Synthetic Biology The Big Picture Want synthetic genomes
Synthetic Biology
The Big Picture • Want synthetic genomes to use as ‘biofactories, ’ producing materials useful to humans • Want the minimal genome to use as the building block for synthetic life • Need ability to synthesize genomes to find the minimal genome
Early efforts • 1979 - Synthesis of 207 bp gene of tyrosine suppressor t. RNA (Khorana et al) • 1990 - Synthesis of a 32 kb polyketide gene cluster (Kodumal et al) • 2002 - Full length, infectious polio virus (Cello et al) • 2003 - φX 174 bacteriophage synthesis (Venter et al)
Minimal genome? “The ‘minimal genome’ approach seeks to estimate the smallest number of genetic elements sufficient to build a modern-type free-living cellular organism. ” (Mushegian) Essential set of survival genes
Prerequisites • Knowledge of existing genomes • Define shorter list of key players by dry-lab comparative analysis. • Protein sequence similarities. • Key: homology is the basic concept of any evolutionary analysis
Why Mycoplasma • Part of the mollicutes- generally known as mycoplasma. • Wall-less • Evolved by massive genome reduction • Obligate parasites • Smallest known genome of any free living organism capable of growing in axenic culture • Lack genomic redundancy
Testing for non-essential genes • Used transposon mutagenesis to systematically disrupt genes • Looked for mutants that survived after 4 weeks (in order to detect slow growing mutants) • Detected about 100 nonessential genes • Statistically approaching saturation
What genes are nonessential? • 48% of genes found were hypothetical proteins or encoded proteins of unknown function • Some of those that were identified: DNA metabolism, transporters, recombination, DNA repair • Some genes identified in the study may be essential for long term survival. Metabolic genes… DNA repair… • Found more genetic redundancy than previously thought
Why care about artificial chromosomes? • 100 genes are not essential, but… • In combination? • Need to be able to efficiently assemble reduced genomes with combinations of these genes missing
http: //www. youtube. com/watch? v=i. Q 1 VNEgc. W E 8
M. Genitalium JCVI-1. 0 • Contains functional copies of all wild type genes except MG 408 • MG 408 disrupted by antibiotic marker to block pathogenicity and allow selection • “Watermarks” in intergenetic regions • Designation?
5 -7 kb cassettes • Partition up the 583 kb M. genitalium genome into 101 pieces. • Overlaps 80 -360 bp • Boundaries between genes- why? • Insertion of aminoglycoside resistance into MG 408
The Artificial Chromosome: How Did They Do It? • In the past… showed 5 -7 kb fragments could be assembled de novo. • Take these small fragments and join them invitro to make larger assemblies… • …And larger… • Until you can synthesize a ~583 kb genome
Five Stage Assembly
In-Vitro Recombination and Vector Insertion
In-Vitro Recombination • 3’ exonuclease “chew-back” using T 4 polymerase without d. NTPs • Annealing • Repair gaps w/ Taq pol. and Taq ligase
Vector Prep and Insertion • To prepare the BAC using primers with “tails” homologous ends of the A assembly. • Also engineer in Not 1 sites. More about that later… • Clone into E. coli and amplify plasmid copies
What About The Not 1 Sites?
In-Vivo Recombination In Yeast • ½ genomes assembled by in-vitro recombination did not work well. Why? • TAR cloning: homologous recombination invivo • ¼ genomes combined to form whole genome in circular p. TARBAC 3 vector • This is cool because you are combining 6 pieces of DNA at once • One of the ¼ genomes is cut. Why?
Whole Genome: QC and Recovery • Screened yeast transformants by PCR + Southern Blot • Positive clones tested for stability by Southern Blotting of subclones • Selected one of the clones for shotgun sequencing – Isolate and enrich artificial chromosome – Purify – Shotgun sequence: 7 x coverage
ERROR! Errors in sequence supplied to contractors Errors in cassettes synthesized by contractors From repair of assembly junctions From propagation of assemblies in E. coli and yeast • Sequenced assemblies at various stages: mostly correct, a few errors. These were corrected • •
Bioethics “…progress in science and technology often outpaces the relevant ethical, legal and moral discourse, and regulation…” (Gabrielle et. al)
Great promise. . . • • • Renewable fuel sources Pharmaceuticals Chemical detoxification Environmental control Beneficial microbes
. . . or great risk? • • Synthetic pathogens Genetic transfer (similar to GMO arguments) Economic risk Patent/ownership
Regulation • Self-governance • US National Science Advisory Board for Biosecurity (NSABB) • Trade regulation (one for you, two for me)
Isolation • Physical isolation • Biological isolation – New genetic code – Engineered nutrient dependency – Programmed cell death – Microbial ‘bouncers’ – Remove genetically mobile elements
NSABB’s ‘Experiments of Concern’ • Include experiments that might create knowledge, products or technologies that could enhance the harmful consequnces of a biological agent of toxin; disrupt immunity or the effectiveness of an immunization without clinical and/or agricultural justification; introduce resistance of a biological agent against useful prophylactic or therapeutic interventions, or facilitate their ability to evade detection methodologies; increase the stability, transmissibility or the ability to disseminate a biological agent or toxin; alter the host range or tropism of a biological agent or toxin, enhance the susceptibility of a host population; generate a new pathogenic agent or toxin; or reconstitute an eradicated or extinct biological agent (NSABB, 2007).
References • Gibson, D. G. Et al. (2008) Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. • Glass, J. I. et al. (2005) Essential Genes of a Minimal Genome. PNAS 103, 425 -430 •
Questions?
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