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Oxidative Burst Doug Tischer 1. General Idea Signal Galactose oxidase Catalase Buffers the cell from any leaky GOase expression = H 2 O 2 = H 2 O = O.

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Presentation on theme: "Oxidative Burst Doug Tischer 1. General Idea Signal Galactose oxidase Catalase Buffers the cell from any leaky GOase expression = H 2 O 2 = H 2 O = O."— Presentation transcript:

1 Oxidative Burst Doug Tischer 1

2 General Idea Signal Galactose oxidase Catalase Buffers the cell from any leaky GOase expression = H 2 O 2 = H 2 O = O 2 State 1 State 2 2 (Bad cell) No signal Galactose oxidase Catalase

3 System Design luxRGOasetetR LuxR pTET katE+ssrA 3

4 System Response GOase TetR KatE H 2 O 2 Stimulus (AHL) Time (min) 0 30 60 90 120 150 Relative Concentration 0% 100% 1.KatE degradation lags behind GOase production 4

5 Parts Breakdown luxRGOasetetR lux pR pTET katE+ssrA F2622 lacI + pL Q04400 B0015B0034 BBa_M0050Genomic PCR Q04400 From Arnold lab 5

6 Status PartStatusWhat’s Next? F2622 (luxR quorum sensing) Adding upstream of RBS. B0034 (RBS)Mini-preped. B0015 ( double transcriptional terminator) On plate. Q04400 (tetR inverter)Adding upstream of RBS. pSB1A2 (BioBricks backbone vector) Mini-preped. katE (catalase)Cloned into pSB1A2. Confirm orientation. Site directed mutagenesis. GOase (produces H 2 O 2 )In non-biobrick, IPTG inducible vector. Confirm in vitro & in vivo activity. Site directed mutagenesis. 6

7 Connection to other projects 1.Source of galactose could be from lactose breakdown. 2.Cells could be triggered to adopt this fate from SSM. 3.ROS species could be detrimental to vitamin synthesis (among many other things). 4.Vitamins could be harmful to ROS generation if they are antioxidants. 7

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