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-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 P n

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 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

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

12. 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

13. 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)

14. 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

15. I. MECHANISMS OF ANTIBIOTIC RESISTANCE IN MRSA

16. 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)

17. blaZ produces β-lactamase Homologous to mecA Induced in the presence of β-lactams Produces enzyme β- lactamase, which hydrolyzes β-lactam ring

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

19. II. GENETIC SYSTEM DESIGN

21. 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

22. 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

23. 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

24. Corilagin Synthase Gene from Arctostaphylos uva- ursi Diterpenoid that potentiates methicillin by inhibiting PBP2a cross-linking Increases cell damage Lowers MIC

25. 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

26. 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

27. III. RESEARCH AND DEVELOPMENT

28. 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

29. 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