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C. jejuni Full ORF Expression Clones

C. jejuni Full ORF Expression Clones

Campylobacter jejuni is the most commonly recognised cause of bacterial gastroenteritis in humans. Initial difficulties in culture requirements, and attempts at genetic modification held back the progress in understanding this organism compared to other enteric pathogens. 

The sequencing of 1.64 million nucleotides in the C. jejuni genome at the Wellcome Trust Sanger Institute has heralded a new era in C. jejuni research. Using this sequence information, along with 31 predicted ORF's by TIGR, Jodi Parrish (Russell Finley's lab - Wayne State University School of Medicine) has created a C. jejuni ORF library.

Campylobacter jejuni is the most commonly recognised cause of bacterial gastroenteritis in humans. Initial difficulties in culture requirements, and attempts at genetic modification held back the progress in understanding this organism compared to other enteric pathogens.

The C. jejuni ORF library is a proteome-scale expression clone set comprising of more than 1,600 ORF's. The clones were generated by efficiently utilizing the native E. coli recombination system.

Recombination sites have been confirmed by sequencing, and most of the clones have been 5' sequence verified. The ORFs range from 45 - 4020bp in length. After expression, affinity purification can be performed on the proteins using either GST or His-tags.

The clone set has many applications, including protein interactions, activity, and structural studies. Furthermore, the clones can be utilised for gene expression patterns by micro-array analysis. The process of endogenous recombination in E. coli facilitates the insertion of the ORF in the correct 5' to 3' direction. The antibiotic resistance gene, encoded on the plasmid, allows for the selection of the new construct.

Protocols / Clone Handling

Plates- contain LB broth with 8% glycerol and ampicillin. These are sent on Dry Ice. These should be stored at -70°C.

Individual clones pools have been streaked onto LB agar containing ampycillin (100µg/ml). Please store them at 4°C (not in a freezer). As soon as possible, transfer the culture to LB broth containing ampicilllin + 8% glycerol and incubate overnight at 37°C (do not try to transfer single colonies, instead scoop out the whole bacterial culture using a disposable inoculation loop). Incubate overnight at 37°C for subsequent freezing at -70°C.

These clones are for research purposes only.

References

We are grateful to Russell Finley & Jodi Parrish (Wayne State University School of Medicine) for the gift of this library and associated database.

Please acknowledge the Wayne State University School of Medicine and Source BioScience LifeSciences in any resulting publications.

Please also cite the following paper:

Parrish J, Limjindaporn T, Hines JA, Liu J, Liu G, and Finley RL, Jr. High-throughput cloning of Campylobacter jejuni ORFs by in vivo recombination in Escherichia coli, J. Proteome Research, May-June 2004; 3(3): 582-586.


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