al., 2010 Science 329, Synthesis of the Mycoplasma mycoides JCVI-syn1.0 Genome

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

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 non- essential 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

M. mycoides wild type1089 kb M. mycoides JCVI-syn 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) 980 kb M. mycoides JC syn1.0 – 6RM(12 genes, 17 kb) 928 kb – 6 IS (12 genes, 9 kb) – ICE (44 genes, 71 kb) – D5 deletions (52 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 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. Design and synthesis of a “Hail Mary” genome

“Hail Mary Deletions” mapped onto M. mycoides JCVI-syn1.0 West Coast Design

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 “Hail Mary” Minimal Genome Construction 1/8 th molecules can be individually tested for functionality and mixed with subsequent designs

KEEP DELETE Amino acid biosynthesis 0 4 Biosynthesis of cofactors 9 2 Cell envelope2892 Cellular processes 3 8 Central intermediary metabolism 7 8 DNA metabolism 3232 Energy metabolism2835 Fatty acid and phospholipid metabolism 7 6 Hypothetical proteins Mobile and extrachromosomal element fcns 014 NULL(tRNAs, rRNAs, RNAs)49 0 Protein fate2223 Protein synthesis Purines19 7 Regulatory functions 9 8 Signal transduction 314 Transcription14 4 Transport and binding proteins3533 Unknown function2147 Yeast vector and markers 4 0 ___________________________________________________________ TOTAL Hail Mary Genes by functional category

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? Key promoterterminatortRNA gene 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. M. mycoides JCVI-syn 1.0

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 Funding from DARPA Living Foundries Synthetic Genomics Inc. DOE GTL program  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

DNA synthesis is getting easier, faster, and cheaper Dollars per basepair Year $0.15 / bp Agilent