diritapresentation

Lab Meeting: Isolation and sequencing of transformed C. jejuni

mutants

Jessica Williams

Mentor: Jessica Beauchamp University of Michigan Phd Candidate

DiRita Laboratory

4. 22. 2015

gDNA E. coli C. jejuni 1.010 -9

1.010 -8

1.010 -7

1.010 -6

1.010 -5

1.010 -4

1.010 -3

1.010 -2

Tran

sfor

mat

ion

Effic

ienc

yTransformation in C. jejuni is restricted

Experimental Question

What mechanism allows Campylobacter jejuni to transform E. coli DNA?

• Can we find a mutant that transforms E. coli DNA and identify which gene the transposon has interrupted?

Transformation Assay

1. Re-suspend bacteriato OD600 = 0.5

3. Plate dilutions on antibiotic containing media

2. Incubate 0.5 ml cells with 1μg of DNA for 4 hours at 37°C and 5% CO2

Transformation AssayControls:C. jejuni genomic DNA with a Kan resistance cassette

Experimental:Digested E. coli plasmid DNA- PSK plasmid backbone - Kan resistance cassette inserted into ZupT - Linearize plasmid DNA to ensure resistance is due to

recombination onto the chromosome

Plasmid with Kan cassette flanked by ZupT

Summary (thus far)• Completed plates 60 – 41• Successful transformation seen in 54, 53, 51, 43• 86 hits • Will continue screening library

Regrow mutant colonies: • Chloramphenicol and kanamycin resistant

- Transposon has CM resistance cassette- Mutants have Kan cassette

• Nalidixic acid as selection mechanism

Confirm Transformation

WT 51-1 53-1 53-2 53-12 53-13 54-1 54-2 54-12 54-13 54-1410 -8

10 -7

10 -6

10 -5

10 -4

10 -3

Transposon Mutant TE

Tran

sfor

mat

ion

Effic

ienc

y

gDNAECNo DNA

Transformation Efficiency of Mutants (Naladixic Acid selection)

Re-testing 54-12

Isolating Transposon Insertion

• Prepped gDNA from successful transformants• 2 methods: Ligation and Arbitrary PCR

– More sequence with Ligation

Arbitrary PCR

Ligation Method

• Use RsaI to chop genome• Clean up and precipitate DNA • Mix with pGEM and ligase • If transposon is taken up will be resistant to CM

pGEM

DNA fragment with Transposon

Hits Summary Mutant # Method Gene ID Gene Function54-1 Ligation 81-176_0179 znuA: zinc uptake protein

Arbitrary PCR 81-176_0179 znuA: zinc uptake protein

54-2 Arbitrary PCR 81-176_pVir0053 virB4: ATPase

54-3 Ligation CJJ81176_1548 methyl-accepting chemotaxis protein

54-4 Ligation CJJ81176_1548 methyl-accepting chemotaxis protein

54-5 Ligation CJJ81176_1080 ileS: isoleucyl-tRNA synthetase

54-10 Ligation Cjj_1500 fdhD: formate dehydrogenase accessory protein

54-12 Arbitrary PCR 81-176_pVir0028 Hypothetical Protein

53-2 Ligation 81-176_0939 pckA: phosphoenolpyruvate carboxykinase

Arbitrary PCR CJJ81176_0938 argH: argininosuccinate lysase

51-1 Arbitrary PCR CJJ81176_0938 argH: argininosuccinate lysase

Retesting ∆znuA

Future Experimentation

• Continue screen – ID current hits and continue with library – Leaning away from rescreening hits and focus on

identification of insertion

Questions?