evolution was carried out on a Pseudomonas aeruginosa PAO1 strain
of bacteria after screening of 28 known hydrocarbon utilizers for
activity on alkanes of various chain lengths. PAO1's activity was
improved from (0.6cm/24h at 37 C) to (0.9cm/24h at 37 C). Only one
round of evolution was needed to achieve this improvement. After
making the library by error prone PCR, the mutated products were
purified using QIAquick DNA Purification Kit and then cloned back
into the source strain and into E. Coli with TOPO Cloning Kit from
Invitrogen. The most active strains P. putida and B. subtilis, which
will be used in the rest of the project have not been mutated as
the current primers only amplify PAO1's AlkB gene.
assay using zones of clearing was developed to screen for hydrocarbon
degrading activity. Of the 28 strains selected, 12 had significant
(>0.5cm/24h at 37 C) activity on C20, the largest substrate screened.
Primers for the PCR were designed by using NCBI nucleotide data
for Pseudomonas aeruginosa PAO1 and BLAST of Alkane Monooxygenase,
the first enzyme in the hydroxylation pathway. The upstream primer
used was 5'ggcacccgaagcttccgtttcc 3' the downstream primer used
was 5'gtctgagaattctcctccc 3'. With HindIII and EcoRI restriction
Screening of the wild
type and mutant strains was done on M9 medium without sucrose, sprayed
with a thin layer of target substrate (0.5g/plate), strain success
was measured by the presence and rate of zone of clearing. M9 media
was used because it limits the carbon source to the target substrate.
Napthalene, Catechol, C10-C15 and C20 substrates were screened.