Transcript Artificial White Blood Cell

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Artificial White Blood Cell
Doug Tischer
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The Big Idea
• Our engineered cells will “explode” when they
detect a “bad” cell, killing both itself and the
nearby bad cell.
• The “explosion” is a sudden burst of H2O2,
similar to a neutrophil’s oxidative burst.
• The bad cell will be detected when it attempts to
conjugate with our cell. This ensures it is
physically close, and will be killed by the
oxidative burst.
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Genetic Construction
cre
(riboregulated)
loxP
loxP
trypsin
xod
• Cre recombinase translation is allowed only in the presence of
tetracylcine resistance transcripts.
▫ Can riboswitches also be unlocked by ssDNA, as occurs during
plasmid transfer during conjugation?
• Xanthine oxidase requires trypsin processing to be active.
• Trypsin expression must be tightly controlled. Any bacterial
promoter would be to leaky.
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Mechanism
1. The cells
2. Conjugation
“Bad Cell”
tetR
“Engineered WBC”
tetR
trypsin
xan. oxidase
(inactive)
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Mechanism Cont.
3. Hydrogen Peroxide Production
tetR
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2H2O2
H2O2
H2O2
H2O2
H2O2
xan. oxidase
(active)
4. Death!
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Advantages
• Xanthine oxidase naturally requires trypsin
processesing to be active.
• H2O2 is naturally used by neutrophils to kill
bacteria.
• Conjugation typically lasts ~100 minutes. The
bacteria should be close together long enough
for the H2O2 to be produced in large amounts.
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Alternatives/Improvements
• Better detection system. Riboswitches can be
leaky, but would allow for detection of a wide
variety of antibiotic resistant strains.
• Alternatives to xanthine oxidase
▫ Glucose oxidase: dimeric, well studied
▫ NADH oxidsase: produces H2O2 outside cell
membrane. 7 subunit integral protein.
▫ Induce phage to kill other bacteria.
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References
• Stirpe, F., and Della Corte, E., Regulation of Rat
Liver Xanthine Oxidase. J. Biol. Chem., 244,
3855-3863 (1969).
• Harrison, R., Structure and Function of
Xanthine Oxidoreductase: Where are we now?
Free Radical Biology & Medicine., 33, 774-797
(2002).