BIOMI 404 Prelim 1
February 27, 2008
1a) Recombinant-based in vivo expression technology is used to figure out which
bacterial promoters are active during infection
. A plasmid is made that carries
pieces of bacterial genomic DNA fused to a reporter gene; in this article, the reporter
gene is the resolvase gene (
) that creates the sucrose-resistant selectable phenotype.
The sucrose-resistant phenotype is created when resolvase excises a cassette flanked by
res sites and containing neo-sacB. The resolvase gene is only transcribed, however, if a
cross-over recombination event happens that puts
within an active promoter or
operon; the crossover happens by homologous recombination, and a second wild-type
copy of the viral gene of interest exists so that a mutant phenotype is not created.
Scientists can then isolate bacteria that were only sucrose resistant
the viral DNA that was fused with the
, making a transcriptional unit with
Signature-tagged mutagenesis works by taking a mutant pool of bacteria and introducing
this pool to a mouse model. The bacterial mutant pool is created via transposon
mutagenesis: a transposon with random oligomers (which serve as an individual tag) and
with a selectable marker, such as antibiotic resistance, is introduced into the bacterial
strain. The transposon inserts itself into the bacterial genome, making a specific mutant.
Each colony made by this method represents a different mutation. Then, an animal is
inoculated with a mixture of all the mutant, transposon-containing colonies. After
infection, a specific organ is removed, and bacteria are recovered. Then, DNA is removed
from colonies that grew, and PCR primers are used to amplify the tags for each
transposon; the tags are also radiolabeled. The radiolabeled tags are then used to probe
the original mutants. If a mutant does not hybridize with a tag, that means that the tag
was not isolated, and that that specific mutant had a gene required for survival and
infection of the host. The DNA around the transposon of the non-surviving mutant can
than be analyzed to see which gene was interrupted.
Advantages: Requires few animals, since many mutants can be studied within one
Disadvantages: Can only look at 96 mutants at a time (since the chips have 96
wells). Genes that are required for pathogenicity (but not survival) can be missed.
Also, if a gene produces a toxin, this gene will also be missed.
induced antigen technology (IVIAT) is used to figure out which host-produced
antibodies are made to antigens produced
only. This is done by first extracting
serum from an infected patient, and also growing the bacterium
. When you mix
the patient serum and the
-grown microorganism, patient antibodies will bind to
bacterial antigens; any antibody that did not bind anything must bind antigen that is only
. At the same time, an expression library must be made by cutting up the