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