Synthetic biology

Genome engineering

Chris Yellman, U. Texas CSSB

What is synthetic biology?

synthesis: the combination of two or more parts to make a new productgreek: 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

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)

2ability 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)

What makes synthetic biology interesting?

1 make useful natural products- insulin- artemisin (current best anti-malarial drug)- ethanol, other bio-fuels

2 make new model systems

3 intervene in biological systems to figure out how they work, for example rearrange the genes in a bacterial operon

4 understand the limitations of evolution and perhaps augment biology with additional amino acids or protein coding

5 understand the origins of life – can we make a completely artificial cell?

Can we make life abiotically (from non-living material)?

Jack Szostak’s model of a protocell

using redundant codons to expand the genetic code

non-natural amino acids can be incorporated into proteins

DNA synthesis: the basis for much of synthetic biology

1 oligonucleotides

2 genes

3 chromosomes

4 genomes

DNA of almost any size can now be made entirely in vitro

oligonucleotide synthesis

entire yeast chromosomes have been made in vitro

Dymond et al., 2011, Nature, Saccharomyces Genome Database

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

DNA assembly: making meaningful parts

1 genes, promoters and terminators can be assembled to make operons or bring the genes under different regulation

2 centromeres and origins of replication are included to give synthetic DNA the properties of native chromosomes

3 genomes can be assembled to mimic known genomes or to create completely artificial new genomes with genes from different species

“Recombineering”

1 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

2 E. coli and yeast both repair their genomes by homologous recombination

3 using live organisms “in vivo” takes advantage of natural enzyme activities, DNA repair and proofreading processes, etc.

4 in vivo hosts have different properties

Escherichia coli

Saccharomyces cerevisiae

Roberta Kwok, 2011 Nature

Recombineering using E. coli and yeast

1 synthesize by PCR

2 Use E. coli with phage enzymes that promote homologous recombination

3 multiple linear pieces of DNA are co-transformed into the bacteria, where they are assembled by the endogenous enzymes

4 we can also modify the native chromosomes of bacteria and yeast

Gibson assembly (NEB), an in vitro method

Assembly in vitro using purified enzymes “one pot”.

Works for multi-piece assemblies.

Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospectingFu et al., 2012 nature biotechnology

Ryan E Cobb & Huimin Zhao nature biotechnology, 2012

Court lab, NIH

multi-piece assembly of ds PCR fragments in yeast

• ~ 150 yeast colonies

• 10/30 are full assemblies by PCR analysis and NotI digestion

• 6/10 assemblies are correctby sequencing

pSENCEN-ARS-natR5688 bp

CEN-ARS-natR2 kb

B0014 terminator~200 bp

spacer ~300 bp

LasI ~700 bp

pLac ~180 bp

tetR terminator~300 bp

p15Aori-camR2.4 kb

• all f

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

Genome engineering

native bacterial “immunity” to phages

title

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…