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Rewriting the Genetic Code
BLI Biological Research 2013
Synthetic Biology Research Project
Sejal Jain
Replacing TAG with TAA
• In 2011, Farren J. Isaacs of Yale University
and Peter A. Carr of MIT site-specifically
replaced all 314 TAG stop codons in E.
coli with TAA stop codons
• Testing for translational/genomic changes
despite functional similarity
• Chromosome as an “editable and
evolvable template”
Redundant Stop Codons
• RF1 recognizes UAA
and UAG, while RF2
recognizes UAA and
UGA
• If maintained viability
without TAG (and
RF1), TAG would no
longer encode a stop
codon, rendering it
“blank”
Long-Term Goals
• If genome were
engineered to no longer
recognize TAG as a stop
codon, “blank” TAG could
be reprogrammed to
encode amino acidsincluding synthetic ones
• Confer immunity to
bacterial DNA
• Rewriting entire genome
by manipulating existing
code
MAGE Codon Swap
• Multiplex automated
genome engineeringused for TAG-TAA swap
• Pools of water contained
E. coli, single-stranded
DNA fragments
(sequenced in
accordance with 314 TAG
points), and viral
enzymes; underwent
electrical charge to allow
DNA to pass through
bacterial membranes
CAGE Recombination Technique
• After MAGE and sequencing/PCR to confirm
gene modification results, 32 strains with 10
different switch points were isolated
• Conjugative assembly genome engineering
• Uses bacterial conjugation to allow
systematically paired strains to swap DNA
until one strain contains all of the 314
necessary fragments (complete TAG-TAA
swap)
Systematic CAGE
• Donor strain contains oriT-kan cassette,
combining oriT conjugal gene with
kanamycin resistance gene, positive
selection gene, and F plasmid
– cassette easily integrated in any locus on E.
coli genome
• Recipient strain contains positive-negative
selection gene Pn
How the CAGE
system works
Positive and
positivenegative
selections
applied after
conjugation
ensure that
recombinant
strain contains
TAA while
retaining the
other regions
of recipient
genome
Hierarchal CAGE
• After first round of CAGE,
16 strains with twice as
many TAG-TAA changes
produced
• Second stage produced
eight such strains
• Obtained four strains
produced that
theoretically can be
recombined to form one
• Each of the four have 80+
genetic modifications
Frequency map of oligo-mediated TAG::TAA codon
replacements and genetic marker integrations across the
E. coli genome at each replacement position
Bacteria Inhibiting Antibiotic
Resistance in methicillin-resistant
Staphylococcus aureus
BLI Biological Research 2013
Synthetic Biology Design Project
Sejal Jain
What is MRSA?
• A bacterium that has developed extreme
resistance to β-lactam antibiotics
• 40-50% of strains are resistant to newer,
semisynthetic menicillin and vancomycin
• Transmitted through surface contact
• Rampant in hospitals, prisons, nursing
homes
• Patients suffer periodic relapses
The Antibiotic Paradox
• When treated, a few
develop resistance
(mutation or gene
transfer)
• Too much antibiotic
use/too strong
antibiotics -> loss of
drug potency (selects
for more resistant
strains)
Project Goals
• Create a synthetic genetic system in a
bacterium that will synergistically work with
current antibiotics to inhibit antibiotic
resistance
• Lower MIC of drugs- preserve potency
• Mitigate natural selection and horizontal
gene transfer
I. MECHANISMS OF
ANTIBIOTIC RESISTANCE IN
MRSA
SCCmec and the mecA resistance gene
• SCCmec is a genomic
island
• mecR1/mecR2- encode
signal transmembrane
proteins
• MecI- repressor protein
• mecA encodes for
PBP2a (low affinity for
β–lactams,
transpeptidase activity)
blaZ produces β-lactamase
• Homologous to mecA
• Induced in the
presence of β-lactams
• Produces enzyme βlactamase, which
hydrolyzes β-lactam
ring
NorA MDR Efflux Pump
• In the cytoplasmic
membrane
• Uses active
transport to “pump”
out toxic
substances (efflux)
• Multi-drug
resistance
II. GENETIC SYSTEM DESIGN
agr quorum sensing device
• agrBDCA operon
encodes 2component system
• In this design, agrD
and agrB (AIP
synthesis genes)
omitted
• P3 promoter used to
promote inhibitor
sequences instead of
RNAIII
ALO1
• Produces D-Arabino- 1,4-Lactone Oxidase
(ALO)
• Not naturally produced in E. coli
• Catalyzes terminal step in biosynthesis of
D-erythro ascorbic acid (EASC)
• Ascorbate inhibits β-lactamase through
induction of BlaI
Cyslabdan Synthase
• Gene from Streptomyces K04-1044
• Cyslabdan is a labdane-type diterpene, or
protein
• Inhibits transpeptidase activity by inducing
repressor protein FemA
• Prevents MRSA from forming cell walls
even with PBP2a
Corilagin Synthase
• Gene from
Arctostaphylos uvaursi
• Diterpenoid that
potentiates methicillin
by inhibiting PBP2a
cross-linking
• Increases cell
damage
• Lowers MIC
Columbus gene
• Encodes for HMG-CoA
• Synthesizes a protein
called geranylgerynal
pyrophosphate
• Undergoes a diterpene
metabolic pathway that
forms totarol
• Totarol is an EPI
inhibiting NorA
ACL5 antibiotic resistance gene
• Constitutively
expressed
• Ensures that bacteria
won’t die in presence
of β-lactam
• Encodes for
spermine, which
inhibits transport
through porins in OM
III. RESEARCH AND
DEVELOPMENT
Issues/Questions
• Exact genomic sequences producing
corilagin/cyslabdan
• Development of BioBricks
• Determine amount of EASC needed for
MIC of ascorbate
• Make sure spermine binds to β-lactam
porins only
• Specifically target MRSA AIPs
Applications
• Synergistic use with antibiotics will
decrease dependence on stronger
antibiotics (defeats antibiotic paradox)
• Can be applied topically on skin (MRSA
resides in cutaneous/subcutaneous levels)
• Can be used preventatively on surfaces
e.g. intravenous medical equipment