Synthetic biology Genome engineering

1. Synthetic biology Genome engineering Chris Yellman, U. Texas CSSB

2. What is synthetic biology? synthesis: the combination of two or more parts to make a new product greek: synthetos, “put together, constructed, compounded” examples: rearrangements of existing DNA sequences to make new genes, gene fusions, new regulatory elements production of chemicals and drugs with biological activity synthetic insulin (a peptide hormone) made in yeast or E. coli antibodies, such as anti-toxins for snake venom genome synthesis or genome rearrangement: can make genomes that have never existed

3. What makes synthetic biology possible? 1 sequence data from natural genomes - bacteriophages and other viruses - bacteria such as E. coli but many others as well - eukaryotes from yeast to humans (full evolutionary spectrum) • ability to synthesize DNA, RNA, proteins - oligonucleotides, entire genes - peptides (pieces of protein) 3 purified enzymes • DNA polymerase for PCR amplification of DNA from templates • restriction endonucleases to cut DNA at specific sites 4 model organisms with well understood biology • E. coli, a prokaryote, phages and viruses • Saccharomyces cerevisiae (yeast), a eukaryote • Drosophila (fruit fly), C. elegans(worm), mouse, human cells (stem cells, other cell lines)

4. What makes synthetic biology interesting? • make useful natural products • insulin • artemisin (current best anti-malarial drug) • ethanol, other bio-fuels • make new model systems 3 intervene in biological systems to figure out how they work, for example rearrange the genes in a bacterial operon • understand the limitations of evolution and perhaps augment biology with additional amino acids or protein coding • understand the origins of life – can we make a completely artificial cell?

5. Can we make life abiotically (from non-living material)? Jack Szostak’s model of a protocell

6. using redundant codons to expand the genetic code non-natural amino acids can be incorporated into proteins

7. DNA synthesis: the basis for much of synthetic biology DNA of almost any size can now be made entirely in vitro • oligonucleotides • genes • chromosomes 4 genomes

8. oligonucleotide synthesis

9. entire yeast chromosomes have been made in vitro Dymond et al., 2011, Nature, Saccharomyces Genome Database

10. an entire synthetic genome: M. mycoides JCVI-syn1.0 1 kb assemblies in vitro from oligonucleotides 10 kb assemblies in yeast 100 kb assemblies in yeast assembly of the 1.1 mb genome in yeast on a CEN-ARS plasmid Gibson et al., 2010, Science

11. DNA assembly: making meaningful parts • genes, promoters and terminators can be assembled to make operons or bring the genes under different regulation • centromeres and origins of replication are included to give synthetic DNA the properties of native chromosomes • genomes can be assembled to mimic known genomes or to create completely artificial new genomes with genes from different species

12. “Recombineering” • based on homologous recombination in vivo or in vitro • nucleotide base pairing is one of the most fundamental principles in biology • can occur between DNA and/or RNA strands • E. coli and yeast both repair their genomes by homologous recombination • using live organisms “in vivo” takes advantage of natural enzyme activities, DNA repair and proofreading processes, etc. • in vivo hosts have different properties

13. Escherichia coli

14. Saccharomyces cerevisiae Roberta Kwok, 2011 Nature

15. Recombineering using E. coli and yeast • synthesize by PCR • Use E. coli with phage enzymes that promote homologous recombination • multiple linear pieces of DNA are co-transformed into the bacteria, where they are assembled by the endogenous enzymes • we can also modify the native chromosomes of bacteria and yeast

16. Gibson assembly (NEB), an in vitro method Assembly in vitro using purified enzymes “one pot”. Works for multi-piece assemblies.

17. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting Fu et al., 2012 nature biotechnology Court lab, NIH Ryan E Cobb & Huimin Zhao nature biotechnology, 2012

18. multi-piece assembly of ds PCR fragments in yeast

19. Genome engineering moving genes and pathways between species creating mutant libraries of genes to study the genetic basis for diseases bio-prospecting for useful enzymes or other molecules

20. native bacterial “immunity” to phages

21. title

22. How CRISPR/Cas9 will change eukaryotic biology inducing double-strand breaks leaves damage that gets repaired by the cells the repair process can be used to insert new DNA new DNA can be disease alleles of genes, GFP fusions to genes, etc…