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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 - PowerPoint PPT Presentation
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Rewriting the Genetic Code
BLI Biological Research 2013Synthetic 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 acids- including synthetic ones
• Confer immunity to bacterial DNA
• Rewriting entire genome by manipulating existing code
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
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 worksPositive and positive-negative 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 2013Synthetic Biology Design
ProjectSejal 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 β-lactamase, 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 2-component 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 uva-ursi
• 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