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