Post on 16-Dec-2015
Lambda phage killing: infection and “parts”
N Kuldell for 20.020
Spring 2009
Heidelburg iGEM 2008: ecolicense to kill
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Part 2: DNA transfer, genetically programmed self-assembly and “parts”
Background information
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DNA transfer
natural context: 3 mechanisms
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http://www.scq.ubc.ca/wp-content/uploads/2006/08/HorizontalTransfer.gif
“Transduction” by bacteriophage
www.phagebiotech.com/
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Protein coat encapsulates nucleic acids
Bacteriophage: viruses that infect bacteria
INFECT
REPLICATE
ASSEMBLE
RELEASE
“Temperate” bacteriophage lambda
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
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lytic
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
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lytic
lysogenic
“Temperate” bacteriophage lambda
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
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“Temperate” bacteriophage lambda
Some great ??s
What guides lytic/lysogenic decision?
What keeps lysogen stable?
What triggers lysogen to lytic cycle?
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
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“Temperate” bacteriophage lambda
Some great ??s the Heidelberg team asked:
Can we infect prey from lysogen (=predator)?
Can we keep lysogen from lysing itself?
Can we monitor lysis and lysogeny?
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
“Temperate” bacteriophage lambda
Some great ??s the Heidelberg team asked:
Can we infect prey from lysogen (=predator)?
Can we keep lysogen from lysing itself?
Can we monitor lysis and lysogeny?
Genetically programmed infection iGEM context: phage infection via conjugation
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Genetically programmed infection iGEM context: phage infection via conjugation
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http://escience.ws/b572/L18/L18.htm
io.uwinnipeg.ca
Genetically programmed infection iGEM context: phage infection via conjugation
What “parts” are needed
Pilus…. “F’”
Origin for transfer… “oriT”
Selectable marker… “CamR”
Model predicts: 10 killer cells kill 10^9 prey cells “in silico”
F’oriT
CamR
“Part” is a genetically-encoded, human defined function
Genetically programmed infection iGEM context: phage infection via conjugation
What “parts” are needed
Pilus…. “F’”
Origin for transfer… “oriT”
Selectable marker… “CamR”
F’oriT
CamR
“Part” is a genetically-encoded, human defined function
I714030
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
“Temperate” bacteriophage lambda
Some great ??s the Heidelberg team asked:
Can we infect prey from lysogen (=predator)?
Can we keep lysogen from lysing itself?
Can we monitor lysis and lysogeny?
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
“Temperate” bacteriophage lambda
Some great ??s the Heidelberg team asked:
Can we infect prey from lysogen (=predator)?
Can we keep lysogen from lysing itself?
Can we monitor lysis and lysogeny?
Genetically programmed bi-stable switch natural context: epigenetic regulation
Genes Dev. (2004)18(17): 2086–2094. doi: 10.1101/gad.1226004.
lysogeny
lysis
Genetically programmed bi-stable switch iGEM context: flip and hold in one state
Genes Dev. (2004)18(17): 2086–2094. doi: 10.1101/gad.1226004.
lysogeny
promoter RBS cI ORF transcriptional terminator
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
“Temperate” bacteriophage lambda
Some great ??s the Heidelberg team asked:
Can we infect prey from lysogen (=predator)?
Can we keep lysogen from lysing itself?
Can we monitor lysis and lysogeny?
http://escience.ws/b572/L17/L17.htm
INFECT
REPLICATE
ASSEMBLE
RELEASE
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
“Temperate” bacteriophage lambda
Some great ??s the Heidelberg team asked:
Can we infect prey from lysogen (=predator)?
Can we keep lysogen from lysing itself?
Can we monitor lysis and lysogeny?
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Fluorescence vs bioluminescence
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AI-2AI-1
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Fluorescence vs bioluminescence
AI-2AI-1
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http://www.lifesci.ucsb.edu/~biolum/chem/
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Fluorescence vs bioluminescence
AI-2AI-1
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http://www.lifesci.ucsb.edu/~biolum/chem/
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One last thing about fluorescence
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Genetically programmed infection iGEM context: phage infection via conjugation
What “parts” are needed
Pilus…. “F’”
Origin for transfer… “oriT”
Selectable marker… “CamR”
Stable lysogen… “cI”
Moniter… “GFP”
F’oriT
CamR
Predator cellstable lysogen that infects by conjugation
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+ CamR
+ GFP to follow process