Rewriting the Genetic Code
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Transcript of Rewriting the Genetic Code
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Rewriting the Genetic Code
BLI Biological Research 2013Synthetic Biology Research Project
Sejal Jain
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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”
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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”
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Long-Term Goals• If genome were
engineered to no longer recognize TAG as a stop codon, “blank” TAG could be reprogrammed to encode amino acids- including synthetic ones
• Confer immunity to bacterial DNA
• Rewriting entire genome by manipulating existing code
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MAGE Codon Swap• Multiplex automated
genome engineering- used 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
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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)
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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
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How the CAGE system worksPositive and positive-negative selections applied after conjugation ensure that recombinant strain contains TAA while retaining the other regions of recipient genome
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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
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Bacteria Inhibiting Antibiotic Resistance in methicillin-resistant
Staphylococcus aureus
BLI Biological Research 2013Synthetic Biology Design
ProjectSejal Jain
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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
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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)
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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
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I. MECHANISMS OF ANTIBIOTIC RESISTANCE IN MRSA
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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)
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blaZ produces β-lactamase
• Homologous to mecA
• Induced in the presence of β-lactams
• Produces enzyme β-lactamase, which hydrolyzes β-lactam ring
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NorA MDR Efflux Pump• In the
cytoplasmic membrane
• Uses active transport to “pump” out toxic substances (efflux)
• Multi-drug resistance
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II. GENETIC SYSTEM DESIGN
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agr quorum sensing device• agrBDCA operon
encodes 2-component system
• In this design, agrD and agrB (AIP synthesis genes) omitted
• P3 promoter used to promote inhibitor sequences instead of RNAIII
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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
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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
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Corilagin Synthase• Gene from
Arctostaphylos uva-ursi
• Diterpenoid that potentiates methicillin by inhibiting PBP2a cross-linking
• Increases cell damage
• Lowers MIC
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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
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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
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III. RESEARCH AND DEVELOPMENT
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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
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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