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presentation - Expression of the Major Porin Gene mspA in...

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Unformatted text preview: Expression of the Major Porin Gene mspA in Mycobacterium smegmatis By Heather Crozier Wenmei Zhang http://www.structbio.uni-freiburg.de/ Mycobacteria smegmatis Fast-grow Mycobacterium Gram +, aerobic High percentage of C and G Unique cell walls H http://en.wikipedia.org/wiki/Mycobacterium http://www.pitt.edu/~biohome/faculty/hatfull.html M. Smegmatis genome M http://wishart.biology.ualberta.ca/BacMap/graphs_cgview.html Sample Preparation E. coli DH 5 - LB medium, 37C M. smegmatis SMR5 - Middlebrook 7H9 liquid medium (0.2 % glycerol, 0.05% Tween 80) - Middlebrook 7H10 agar (o.2% glycerol) - 37C with shaking M Result 1: Identification of the mspA promoter Step 1: TSP determination of mspA gene H RNA Preparation H Primer Extension Experiment TSP M of mspA at G -135 position http://jb.asm.org/cgi/content/full/189/3/958 Nucleas S1 mapping H TSPs of mspA at -135 and -153 positions Upstream region of the mspA gene M http://jb.asm.org/cgi/content/full/189/3/958 Result 1: Identification of the mspA promoter Step 2: A potential promoter sequence upstream of the main TSP at position -135 M&H Point Mutaion of Promoter Position 1 2 3 4 Conservation (%) 100 93 50 57 Wild-type T A T G Mutated C C T G 5 43 T T 6 71 T C M -galactosidase activity measurements M http://jb.asm.org/cgi/content/full/189/3/958 Result 2: Long 5' upstream needed for full function of the mspA promoter Compare to the activities: A. B. C. 500 << 600 < 700 1100 600 up <500 << 1100 1100 = max 500 + 200 < 700 M http://jb.asm.org/cgi/content/full/189/3/958 Result 3: Size and Half-life of the mspA Transcript Half-life H RNA probe construction H Northern Blot Size A: 1 = mspA mutant strain MN01 2 = WT strain SMR5 Half-life B & C: Half-life = 6 min D: Replacement of original promoter lead to decrease of gene expression H&M http://jb.asm.org/cgi/content/full/189/3/958 Result 4: Factor 1 Growth Phase H http://jb.asm.org/cgi/content/full/189/3/958 Result 5: Factor 2 Environmental Signals Nutrient limitations C N mspA mRNA level P Temperatur e 28-37 42 Osmolarity Chemicals EtO H pH NaC Glucose H2O2 l __ M&H http://jb.asm.org/cgi/content/full/189/3/958 Result 6: Factor 3 pH Value Under pH = 4.5 A: no WT mspA B: no WT/mutant mspA (own promoter) mutant mspA (replaced promoter) C: no lacZ (own mspA promoter) H http://jb.asm.org/cgi/content/full/189/3/958 Summary of Methods M Conclusions Change the porin gene expression responsed to different environmental signals. Original 5' upstream region was required for the stability and full function of porin gene. M Questions ? ...
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