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1 Artificial White Blood Cell Doug Tischer 2 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. 3 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. 4 Mechanism 1. The cells 2. Conjugation “Bad Cell” tetR “Engineered WBC” tetR trypsin xan. oxidase (inactive) 5 Mechanism Cont. 3. Hydrogen Peroxide Production tetR H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2H2O2 H2O2 H2O2 H2O2 H2O2 xan. oxidase (active) 4. Death! 6 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. 7 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. 8 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).