Amarilis Dyer: Undergrad research project

Here is what one of our undergraduate students (Amarilis Dyer) did during the 2013-14 academic year with funding from the Biology department in the lab of Dr Dayle Daines:

A new cis-complementation system for nontypeable Haemophilus influenzae (NTHi) isolates

The objective of this experiment was to design and construct a cis-complementation system for clinical isolates of nontypeable Haemophilus influenzae (NTHi).Two strains were used in this study: a nasopharyngeal isolate from a child with chronic otitis media, and  a blood isolate from a child with meningitis. The green fluorescent protein gene, gfp, was the reporter used in these experiments to insert as a single copy. A pseudogene in the NTHI chromosome was chosen as the recipient site for cis-complementation.  A set of forward and reverse primers were created that annealed to the 5’ end of the gene (first arm), amplifying 784 base pairs by Polymerase Chain Reaction (PCR), and another set that bound to and amplified 791 base pairs of the 3’ end of the gene by PCR (second arm). The purpose for cloning the first and second arm of the gene was to know exactly where the inserted DNA was located.

To create strains expressing gfp, a spectinomycin antibiotic resistance cassette was added as a selection marker. Once the antibiotic cassette was inserted, a gfp gene controlled by a strong promoter was amplified and inserted between the two arms of the psuedogene. The reason for the insertion of the reporter gene was to run assays and determine if the gene was present.  The antibiotic cassette was used to insure that the gene would stay in. This resulted in the antibiotic resistance cassette and the reporter gene being flanked by NTHi DNA that is homologous to the pseudogene locus.  This construct was introduced into the two NTHi strains by homologous recombination.  The antibiotic-resistant transformants were tested for GFP expression using fluorescence microscopy. By amplifying the flanking regions and sequencing the products, we determined that the location of the gfp gene was within the pseudogene. An immunoblot was performed to confirm gfp expression in both strains. Our next steps include using this approach to complement existing NTHi deletion mutants in cis.