strains

NonModel Developing a Framework for the

Genetic Manipulation of Non-Model and Environmentally Significant Microbes

Yale University iGEM Colin Hemez, Lionel Jin, Danny Keller,

Dan Shapiro, Jessica Tantivit, Erin Wang, Holly Zhou

2015 iGEM Giant Jamboree Sunday, September 27

Why Engineer Non-Models?

“. . . the applica,on of synthe,c biology methods and techniques needs to be broadened to a larger group of

organisms to fully reach its poten,al.”

– Ramey et al. Acs. Synth. Bio. 2015

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Problems we chose to address:

Lipid bioduel-producing cyanobacteria and

super-rhizobia

What We Developed: A framework for building expertise in non-model prokaryotes

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Choose Strain

Grow

Select

Transform

Modify Genomes

Repor?ng Assays

Multiplex Automated Genome Engineering (MAGE) in E. coli

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Grow Cells

Transform Oligos

Recover Cells

Induce λ-‐Red

Screen for Phenotypes

Wang, H. et al. Nature 2009.

CRISPR-Cas9 Systems (Syn bio’s favorite new search-and-destroy tool)

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5’

3’

3’

5’

5’

3’ gRNA

Target DNA

Cas9

Hsu, P., Lander, E. & Zhang, F. Cell 2014.

Let’s Run Through the NonModel Framework

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Yale iGEM 2015

Choose Strain

Grow

Select

Transform

Modify Genomes

Repor?ng Assays

Choose Strain Grow Select Transform Modify

Genomes Repor?ng Assays

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Meet Our Strains Synechococcus sp. PCC 7002

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Sinorhizobium melilo1 1021

Rhizobium tropici CIAT 899

Mar?nez-‐Romero E, Segovia L et al. Int. J. of System. Biotech.1991. Ruffing A. Front. Bioeng. Biotechnol. 2014.





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•  Strains: –  Cyanobacteria: Synechococcus 7002 –  Rhizobia: Sinorhizobium melilo= 1021,

Rhizobium tropici •  Growing media:

–  Cyanobacteria: A+ Media –  Rhizobia: Tryp?c Soy Broth & LB

Growth of PCC7002 with Glycerol Supplemented (OD 730nm vs. Time)

•  Doubling ,mes: –  PCC7002: 4 hours –  Rhizobia: 5-‐7 hours



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Growth of PCC7002 in A+/Kanamycin Media (OD 730nm vs. Time)



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INNATE ANTIBIOTIC RESISTANCES IN RHIZOBIUM STRAINS

An,bio,c R. tropici CIAT S. melilo, 356 S. melilo, 370 S. melilo, 371

Culture Solid Liquid Solid Liquid Solid Liquid Solid Liquid

Streptomycin NO NO NO YES NO YES NO YES

Carbenicillin YES NO NO NO NO NO NO YES

Kanamycin NO NO NO NO NO NO NO NO

Rifampicin NO NO NO NO NO NO NO NO

Spec?nomycin NO NO NO NO NO NO YES YES



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Transformation of PCC7002 by

Natural Competency



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Transformation of Rhizobium Strains by Conjugation and Electroporation



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Quan?fy expression of promoters using reporter gene

Assemble to gene of interest

Transform construct and induce expression



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Promoters drive expression in E. coli

Citrine TerminatorPromoter

•  Improved characteriza?on of Anderson promoters •  Submibed 7 new biobricks



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Tested promoters S. meliloti – 3 usable promoters

•  Anderson Strong, Anderson Medium and tac drive expression Citrine TerminatorPromoter



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Recombinases •  Iden?fied 16 recombinases using BLAST – submibed 3 as biobricks

-‐ lambda beta -‐ 9 rhizophage recombinases -‐ 6 cyanophage recombinases

•  Successfully assembled Anderson Medium to 6 different recombinases and transformed into S. melilo=

•  Next step: MAGE



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DNA repair machinery knockout ! Increase MAGE efficiency •  Successful mutS KO by natural transforma?on with linear DNA

mutS

kanRFRT FRT

Upstream Homology

Downstream Homology

∆mutSWT



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Iden?fy selectable marker

Iden?fy screenable marker

Quan?fy protocol efficiency

Generalize to desired func?on

MAGE •  Selectable marker – an?bio?c resistance •  Screenable marker – fluorescence/morphology change

CRISPR •  Knockout gene ! Confer resistance



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5’ 3’

FRT Sites

kanR mCit ampR

TAG TAA

1 kb Upstream 1 kb Downstream

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Modeling A PCR mutation predictor for higher fidelity BioBrick amplification

Sharifian, Hoda. Errors induced during PCR amplifica?on. Swiss Federal Ins?tute of Technology, Department of Computer Science (2010). hbp://dx.doi.org/10.3929/ethz-‐a-‐006088024

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40

Fide

lity

n

Fidelity vs number of PCR cycles

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Human Practices and Outreach iGEM and LGBTQ+

Summer Science Research Institute and Science Café

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Collaborations Validating the common themes in non-model organism research

Cornell

Northeastern

La Verne

Utah State

Concordia

British Columbia

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Recap Many problems could be

solved with synthe?c biology applica?ons in non-‐model organisms

Let’s make those organisms easier to

gene?cally engineer!

Many thanks to our sponsors

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Thank You! iGEM Lab Researchers

Colin Hemez

Lionel Jin

Danny Keller Dan Shapiro

Jessica Tan?vit

Erin Wang

Holly Zhou

Special Thanks

Maria Moreno

Modeling Team

Joe Lanzone

Andrew Saydjari

Research Coordinator

Ariel Hernandez-‐Leyva

Board

Ed Kong Alex Buhimschi

Stephanie Mao

Yamini Naidu

Graduate Mentors

Natalie Ma

Jaymin Patel

Corey Perez Paul Muir

Faculty Sponsors

Steve Dellaporta

Farren Isaacs

Questions?

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Supplements

Anderson Promoters -‐  Picked 3 promoters from previously exis?ng biobricks -‐ Strong, Medium, Weak

-‐  J23100, J23111, J23114 -‐  Assembled to citrine and T7 terminator to generate new biobricks

-‐  K185002, K185003, K185004 -‐  Characterized in E. coli and S. melilo=

BioBricks - Improved Characterization + New Constructs

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New Inducible Promoter Constructs -‐  bacA, melA, tac, lac → Assemble to citrine and T7 terminator

-‐  K185000, K185001, K185005, K185006

-‐  Characterized in E. coli and S. melilo= New Recombinases -‐  Rhizobium phage recombinases gp32-‐based cyanophage recombinase

BioBricks - New Constructs

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•  mutS func?ons in the DNA mismatch repair pathway –  Highly conserved system from prokaryotes to higher eukaryotes

•  MutS protein recognizes a DNA mismatch •  Since DNA mismatch is necessary for the MAGE mechanism, a

mutS KO is essen?al for high mutagenesis efficency

Why Knock Out mutS?

Acharya. Molecular Cell. 2003

Flp-‐FRT Recombina,on: A Knockout Technique Adapted from the Saccharomyces cerevisiae 2µm Plasmid

•  2µm plasmid confers no known evolu?onary advantage to yeast cells –  Plasmid “flips” between two inverted

isoforms

•  “Flipping” ac?on is catalyzed by Flp recombinase recognizing 48 bp flippase recogni?on target (FRT) sites

•  Flp either deletes or inverts sequence between FRT sites, depending on FRT site orienta?on

Ava I

Ava I

Pst I

Pst I Ava I

Ava I

B

A

2µm Plasmid Isoforms

599 bp inverted repeats Cox. Proc. Natl. Acad. Sci. 1983. Schweizer. J. Mol. Microbio. and Biotech. 2003.

•  Conserved 48 bp sequence –  3x 13bp symmetry sequences

–  8bp asymmetry element containing Xba I restric?on site –  1bp spacer

The FRT Site

5’-GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3’

3’-CTTCAAGGATTAGGCTTCAAGGATTAGAGATCTTTCATATCCTTGAAG-5’

Schweizer. J. Mol. Microbio. and Biotech. 2003.

Xba I

Effect of Asymmetric FRT Site Element on Flp Ac,on

Schweizer. J. Mol. Microbio. and Biotech. 2003.

leads to…

FRT FRT Sequence

leads to…

•  DNA between FRT sequences poin?ng in the same direc?on is deleted

•  DNA between FRT sequences poin?ng in opposite direc?ons is inverted

Surrounding DNA Surrounding DNA

FRT FRT Surrounding DNA Surrounding DNA

•  Two components are needed, regardless of species: –  A selectable marker gene flanked by same-‐direc?on FRT sites

–  Source of Flp recombinase

Implementa,on of Flp-‐FRT for Gene Knockouts in Bacteria

In E. coli •  λ-‐Red recombinase replaces KO

target with marker/FRT sequence (35-‐50 bp homology sequences)

•  Flp gene (under heat-‐inducible promoter) is transformed on a heat-‐curable plasmid

In Cyanobacterium •  Na?ve recombina?on

mechanisms replace KO target with marker/FRT sequence (1 kb homologies)

•  Flp gene (under Cu-‐limi?ng or IPTG-‐induced promoter) is transformed on a heat-‐curable plasmid

Datsenko and Wanner. PNAS. 2000. Schweizer. J. Mol. Microbio. and Biotech. 2003. Tan et al. Appl. Microbiol. Biotechnol. 2013.

•  Necessary Components: –  A selectable marker gene flanked by same-‐direc?on FRT sites

–  Source of Flp recombinase

Strategy for mutS Knockout in Sinorhizobium melilo1 1021

In Cyanobacterium •  Na?ve recombina?on

mechanisms replace KO target with marker/FRT sequence (1 kb homologies)

•  Flp gene (under Cu-‐limi?ng or IPTG-‐induced promoter) is transformed on a heat-‐curable plasmid

In Rhizobium •  Homologous recombina?on

mechanisms integrate plasmids containing marker/FRT sequences between KO target

•  Flp gene (cons?tu?ve expression) is transformed on a plasmid with sucrose sensi?vity cassebe

House et al. App. Env. Microbiol. 2004. Tan et al. Appl. Microbiol. Biotechnol. 2013.

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