Reverse genetics on a non-model organism

Reverse genetics

• Gene in hand. What’s its function?

Thiomicrospiras

JANNASCH (H.W.), WIRSEN (C.O.), NELSON (D.C.) and ROBERTSON (L.A.): Thiomicrospira crunogena sp. nov., a colorless, sulfur-oxidizing bacterium from a deep-sea hydrothermal vent. Int. J. Syst. Bacteriol., 1985, 35, 422-424.

Enterics

Sodalis glossinidius str. morsitans Photorhabdus luminescens subsp. laumo...

Blochmannia/Buchnera

Haemophilus spp.

Vibrio/Photobacterium spp.

Aeromonas salmonicida subsp. salmonic... Aeromonas hydrophila subsp. hydrophil...

Shewanella spp.

Idiomarina loihiensis L2TR Pseudoalteromonas haloplanktis TAC125...

Psychromonas ingrahamii 37 Pseudoalteromonas atlantica T6c Colwellia psychrerythraea 34H

Pseudomonas spp.

Saccharophagus degradans 240 Hahella chejuensis KCTC 2396

Chromohalobacter salexigens DSM 3043 Alcanivorax borkumensis SK2

Marinobacter aquaeolei VT8 Marinomonas sp. MWYL1

Psychrobacter spp.

Acinetobacter spp.

Xanthomonas/Xyllela spp.

Dichelobacter nodosus VCS1703A Legionella spp.

Methylococcus capsulatus str. Bath Coxiella spp.

Nitrosococcus oceani ATCC 19707 Halorhodospira halophila SL1

Alkalilimnicola ehrlichei MLHE1 Thiomicrospira crunogena XCL2

Candidatus Vesicomyosocius okutanii HA Candidatus Ruthia magnifica str. Cm (... Francisella spp.

Sulfurovum sp. NBC371 Campylobacter jejuni subsp. jejuni NC...

Wolinella succinogenes DSM 1740 Thiomicrospira denitrificans ATCC 33889

Candidatus Carsonella ruddii PV35

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9468

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• Competence?• Conjugation?• What works on the

relatives?

Genetically manipulating a nonmodel organism

Genetically manipulating a nonmodel organism

• Take a comprehensive EKS approach– Competence

• Electroporation– Buffers, voltages, growth stage

• Chemical competence– Buffers, growth stage, heat shock conditions

• Natural competence– Growth stage

• Vector

– Conjugation• Mating with E. coli = success!

– pRL27 for random mutagenesis– pLD55 for site-directed mutagenesis

Things to tweak for mating

• Growth stage of recipient cells– Exponential, stationary?

• Mating medium – FW heterotroph + SW lithoautotroph = TLA?– TASW + LB, 30oC

• Mating interval (o/n)• Recovery interval (o/n)• Strength of initial counterselection

– Antibiotic conc’n

Functional genomics projectMicrobial physiology (MCB 4404L)

• Seniors• 50 students (2X25)• Bio/Microbio/BMS majors

• 2 student assistants (volunteers from class)

Our system: Thiomicrospira crunogena

Chemolithoautotroph *requirements for growth: O2, thiosulfate, CO2, ammonia or nitrate, phosphateMotile14 methyl-accepting chemotaxis protein (MCP) genes *14 ‘noses’ to sense nutrients or toxins *what does each MCP detect????

Steps to find what each MCP detects

1. Characterize chemotaxis in wild-type 2. Make 14 mutant strains3. Screen the phenotype of the mutant strains4. Write this up in a lab report.

1. Characterize chemotaxis in wild-type T. crunogena

• We tested for chemotaxis toward:– High O2

– Low O2

– Thiosulfate– Phosphate– Nitrate– Ammonia– Bicarbonate

Chemotaxis assay

No nu-trients added

N2 O2 10 mM

HCO3-

10 mM

PO4-3

10 mM

S2O3-2

10 mM

NO3-

10 mM NH4

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Series1

Cell suspension

Chemotaxissolution

2. Make 14 mutant strains, each with one of its methyl-accepting chemotaxis genes interrupted

2. Making 14 MCP mutant strains using site-directed mutagenesis

1. Amplify target genesfrom T. crunogena gDNAvia PCR

2. Ligate PCR product intoworkhorse plasmid (pRC3.1); TCCE 3. Subject plasmid to Tn5-mediated

mutagenesis in vitro; TCCE

4. Screen clones for Tn5-interruptedtarget gene

5. Amplify interrupted target genes via PCR6. Ligate interrupted genes

into mating plasmid (pLD55); TCCE

7. Mate into T. crunogena

1. E. coli (blue) carrying a plasmid (black), which carries a plasmid that contains a methyl-accepting chemotaxis gene interrupted by a transposon (yellow) that contains a kanamycin resistance gene (red), is mated with T. crunogena (pink)

E. coli T. cruno E. coli

2. The transposon (yellow) cannot hop off the plasmid, as this plasmid does not express a transposase enzyme. Instead, the RecA protein catalyzes homologous recombination between the mutated gene on the plasmid and the wild-type gene on the chromosome, conferring kanamycin resistance on the recipient cell.

T. cruno

RecA

Second selection to remove wt copy

• Fusaric acid to impair TetR cells– Selects for double recombinants

3. Screening the phenotype of the 14 MCP mutant strains

• Redo the chemotaxis assay, but use the mutant strains of T. crunogena instead of wild-type

• Does chemotaxis change in any of the mutants?• Can we correlate a nutrient to a particular MCP?

Next semester

• Pick up where we left off• Mate into T. crunogena• See if chemotaxis behavior changes• Functional complementation in E. coli