1 / 29

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

calida
Download Presentation

Rewriting the Genetic Code

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related