High-Throughput Mutant Screening in Vibrio cholerae via Transposon Sequencing

Abstract

Transposon mutagenesis greatly facilitates the study of gene function in microorganisms ranging from viruses to fungi. Traditionally, one would study individual transposon mutants with interesting phenotypes one mutant at a time. Here, we describe methods for the study of tens of thousands of transposon mutants in parallel in the bacterial pathogen Vibrio cholerae using transposon-sequencing. The first section outlines methods for making a saturated transposon mutant library. The second section outlines methods for massively parallel sequencing of the transposon junctions. The third section outlines methods for analyzing the sequence data to calculate the fitness contribution of genes.

FIGURE 1.

Illustration of plasmid pDL1093 used for in vivo transposition in Gram-negative bacteria.

FIGURE 2.

Illustration of polymerase chain reaction (PCR) products that capture a transposon–chromosomal junction sequence. (A) A product from the first PCR showing chromosomal DNA as a poly(N) sequence flanking the inverted repeat-left (IR-left) of the mTn10. For convenience, the sequence has been inverted relative to that shown in Figure 1. (B) A product from the second, nested PCR showing the index 1 (i7) sequence of primer BC45. The sequencing primer (olj386) used in Step 54 is shown.