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Mutagenesis studies In order to better understand this organism, my research involved attempting to systematically create a comprehensive bank of H. influenzae mutant strains so that we could study the effects of the mutation to try to elucidate each gene's function. Here is the basic outline of the research: Construction of the Haemophilus influenzae Rd KW20 insertion libraries. a-b. Restriction digestion or sonicated fragments of the Haemophilus genome were cloned into the AscI site of the pASC series of cloning vectors. c. Non-essential regions of the vector backbone were removed by sequential digestion/religation steps with ApaI and NotI. d. The library was mutated by in vitro mutagenesis using the transposons Tn7 or Tn5. e. The site of transposon insertion was mapped by dideoxynucleotide sequencing from unique primers within the transposon across the insertion junction. In later libraries, steps C and D were reversed (post-mutagenesis minimalization) or chromosomal DNA was cloned directly into a minimal vector lacking the lacZα sequence (pASC18MIN). Abbrieviations used: lacZα, β-galactosidase gene; ori, ColE1 origin of replication; bla, ampicillin resistance gene; km, kanamycin resistance gene
Results: In vitro transposition with Tn7 and Tn5 was employed with varied success in creating the library of mutagenic constructs. In total, 1290 plasmids were sequenced and the sites of 1426 Tn7 or Tn5 insertion events were mapped. Of these, 1024 (71.8%) were mapped to H. influenzae chromosomal DNA and were located in 246 of the 1710 Rd KW20 protein coding genes (14.4%). I was able to notice a statistically significant pattern emerging in the transposon insertions that identified a previously unrecognized bias for the insertion of this transposon. Since this is the subject of paper I'm currently writing, I'll not be discussing those findings on this site for a while.
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