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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?