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.

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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.

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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 kBT1 kBT =1 pN nm

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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

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HistoryIdeas 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.

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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

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Alexander Kamenev Boris Shklovskii

Theory:

John Broadhurst David ThomasJoachim Mueller

Experiment:

Biophysics Group

Vincent Noireaux 7

Alex Kamenev

1. Populations dynamics, as an example of non-equilibrium statistical mechanics.

2. Transport through ion channels, as an example of 1D physics.

Predator Population

Pre

y P

op

ula

tio

n

8

rR

Electrostatic theory of viral self-assemblyBoris Shklovskii

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Thomas LabSpectroscopic Probes of Muscle Protein Structure and Dynamics

Ca2+

Actin

CardiacCalciumPump

Phospho-lamban

Spectroscopic Probe Methods: Electron paramagnetic resonance (EPR) Nuclear Magnetic Resonance (NMR) Time-resolved fluorescence and phosphorescence

Probes

NO NH

C

O

CH2I

I

HOI

O

ICOOH

O

N = C = S

•

I

ATP

ADP + Pi

ATP

ADP + Pi

Myosin

Ca2+

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J. Broadhurst

Magneto encephalography (MEG)

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?)

 

Study the location in the human brain of the processors of external stimuli.

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.

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Mueller Lab: Fluorescence Fluctuation Spectroscopy (FFS)

Two-photon EffectSingle-molecule microscope

objective

Two-PhotonSpot

FFS in cells

104 105 1062000

3000

4000

5000

6000

7000

8000

9000

10000102 103 104

Concentration (nM)

dimer

monomer

RAR LBD TR4

app(c

psm

)

Intensity (Counts per second)

Watch Protein Interactions in Living Cells:

Photon Count

Statistics

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Image of a cell assembling viral-like particles. We study assembly process of retroviruses, such as HIV-1.

Joachim Mueller: Protein Assemblies and VirusesJoachim Mueller: Protein Assemblies and Viruses

Light burst from single molecules passing through tiny optical volume

Harvest viral particles

Microfluidics of viral particles

0 100 200 300 4002000

2500

3000

3500

4000

4500

5000

5500

Inte

nsity

(cp

s)

time (sec) • protein coat contains holes• the hole density varies• below percolation threshold

FluctuationAnalysis

ViralParticle

2-photon spectroscopy

FluctuationAnalysis

Construct physical model of assembly pathway

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Vincent Noireaux

● information processes (synthetic genetic circuits).● biopolymer self-assembly at the membrane: cell division, motility, nano by bio.● artificial cell system.

Artificial cell system.Self-assembly of proteins/biopolymers.

20μm

Reconstitution of genetic circuits in vitro.Coarse-grained model of circuits.

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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

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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.

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DNA sequencing and synthesis

Sequencing of bacterium genome: 1 week (5 Mb)

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Information

man-made

nature - evolution 18

New interdisciplinary fields

● system biology: (1) understanding the structure of the system, such as gene regulatory

networks.(2) understanding the dynamics of the system, both quantitative and

qualitative analysis.(3) understanding the control methods of the system.(4) 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.

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Vincent Noireaux, UMN

Molecular programming in a test tube: synthetic gene

circuits, phage synthesis and artificial cell.

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1Introduction – Motivations

• The three components of cellular life.• the bottom-up approach to living systems.

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Living cell(bacteria E. coli)

DNA RNA proteins

Nutrients

1 μm(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

Genome (DNA)

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Information Compartment

Metabolism

Each part is essential.Each part is made of molecular machineries.

Cell: the basic unit of life

Unique property: self-reproduction.

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Synthetic biology eraThe 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.

Synthetic biology platforms

in vivo

in silico

in vitro

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Synthetic biology in a test tube(cell-free synthetic biology)

● 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.

DNA mRNA proteinTXTX TLTL

Constructing living systems in a test tubefrom the DNA program.

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2Small gene circuits in a test tube

(DNA mRNA protein)n

TXTX TLTL

circuits

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Transcriptional activation cascade

P70σ28 P28 deGFP

σ70

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AND gate S54-NtrC

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Multiple stage cascade

P70σ38 P19

σ28P38σ19 P28 T7rnap PT7 deGFP

σ70

Loss of specificity

€

τm =12 ±2 min

Leak!

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P70σ38 P19

σ28P38σ19 P28 T7rnap PT7 deGFP

σ70

Leak attenuationSpecificity

€

τm =6 ±1min

Multiple stage cascade

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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

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● What is the real capacity of the system to construct circuitsand 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.

Genome scale circuits(information and self-organization)

50-60 genes

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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.

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genome mRNA phageTXTX TLTL

Phage T7 synthesis in a test tube

● TEM image● 5-6 hours of incubation● batch mode reaction

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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.

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T7 - E. coli Infection testNo difference observed between in vivo and in vitro

synthesized phages.

● phages per cell ≈ 100.● phage cycle ≈ 25 min.● E. coli division ≈ 30 min.