Transcript E. coli - Marcotte Lab

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 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
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)
2 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)
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
DNA of almost any size can now be made entirely in vitro
1 oligonucleotides
2 genes
3 chromosomes
4
genomes
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 bioprospecting
Fu et al., 2012 nature biotechnology
Court lab, NIH
Ryan E Cobb & Huimin Zhao nature biotechnology, 2012
multi-piece assembly of ds PCR fragments in yeast
p15A ori-camR
2.4 kb
pSENCEN-ARS-natR
5688 bp
tetR terminator
~300 bp
pLac ~180 bp
CEN-ARS-natR
2 kb
•
all fragments are PCR products,
with 36-42 bp overlaps
•
~ 150 yeast colonies
•
10/30 are full assemblies by
PCR analysis and NotI digestion
•
6/10 assemblies are correct
by sequencing
LasI ~700 bp
B0014 terminator
~200 bp
spacer ~300 bp
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
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…