Bethany Yachuw Phages Phage life cycles Transcription determines life cycle RNA Polymerase Repressor Antirepressor Find the putative antirepressor Yuan et al 2014 Via conceptual translation ID: 611178
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Slide1
Antirepressor identification in bacillus bacteriophage phicm3
Bethany YachuwSlide2
PhagesSlide3
Phage life cyclesSlide4
Transcription determines life cycle
RNA Polymerase
Repressor
AntirepressorSlide5
Find the putative antirepressor
Yuan et al (2014
)
Via conceptual translation
putative antirepressor (gp37)
Promoter, Operator, and Repressor were not annotated
F
ind promoter, operator, and repressor
Via bioinformatics Slide6
Find and test repressor - Plasmids
Insert putative repressor gene here with promoter, operator
Reporter gene (on other plasmid) is
lacZ
which codes for
β-
galactosidase
Slide7
Find and test repressor – plasmids
Add promoter and operator here at the
HindIII
site
Delete downstream
HindIII
site, so that the plasmid can be reopened
Plate control and bacteria with repressor gene
Lytic Cycle, not the
repressorLysogenic Cycle, it is the repressorSlide8
IPTG and onpg –
Further test repressorSlide9
Hopeful ResultsSlide10
test antirepressor - plasmids
Repressor
Antirepressor Slide11
IPTG and onpgSlide12
Hopeful ResultsSlide13
spectrophotometer
Measure the yellow color made
Convert to Miller Units (measures β-
galactosidase
activity)
1
Miller Unit = low activity
1000+ Miller Units = high activity
t = reaction time in minutes v = volume in mLSlide14
Questions?