By Pontus Nordenfelt, Mattias Collin
This quantity discusses a variety of tools and protocols used for the experimentation of a variety of bacterial species, corresponding to Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumonia, Listeria monocytogenes, and Mycobacterium marinum. Bacterial Pathogens: equipment and Protocols is split into 6 components: half 1 describes diversified ways to settling on and characterizing bacterial effector molecules; half 2 bargains with structural biology of bacterial pathogenesis and the way to beat folding and balance issues of recombinantly expressed proteins; half three information technique that identifies micro organism in complicated groups and the way genomes of bacterial pathogens have developed; half four displays at the speedy improvement of complicated imaging suggestions that handle questions about molecular houses of person dwell micro organism, ultrastructure of surfaces, and subcellular localization of bacterial proteins; half five describes equipment from in vitro and in vivo modeling of bacterial infections; and half 6 explores how bacterial pathogens are the genuine specialists of the immune approach. Written within the hugely profitable Methods in Molecular Biology sequence layout, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and pointers on troubleshooting and keeping off identified pitfalls.
Cutting-edge and entire, Bacterial Pathogens: tools and Protocols is a precious source for someone who's drawn to this attention-grabbing and evolving field.
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Extra resources for Bacterial Pathogenesis: Methods and Protocols
10. 1 M): 871 mg PMSF, add isopropanol to 50 mL in a 50 mL conical. Store at room temperature. Another protease inhibitor may be substituted. 4 DRaCALA Screen 1. Radiolabeled ligand in binding buffer: calculate the volume of radiolabeled ligand that will result in 150 μCi activity/50 mL of buffer (see Note 3 regarding radiolabel mixture). To this volume add 5 mL 10× binding buffer and ddH2O to 50 mL in a 50 mL conical. Store at −20 °C until use. 2. 01 % (vol) Tween 20 in H2O: mix 5 μL Tween 20 and 50 mL ddH2O in a 50 mL conical.
PLoS Pathog 10(10), e1004429 7. Hickman JW, Harwood CS (2008) Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69(2):376–389 8. Baraquet C, Harwood CS (2013) Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ. Proc Natl Acad Sci U S A 110(46):18478–18483 9. Leduc JL, Roberts GP (2009) Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv.
If bands are weak, adjusting richness of the media or IPTG levels can improve expression. Incomplete lysis will result in reduced availability of the overexpressed protein for binding. Increasing the amount of lysozyme or adding additional freeze–thaw cycles may help improve lysis. A very viscous lysate will affect spreading of the spot on nitrocellulose. Increasing the DNase concentration can reduce viscosity. 9. Nitrocellulose membrane sheets come sandwiched between sheets of wax paper. When stamping, keep the bottom sheet of the sandwich below the nitrocellulose.