Preparation of Genomic Overexpression Library Protocol

Experiment Summary

This protocol is used to identify/select for Escherichia coli genes that, when overexpressed in Vibrio cholerae Hsp33 (hslO gene) deletion mutant, protect against oxidative heat stress and, by extension, against HOCl-mediated protein damage.

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

4. Plasmid pBR322

5. Plasmid pET11a

6. BamH1

7. BfuCI

8. T4 DNA ligase

9. Gel Extraction Kit

10. Ultracompetent XL10-Gold cells

11. Wizard Plus SV Minipreps kit

12. MacConkey plates

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 Bacterial Genomic DNA kit.

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.

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.

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.

* For research use only. Not intended for any clinical use.

Quick Inquiry

Preparation of Genomic Overexpression Library Protocol

Experiment Summary

Materials and Reagents

Equipment

Procedure

Related Products and Services at Creative Biogene