Physics and Nanotechnology to Study Bacterial Cells � � m B m B � � � � s c F k T m ln m m tot B V V Saturday Morning Physics Lecture Mar. 11, 2017 Jaan Männik
Impact of physics on studies of living systems 1. Application of physics based methods and techniques to experiments with living systems 2. Application of physics based theories to explain life processes.
Bacteria – the unseen majority • Majority of Earth’s biomass are bacteria and archea. They are one of the main determinants how biosphere functions • There are estimated 10 times more bacterial cells in human body than our own cells • Recognizing bacteria and other microorganisms as disease agents have lead human life span to increase twofold over past 100 years. This gain may not be permanent.
Standard microbiology toolbox An agar plate Cell culture tubes The standard microbiology tools are not suitable to follow: 1. How bacteria behave in complex environments 2. Molecular process in individual cells in real time.
Lab-on-a-chip based tools • Well controlled environment for the cells which physical and chemical characteristics can be controlled and manipulated • Compatible with high resolution microscopy of cells (including super-resolution imaging, SEM)
Advantages of lab-on-a-chip platform Device to extract bacterial DNA • In situ biochemical analysis of cells J. W. Hong et al Nat. Biotech. 21 (2003) 1179 • Automation 2 � m F. K. Balagadde et al Science 309 (2005) 137.
Applications of lab-on-a-chip in studies of cells Molecules Cells Organisms Populations Bacteria and tissue Antibiotic resistance interactions Sequencing Ecology, studies evolution Y. Mercy et al. N. Q. Balaban et al. S. Park et al. D.Huh et al. PNAS 104 (2007) 11889 Science 305 (2004) 1622 Science 301 (2003) 188 Science 328 (2010) 1622
Understanding how bacterial cells move in small pores using bio- mimetic lab-on-a-chip devices
Bacterial movement in channels and pores • Most bacteria in different environments live in pores 10 � m and smaller
Can’t do it in patient mouth …
Experimental setting channel 100 � m chamber • Fabricate on the single chip, large number of differently sized and shaped channels. • Monitor bacterial movement through channels (constrictions) using fluorescent microscopy
Microfabrication cleanroom
Microchip fabrication electron beam 1 Use electron beam or PMMA photolithography to write Si pattern of channels 2 Develop resist Repeat the process for different channel heights 3 Reactive ion etch Si wafer 4 Lift-off resist PDMS coated 5 Drill access holes glass coverslip Si Close channels channels
Channels Side view Bacterium D 1 µm 1 µm 1 µm • On single chip, channel width is made to vary between ≈300 nm to 5 � m to using a RIE cryoetch process
Fluorescence UV excitation Exception – jellyfish Aequorea victoria Most materials/objects do not fluoresce when excited with blue light (many materials fluoresce when exited with UV light) � Excitation with blue light allows to selectively observe engineered molecules such as GFP and have very little background from other molecules.
Fluorescent proteins GFP (Green Fluoresccent Protein) Blue light • Insert genetic code for GFP molecule into bacterial genome or in plasmid (short circular DNA that many bacteria carry). Bacteria will synthesize the protein. • Shine blue light on bacteria that express GFP. They will shine green light back. There is little background at green wavelength region.
Gram-negative and Gram-positive bacteria Escherichia coli Gram-negative bacteria cytoplasm lipid membrane Bacteria cell wall Bacillus subtilis cytoplasm Gram-positive bacteria Mollicutes (no cell wall)
Two bacterial species studied Bacillus subtilis Escherichia coli Gram-positive bacterium Gram-negative bacterium E. coli RP437 D D 1 µm 1 µm 1 µm • Superfically looking the two bacterial species are similar but on molecular level they are different than humans are from roundworms.
Bacterial swimming E. coli RP437 1 µm E. coli with fluorescently labeled flagella H. C. Berg group, Harvard University Wikipedia
Introduction: Bacterial motility • Bacterial flagellar motor is a rotary motor superficially similar to DC electrical motor • Rotation speed controlled membrane potential and pH gradient across inner membrane • Bacterial sensory system (chemotaxis receptors through signaling cascade) control direction of rotations
Bacterial motility in channels E. coli RP437 W = 1.2 � m chamber channel chamber 30 • E. coli and B. subtilis bacteria are motile in channels which are only 20 <v> [ � m/s] 30-40% wider than their diameter 10 • In smaller channels bacteria lose their ability to swim but .. 0 0.5 1.0 1.5 2.0 2.5 W [ � m]
Growth and division is bacterial solution to penetrate narrower channels W = 0.6 � m E. coli chamber channel chamber B. subtilis
Different layout of channels • More details of cells visible in the microscope • Channel ceiling soft; Bacteria can deform it
Bacterial movement in sub-micron channels E. coli • Growth in confinement alter drastically E.coli shapes B. subtilis • B. subtilis grows to filaments that buckle and finally divide
Re-emergence of regularly shaped bacteria 0 hrs 5 hrs 49 hrs 10 µm • Over period 1-2 days regular rod-shaped bacteria replace initial population of aberrantly shaped bacteria
Modes of penetration for different channel widths • E. coli but not B. subtilis bacteria are able to grow through channels which widths are smaller than their diameters
Mechanical properties and propagation in narrow channels E. coli B. subtilis cell wall 30-40 nm cell wall � 3 nm channel wall D W channel wall P osm =2-3 atm P osm =26 atm V. R. F. Matias et al Mol. Microbiol 56 (2005) 240 A. Boulbitch et al PRL 85 (2000) 5246 • Cell-wall has high Young modulus but is easily bendable (think of inflated balloon) • The thicker the cell wall the higher the Young modulus and the higher osmotic pressure can bacterium maintain in its interior
Pressure actuated valves to mechanically manipulate cells and sub-cellular structures pressure channel PDMS glass channel for bacteria • Characterize the mechanical properties of bacterial cells • Use it as tool to manipulate sub-cellular structures in a bacterial cell
Mechanical limits of bacterial deformations p max 0 p max = 2 atm p max = 3 atm Step # • Bacteria can be stretched up 25% elastically • Bacteria break open at isolated weak spots at cell poles
Short vs long time response of E. coli 1 3 2 2 1 3 • Elastic deformation after entry in shallow channel • Slow cellular response after prolonged stay in channel
Summary on lab-on-a-chip and squeezing measurements • Lab-on-a-chip platform allows to create biomimetic environments where cell behavior can be studied using high resolution optical microscope and bioanalytical tools. • E. coli and B. subtilis are well adapted to swim in small channels despite their long flagella. Both species retain ability to swim in channels which only 30% exceed their body diameter • Surprisingly, bacteria can get through even narrower channels! For that they use growth and division. • Bacterial growth is robust despite drastic changes in their shapes • Mechanical properties of bacteria determine how small channels they can penetrate.
Understanding how cells are built from molecules up using quantitative high resolution microscopy and modelling
Escherichia coli as living “hydrogen atom” Escherichia coli � E. coli Jacque Monod, “ What is Valid for E. coli is also valid for the elephant ” 1 � m • Bacteria present the simplest systems to understand how the cellular processes unfold using basic physics principles
What is known • Functions of about 70% genes to some degree 1 � m • 50% of protein structures (most based on homology) 2 nm FtsZ MatP tetramer SlmA tetramer
What is not known: from genes to cell DNA lipid ? polysaccharide protein 1 � m How nanometer-scale proteins, DNA and lipids come together and form the micro-size cell?
Cellular organization in bacteria Wikipedia Assembly of molecular Bag with soup of molecules machines • Bacterial cells are highly organized despite their apparent simplicity
envelope • How are chromosomes and cell division apparatus organized in a bacterial cell? • How cell division proteins position relative to nucleoids?
Chromosome organization
Genetic information is tightly packed in the cell Escherichia coli circular chromosome 4.6 � 10 6 basepairs 1 μ� HupA-mCherry labelled chromosome 1.6 mm • Thousand fold compaction of DNA in the cell
Nucleoid cytosol 1 μ� HupA-mCherry labelled chromosome nucleoid • DNA spreads just to a fraction of cell volume (~50%) – the nucleoid (not to be confused with nucleus) • There is no membrane surrounding the nucleoid
What compacts DNA?
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