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Artificial White Blood Cell. Doug Tischer. 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 H 2 O 2 , similar to a neutrophil’s oxidative burst.

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Presentation Transcript

The big idea
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.


Genetic construction
Genetic Construction

cre

  • Crerecombinase 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?

  • Xanthineoxidase requires trypsin processing to be active.

  • Trypsin expression must be tightly controlled. Any bacterial promoter would be to leaky.

(riboregulated)

trypsin

xod

loxP

loxP


Mechanism
Mechanism

1. The cells

2. Conjugation

“Bad Cell”

tetR

tetR

xan. oxidase

(inactive)

“Engineered WBC”

trypsin


Mechanism cont
Mechanism Cont.

3. Hydrogen Peroxide Production

4. Death!

tetR

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

xan. oxidase

(active)


Advantages
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.


Alternatives improvements
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.


References
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).


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