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Rewriting the Genetic Code

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

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Rewriting the Genetic Code

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  1. Rewriting the Genetic Code BLI Biological Research 2013 Synthetic Biology Research Project Sejal Jain

  2. 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”

  3. 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”

  4. 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

  5. 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

  6. 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)

  7. Systematic CAGE • Donor strain contains oriT-kancassette, 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

  8. How the CAGE system works Positive and positive-negative selections applied after conjugation ensure that recombinant strain contains TAA while retaining the other regions of recipient genome

  9. Hierarchal CAGE Frequency map of oligo-mediated TAG::TAA codon replacements and genetic marker integrations across the E. coli genome at each replacement position • 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

  10. Bacteria Inhibiting Antibiotic Resistance in methicillin-resistant Staphylococcus aureus BLI Biological Research 2013 Synthetic Biology Design Project Sejal Jain

  11. What is MRSA? • A bacterium that has developed extreme resistance to β-lactam antibiotics • 40-50% of strains are resistant to newer, semisynthetic menicillinand vancomycin • Transmitted through surface contact • Rampant in hospitals, prisons, nursing homes • Patients suffer periodic relapses

  12. 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)

  13. 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

  14. I. Mechanisms of antibiotic resistance in mrsa

  15. SCCmec and the mecA resistance gene • SCCmec is a genomic island • mecR1/mecR2- encodesignal transmembrane proteins • MecI- repressor protein • mecA encodes for PBP2a (low affinity for β–lactams, transpeptidase activity)

  16. blaZproduces β-lactamase • Homologous to mecA • Induced in the presence of β-lactams • Produces enzyme β-lactamase, which hydrolyzes β-lactam ring

  17. NorA MDR Efflux Pump • In the cytoplasmic membrane • Uses active transport to “pump” out toxic substances (efflux) • Multi-drug resistance

  18. II. Genetic system design

  19. 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

  20. 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

  21. 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

  22. Corilagin Synthase • Gene from Arctostaphylosuva-ursi • Diterpenoid that potentiates methicillin by inhibiting PBP2a cross-linking • Increases cell damage • Lowers MIC

  23. 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

  24. 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

  25. III. Research and development

  26. 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

  27. 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

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