Summer Student Program 2013. First results This is an example. Please insert your picture via slide master! Summer Student Program 2013. First results Dresden, 28.08.2013

Group of Bioinformatics and Biophysics of Nanopores Introduction Wrocław University of Technology Faculty of the Fundamental Problems of Technology Biomedical Engineering Institute of Biomedical Engineering and Instrumentation Group of Bioinformatics and Biophysics of Nanopores

Dr Johannes Raff  Dr Manja Vogel Matthias Suhr Björn Drobot

Outline 1. Introduction Bacterial S-layer Isolates with S-Layer 2. Project: Hole agar diffusion test Metal tolerance test (agar, liquid) S-layer isolation 3. Next steps in project

Project „Isolation and characterization of microbial components for metal detoxification”

Isolates with S-layer

Bacterial S-layer Tickness: 4-30nm Pores ø: 2-8nm Unit cell: 3-40nm Molecular mass: 40-200kDa Raff J. and S. Selenska-Pobell (2006) Nuclear Engineering International 51 (619), 34-36

Hole agar diffusion test Experiments 1. Hole agar diffusion test 2. Metal tolerance test on agar plates 3. Metal tolerance test in liquid

HOLE AGAR DIFFUSION TEST a method for determining the sensitivity of a microorganism to an antimicrobial drug. The zone of growth inhibition is measured around a ditch, hole, or a filter-paper disk containing the drug and located on an agar culture medium seeded with the microorganism in question. Oxford Dictionary of Biochemistry Oxford University Press. Oxford Dictionary of Biochemistry and Molecular Biology © 1997, 2000, 2006

Procedure OD600 = 0.5 ..μl 0,9%NaCl 1ml 1ml 1ml 1ml 30°C Mast -Medium 500μl metal solution 100 μl 900μl 0,9%NaCl 900μl 0,9%NaCl 900μl 0,9%NaCl 1ml 1ml 1ml

Procedure OD600 = 0.5 ..μl 0,9%NaCl 1ml 1ml 1ml 1ml 30°C Mast -Medium 500μl metal solution 100 μl 900μl 0,9%NaCl 900μl 0,9%NaCl 900μl 0,9%NaCl 1ml 1ml 1ml

Data – hole agar diffusion test Tab.1. NB medium ingredients Tab.2. Optical density of precultures. Spectrophotometer Pharmacia Biotech Ultrascpec 100, hole agar diffusion test, 09.08.2013 Substance Concentration Meat extract 5g/L Peptone NaCl 2.5g/L agar 20g/L VE wasser Strain Optical density Volume for OD600 = 0.5 in 1ml A12 0,431 116 μl B12 0,586 85 μl B53 0,488 102 μl B58 0,535 93 μl B62 0,465 108 μl B5T 0,362 138 μl Tab.3. Metal solutions and their concentrations used in experiment Metal solution Concentration Pb(NO3)2 10mM 50mM Cd(NO3)2

Hole test results a) b) d) c) e) Pic.1 The results for JG A12 after 48h incubation: Control plate 10mM Cd-nitrate 50mM Cd-nitrate 10 mM Pb-nitrate 50mM Pb-nitrate

Summary There occurs heavy metal tolerance for all metals in different manner: Pb is tolerated better There occurs heavy metal tolerance for all isolates in different manner: only JG A12 has a growth

METAL TOLERANCE TEST Agar Procedure OD600 = 0.5 ..μl 0,9%NaCl 1ml 1ml Mast -Medium OD600 = 0.5 ..μl 0,9%NaCl R2A medium + Cd-solution 1ml 100 μl 100 μl 100 μl 30°C 900μl 0,9%NaCl 900μl 0,9%NaCl 900μl 0,9%NaCl 1ml 1ml 1ml

Salt solution (10 times concentrated) Data – agar metal tolerance test Tab.1. R2A medium ingredients with addition of Cd-solution. Preparation in the volume of 400 ml Substance Volume Agar 8g Peptone (10%) 40 ml Salt solution (10 times concentrated) Glycerin 2ml CaCl2(100mM) 400 μl Thiamin (50mg/ml) 160 μl Cd-solution See picture VE-wasser 360 ml 1M Cd-solution 1mM Cd-solution 400μl 20μl 100μl 200μl Tab.2. Optical density of precultures. Spectrophotometer Pharmacia Biotech Ultrascpec 100, agar metal tolerance test, 01.08.2013 Strain Optical density Volume for OD600 = 0.5 in 1ml A12 0,505 99 μl B12 0,588 85 μl B53 0,447 112 μl B58 0,322 155 μl B62 0,298 168 μl B5T 0,256 195 μl

METAL TOLERANCE TEST Liquid Procedure 1M Cd-nitrate 0.5mM 1mM 25mM 1M Pb-nitrate 750 μl 15 μl 15 μl 750 μl 30 μl 30 μl NB-Medium NB-Medium 0.5mM 1mM 25mM 0.5mM 1mM 25mM 500 μl 30°C

METAL TOLERANCE TEST Liquid Procedure 1M Cd-nitrate 0.5mM 1mM 25mM 1M Pb-nitrate 750 μl 15 μl 15 μl 750 μl 30 μl 30 μl NB-Medium NB-Medium 0.5mM 1mM 25mM 0.5mM 1mM 25mM 500 μl 30°C

Liquid metal tolerance results b) e) Liquid metal tolerance results a) c) f) Pic.1 The results of liquid metal tolerance test after 24h. JG-B53, Olympus BX-61 Preculture 0.5mM Pb-nitrate 1mM Pb-nitrate 25mM Pb-nitrate 0.5mM Cd-nitrate 1mM Cd-nitrate 25mM Cd-nitrate d) g)

Pb-nitrate Cd-nitrate 24h 48h 24h 48h 0.5mM 1mM 25mM

Summary – liquid metal tolerance test Different metal tolerance for different concenration and kind of metal Pb is better tolerated, Cd is an inhibitor 1mM concentration causes creating the spores 25mM concenration causes the breaking down the cells

S-layer isolation Procedure Preparation of biomass Removal of flagella Mechanical disrupter of the cells Removal of remaining cytoplasmic membrane Preparation of S-layer protein Recrystallization of S-layer protein weighing biomass washing in standard buffer removing flagella with disperser centrifugation Washing pellet in standard buffer 3 times dispersing in standard buffer adding DNAse and RNAse digestion of cell by high pressure homogenizer centrifugation washing pellet in standard buffer 2 times dispersing in standard buffer dilluting with Triton and incubation centrifugation washing pellet 2 times resuspending pellet with standard buffer, lysosyme, DNAse, RNAse incubation centrifugation selecting S-layer by removing contaminations (resuspensions, centrifugation) dialysis

SDS-PAGE One of the technique to seperate proteins. Molecules with a net charge move in an electric field. It is carry out in porous gel (polyacryloamide gel).

SDS-PAGE Mixture of macromolecules Porous gel Electrophoresis direction Electrophoresis 𝜗= 𝐸𝑧 𝑓 𝑓=6𝜋𝜂𝑟 Stryer, L.; Berg, J. M.; Tymoczko, J. L. (2002), Biochemistry (5th ed.), New York: W. H. Freeman

SDS-PAGE SDS is sodium dodecyl sulfate , anionic detergent that disrupts all noncovalent interactions in native protein. Thanks to it proteins can be seperated on the basis of mass .

SDS-PAGE results 1. JG-B58 pellet 1 2. JG-B58 pellet after Homi 4. JG-B58 pellet after Triton 5. JG-B58 pellet 3 after Triton 6. Marker 7. JG-B58 supernatant 3 8. JG-B58 supernatant 1 9. JG-B58 supernatant after Homi 10. JG-B58 supernatant after Triton kDa 200 150 120 100 85 70 60 50 40 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Next steps -liquid metal tolerance test with other isolates and with other metals -reassembly S-layer in metal solution