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Mathematics
Robotics
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Biology
Engineering
Physics
Optics
Measurement Methods in Systems Biology
Widespread Methods:
o GFP Expression
o Fluorescence Imaging
o Two-Hybrid Screen
o Expression DNA Chips
o Quantitative PCR
o Highly Parallel Sequencing
GFP: Green Fluorescent Protein
The green fluorescent protein (GFP) is a protein from the jellyfish
Aequorea victoria that fluoresces green when exposed to blue light.
This process takes place when the protein aequorin, also produced by
A. victoria, interacts with Ca2+ ions thus emitting a blue glow.
The wild-type GFP (wtGFP) from A. victoria has a major excitation peak
at a wavelength of 395 nm and a minor one at 475 nm. Its emission
peak is at 509 nm which is in the lower green portion of the visible
spectrum. In cell and molecular biology, the GFP gene is frequently
used as a reporter of expression. In modified forms it has been used to
make biosensors, and many animals have been created that express
GFP as a proof-of-concept that a gene can be expressed throughout a
given organism.
One of the most powerful uses of GFP is to express the protein in small
sets of specific cells. This allows researchers to optically detect specific
types of cells in vitro (in a dish), or even in vivo (in the living organism).
Due to this widespread usage different mutants of GFP have been
engineered over the last few years: some mutants have been produced
with increased fluorescence and the protein major excitation peak has
been shifted to 490 nm with the peak emission kept at 509 nm (EGFP).
Color mutants have been obtained from the GFP gene as well: in
particular the cyan fluorescent protein (CFP) and the yellow fluorescent
protein (YFP) are two colour variants employed for fluorescence
resonance energy transfer (FRET) experiments.
While most small fluorescent molecules such as FITC (fluorescein
isothiocyanate) are strongly phototoxic when used in live cells,
fluorescent proteins such as GFP are usually much less harmful when
illuminated in living cells. (from: en.wikipedia.org)
Neurons expressing GFP
The Fluorescence Process
Stage 1 : Excitation
A photon of energy hEX creates an excited
electronic singlet state (S1'). In chemiluminescence, S1’ is populated by a chemical
reaction.
Stage 2 : Excited-State Lifetime
For finite time (typ. 1–10 ns), the fluorophore
undergoes conformational changes and is
subject to interactions with its molecular environment.
(a) the energy of S1' is partially dissipated,
yielding a relaxed singlet excited state (S1).
(b) not all excited molecules return to the
ground state (S0) by fluorescence emission:
possibilties are collisional quenching, Fluorescence Resonance Energy Transfer (FRET) and
intersystem crossing.
Stage 3 : Fluorescence Emission
A photon of energy hEM is emitted, returning
the fluorophore to ground state S0. Due to
energy dissipation (S1’ to S1) the energy of
the emission photon is lower (longer wavelength). Difference is called the Stokes shift,
making it possible to detect emission photons
against the huge background of excitation
photons.
Fluorescein
Fluorescence Microscope
http://www.olympusmicro.com/
Two Hybrid Screen
Fields & Song, Nature 340,245 (1989)
Yeast Two Hybrid Screen: Searching for Protein-Protein interactions
Bait
Prey
Two-hybrid screening is a molecular
biology technique used to discover proteinprotein interactions by testing for physical
interactions (such as binding) between two
proteins. One protein is termed the bait and
the other is a prey or library.
For the purposes of two-hybrid screening,
the transcription factor is split into two
separate fragments, called Binding Domain
(BD) and Activating Domain (AD). The BD is
the domain responsible for binding to the UAS
and the AD is the domain responsible for
activation of transcription. The key to the twohybrid screen is that in most eukaryotic
transcription factors, the activating and
binding domains are modular and can
function in close proximity to each other
without direct binding.
The most common screening approach is the yeast twohybrid assay. This system utilizes a genetically engineered
strain of yeast in which the biosynthesis of certain
nutrients (usually amino acids or nucleic acids) is lacking.
When grown on media that lacks these nutrients, the yeast
fail to survive.
In yeast two-hybrid screening, separate bait and prey
plasmids are simultaneously introduced into the mutant
yeast strain. With a certain bait protein, two hybrid
screening can be "directed" to test for protein-protein
interaction with a known protein inserted into prey plasmid.
Alternatively, "library screening" involves pairing bait
protein with millions of different prey plasmids that have
been engineered to produce protein from a unique,
randomly inserted DNA fragment. (from: en.wikipedia.org)
DNA Chips
Highly Parallel DNA Detection Methods
DNA Detection to…
o Define a new ’Microscope’:
RNA Profiles of Cells
o Test for Virus (AIDS...)
o Select medication
o Test for GMO
o Criminal investigations
More Details: http://en.wikipedia.org/wiki/DNA_chip
Highly parallel DNA detection I
A) Spotted Arrays
Rockefeller
Highly parallel DNA detection II
B) On-Chip Synthesis
Affymetrix
Highly parallel DNA detection II
B) On-Chip Synthesis
Affymetrix
Mismatch is in center of 25-base probe
Highly parallel DNA detection III
D) Quantitative PCR (qPCR)
Online Detection of Product
Standard Curve
Quantitative polymerase chain reaction (Q-PCR) is a modification of polymerase chain
reaction used to rapidly measure the quantity of a product of polymerase chain reaction. It is
preferably done in real-time, thus is an indirect method for quantitatively measuring starting
amounts of DNA, complementary DNA or ribonucleic acid (RNA). This is commonly used for
the purpose of determining whether a genetic sequence is present or not, and if it is present
the number of copies in the sample. (en.wikipedia.org)
(c) Roche
Highly parallel DNA detection IV
C) Massive Parallel Signature Sequencing (MPSS)
Individual "cloning" on beads
is performed picking a small
sample from a large pool of
combinations. This ensures
that each bead has a unique
DNA sequence attached.
Brenner et.al. Nature Biotechnology 18:630 (2000)
Highly parallel DNA detection IV
C) Massive Parallel Signature Sequencing (MPSS)
Lynx
Brenner et.al. Nature Biotechnology 18:630 (2000)
+ Searching for unknown sequences
Highly parallel DNA detection IV
Margulies et.al. Nature 437,376 (2005)
Highly parallel DNA detection IV
Margulies et.al. Nature 437,376 (2005)
Highly parallel DNA detection IV
Margulies et.al. Nature 437,376 (2005)
Highly parallel DNA detection IV
Margulies et.al. Nature 437,376 (2005)
Overview Systems Biology
http://en.wikipedia.org/wiki/Systems_biology