Preparation of Genomic Overexpression Library

Materials and Reagents

1. Bacteria donor (E. coli MG1655 wild-type or the ΔhslO deletion)
   
2. Bacteria recipient (Vibrio cholerae O395 ΔhslO deletion)
   
3. GenElute Bacterial Genomic DNA kit (Sigma-Aldrich)
   
4. Plasmid pBR322 (New England BioLab, linearized with BamH1)
   
5. Plasmid pET11a (New England BioLab, linearized with BamH1)
   
6. BamH1 (New England Biolabs)
   
7. BfuCI (New England Biolabs)
   
8. T4 DNA ligase (New England Biolabs)
   
9. Gel Extraction Kit (QIAGEN)
   
10. Ultracompetent XL10-Gold cells (Stratagene)
    
11. Wizard Plus SV Minipreps kit (Promega Corporation)
    
12. MacConkey plates (lab-made)
    
13. LB medium
    
14. TAE buffer
    

Equipment

1. Gel electrophoresis apparatus
   
2. Incubator for bacteria
   

Procedure

1. Prepare genomic DNA from the donor E. coli strains (MG1655 wild-type or the ΔhslO deletion) from 5 ml overnight culture (in LB medium) using the GenElute Bacterial Genomic DNA kit following the manufacturer’s instruction.
   
2. Partially digest the genomic DNA (2 μg) with 0.05-0.2 units of BfuCI in a total volume of 50 μl for 10 min at 37 °C. Then heat inactive the enzyme.
   
3. Analyze digested products on 1% agarose gels (in TAE buffer) and make sure that fragment sizes ranging between 1 and 8 kb.
   
4. Gel purify fragmented genomic DNA with Gel Extraction Kit (some researchers prefer to separate DNA fragments into bins, for example 1-3 kb, 3-5 kb and 5-8 kb).
   
5. Ligate the DNA fragments (approximately 50-80 ng/μl) into linearized pBR322/BamH1 or pET11a/BamH1 plasmid vectors (approximately 80-100 ng/μl) using T4 DNA ligase overnight at 15 °C following the manufacturer’s instruction.
   
6. To propagate overexpressing plasmids library, transform 1-5 μl of ligation results into ultracompetent XL10-Gold cells following the manufacturer's protocol.
   
7. Count colony forming units (CFU) of transformants the next day and calculate the size of the libraries (1 x 10⁵ CFU/ml of cell or more is good library size).
   
8. Combine the transformants by scraping off all colonies from the library and purify the total plasmids using Wizard Plus SV Minipreps kit.
   
9. Transform 1-3 μl of the E. coli genomic library plasmids into recipient Vibrio cholerae ΔhslO deletion.
   
10. Plate the transformants on MacConkey agar and incubate for 24 h at 43 °C to select for clones that rescue the temperature sensitive phenotype of the V. cholerae ΔhslO mutant strain (this mutant cannot grow on MacConkey plate at 43 °C).
    
11. To ensure the growth phenotype, re-streak transformants that formed healthy looking colonies on MacConkey plates and grew for 24 h at 43 °C.
    
12. To eliminate the possibility of mutations in the strain background, purify and re-transform plasmids (that rescured phenotype) of transformants into V. cholerae ΔhslO mutant.
    
13. To identify inserted E. coli gene(s), purify the plasmid and send for DNA sequencing.
    

Acknowledgments

The protocol described here is adapted from one reported previously (Wholey et al., 2012).

References

1. Wholey, W. Y. and Jakob, U. (2012). Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae. Mol Microbiol 83(5): 981-991.