Biophysics – Biological Physics

1. Biophysics – Biological Physics ● nomenclature ● fields of research ● history: old discipline, turning point is recent ● why physicists in biology? ● new multidisciplinary field: system biology, synthetic biology ● biophysics at in the Physics department at UMN ● in vitro gene expression and protocell ● biological physics: education, courses, resources.

2. Biophysics / Biological Physics • Biophysics: • more used among physiologists, biochemists • molecular level • Biological physics: • preferred by physicists • molecular to ecological • Ideas of biophysics or biological physics are the same: • fundamental physics of biological systems and processes. • apply the techniques from physics to understand biological • structure and function.

3. Biophysics, major components / examples • explain biological function in terms of molecular mechanisms • ions channels • protein 3D structures and functions (crystallography) • DNA replication • conversion of external signals to electrical signal • conversion of chemical energy to mechanical force (muscle) ATP hydrolysis = 20 kBT 1 kBT =1 pN nm

4. Biological physics, major components / examples • molecular: channels in membranes, dynamics of chemical reactions • subcellular: transport, signal processing, dynamics of • polymerization, motility, flagellar dynamics • cellular: chemotaxis, swimming, crawling, growth • multicellular: pattern formation, morphogenesis • organism: cardiac dynamics, circadian rhythms, information • processing • evolution / ecology: in vitro evolution, population dynamics

5. History • Ideas are not new: • ● D’Arcy Thompson (1860-1948) talking about cells, tissues, bones, flowers: “Their problems of form are in the first instance mathematical problems, their problems of growth are essentially physical problems.” • ● E. Schroedinger: “what is life?” (1944). • Recent field in terms of research effort: • became clear in the past decade: explosion of meetings, journals. • many departments building research groups in biophysics / biological physics.

6. Why physicists in biology? • What physicists can bring: • quantitative measurements • modeling and testing • reductive approaches • universality of behavior • development of new methods and technologies • in vitro / synthetic approaches

7. Biophysics Group Theory: Alexander Kamenev Boris Shklovskii Experiment: David Thomas John Broadhurst Joachim Mueller Vincent Noireaux

8. Alex Kamenev 1. Populations dynamics, as an example of non-equilibrium statistical mechanics. PreyPopulation Predator Population 2. Transport through ion channels, as an example of 1D physics.

9. Electrostatic theory of viral self-assemblyBoris Shklovskii

10. Thomas Lab Spectroscopic Probes of Muscle Protein Structure and Dynamics ATP ATP ADP + Pi ADP + Pi Ca2+ Ca2+ Cardiac Calcium Pump Myosin Phospho- lamban Actin Probes O Spectroscopic Probe Methods: Electron paramagnetic resonance (EPR) Nuclear Magnetic Resonance (NMR) Time-resolved fluorescence and phosphorescence H • O N C CH2I N I I O HO O I I COOH N = C = S

11. J. Broadhurst Magneto encephalography (MEG) Study the location in the human brain of the processors of external stimuli. Currently work is being done on the identification of different sounds by a part of the brain above and in front of the ear. (This part is known as the auditory cortex, and is located in a fold of the brain called the sylvan fissure). When a sound is received by the ear, it is analyzed into the different frequencies that it contains, before being passed on to the first level of processing. This identifies loudness and the direction of the sound source, and then transmits the information to the second processor, which tries to identify the identity of the sound (Is it a violin, or a cat meowing?) Neurons in the brain activate and produce tiny magnetic fields (10-12 Tesla ). An array of 250 super-conducting magnetometers (squids) are used to measure the fields.

12. Two-Photon Spot objective FFS in cells Two-photon Effect Single-molecule microscope Mueller Lab:Fluorescence Fluctuation Spectroscopy (FFS) Watch Protein Interactions in Living Cells: Photon Count Statistics

13. Joachim Mueller: Protein Assemblies and Viruses Fluctuation Analysis 2-photon spectroscopy Construct physical model of assembly pathway Light burst from single molecules passing through tiny optical volume Image of a cell assembling viral-like particles. We study assembly process of retroviruses, such as HIV-1. Harvest viral particles Viral Particle Fluctuation Analysis • protein coat contains holes • the hole density varies • below percolation threshold Microfluidics of viral particles

14. Artificial cell system. Self-assembly of proteins/biopolymers. 20μm Vincent Noireaux ● information processes (synthetic genetic circuits). ● biopolymer self-assembly at the membrane: cell division, motility, nano by bio. ● artificial cell system. Reconstitution of genetic circuits in vitro. Coarse-grained model of circuits.

15. Vincent Noireaux ● information processes (synthetic genetic circuits). ● biopolymer self-assembly at the membrane: cell division, motility, nano by bio. ● artificial cell system. genome DNA of virus cell-free expression in test tube de novo synthesis of virus

16. Biophysics courses at UMN ● Physics department: - 4911/5081: intro. to biopolymer physics. - 5401: physiological physics. - 5402: radiological physics. ● other courses: - Math 5445: mathematical analysis of biological networks. - Math 8540: topics in mathematical biology. - biology courses.

17. DNA sequencing and synthesis Sequencing of bacterium genome: 1 week (5 Mb)

18. Information  man-made nature - evolution

19. New interdisciplinary fields • ● system biology: • understanding the structure of the system, such as gene regulatory networks. • understanding the dynamics of the system, both quantitative and qualitative analysis. • understanding the control methods of the system. • understanding the design methods of the system, are key milestones to judge how much we understand the system. ● synthetic biology: the design and fabrication of biological components and systems that do not exist in the natural world. Use them either as molecular- scale factories, to make simple computations, deliver vaccines, or to create new hybrid materials. Like system biology, synthetic biology is at a very preliminary stage but physicist could have a significant scientific impact.

20. Molecular programming in a test tube: synthetic gene circuits, phage synthesis and artificial cell. Vincent Noireaux, UMN

21. 1 Introduction – Motivations • The three components of cellular life. • the bottom-up approach to living systems.

22. Living cell (bacteria E. coli) • Genome (DNA): • - 5 millions bases • - 4500 genes • - hundreds gene circuits • Self-reproduce in 30 min. • Capable of: • responding to stresses • sensing the environment DNA  RNA  proteins Genome (DNA) Nutrients 1 μm (E. coli)

23. Cell: the basic unit of life Information Compartment Metabolism Unique property: self-reproduction. Each part is essential. Each part is made of molecular machineries.

24. Synthetic biology era The design and fabrication of biological components and systems that do not exist in the natural world: • to understand gene regulation and make simple computations. • to use them either as molecular-scale factories. • to create new hybrid materials.

25. Synthetic biology platforms in vivo in silico in vitro

26. Synthetic biology in a test tube (cell-free synthetic biology) Constructing living systems in a test tube from the DNA program. ● bottom-up, reductionist and constructive approach. ● no endogenous information. ● no interference and response from an organism. ● more freedom of control and design compared to in vivo. ● molecular programming approach to living systems. TX TL protein DNA mRNA

27. 2 Small gene circuits in a test tube TX TL protein)n (DNA mRNA circuits

28. Transcriptional activation cascade P28 deGFP σ70 σ28 P70

29. AND gate S54-NtrC

30. Multiple stage cascade σ70 σ38 P70 σ19 σ28 P28 T7rnap deGFP P38 P19 PT7 Leak! Loss of specificity

31. Multiple stage cascade σ70 σ38 P70 σ19 σ28 P28 T7rnap deGFP P38 P19 PT7 Leak attenuation Specificity

32. Conclusion: • • constructed and characterized cell-free circuits. • • learned the design rules. • • tuned the dynamics. • • global mRNA degradation rate is critical. • • Shin and Noireaux. ACS Synthetic Biology 2011. Information Compartment Metabolism

33. Genome scale circuits (information and self-organization) • ● What is the real capacity of the system to construct circuits • and living systems? • CFR batch mode: [Protein] = 25-30µM • E. coli: [Protein]ave = 500nM • ● Test the system with genome-sized information. • ● Bacteriophages: • search for genomes composed of ≤ 60 genes. • with molecular biology technically accessible. • condition/bottleneck: complexity of the interaction with the • host beyond TX-TL. 50-60 genes

34. Phage T7 ● lytic coliphage. ● 40 kbp, 60 genes (35 with known functions). ● almost host independent (2 host proteins required). ● has its own RNA polymerase. ● has its own DNA polymerase.

35. Phage T7 synthesis in a test tube TX TL genome mRNA phage ● TEM image ● 5-6 hours of incubation ● batch mode reaction

36. T7 Genome replication ● up to 200 times greater with dNTPs. ● a few billion of functional phages per milliliter synthesized after 5-6 hours of incubation in batch mode.

37. T7 - E. coli Infection test No difference observed between in vivo and in vitro synthesized phages. ● phages per cell ≈ 100. ● phage cycle ≈ 25 min. ● E. coli division ≈ 30 min.