MBB 130.

1: Mol ecul ar Bi ophysi cs Lab LE 1

Methods and Processes
o Plasmid extraction
o Transformation
o Protein expression
o Purification
o DNA and Protein quantification

Plasmid
Extra-chromosomal, autonomously replicating
DNA
Covalently closed circular dsDNA
Non essential but gives competitive
advantage such as antibiotic resistance

Plasmid vectors
Contain an antibiotic resistance marker so you
can select your transformed bacteria
Has a multiple cloning site
TA cloning plasmid: creates overhang in gene
insert. Most plasmid in nature have many RE
sites where you can insert gene of interest

Molecular Biology applications
Cloning different DNA fragments
Protein production

Nucleic acid extraction
3 specific goals
1. Disruption of cell wall and membrane
2. Inactivation of nucleic acid degrading enzymes
(i.e. DNases, RNases)
3. Separation of nucleic acids from other cellular
components

Isolation of Plasmid DNA
Harvest cells by centrifugation
Discard supernatant & remove other media
components that could interfere w/ extraction
protocol or end product it self
o Residual media may interfere w/
downstream steps
Resuspend cells in solution
o Tris-Cl: maintain pH of environment
o EDTA: chelates divalent cations that
activate DNases
o RNase A: contamination may appear as
giant bright smear

Alkaline Lysis method
Lyse cells with Soln 2 (SDS/NaOH)
o SDS denaturing anionic detergent
o Dissolves membranes
o Binds and denature proteins
NaOH
o Double stranded DNA is unwound,
genomic DNA easier to unwind
o Denatures both plasmid and
chromosomal DNA
o Plasmid DNA easily find other strand

Solution 3
Neutralize NaOH (KOAc solution)
o Production of fluffy, white precipitate
! Precipitates SDS-protein complex
o Renaturation of plasmid DNA
o Separation of plasmid DNA from
contaminants via centrifugation

Isolation of plasmid DNA
Alcohol precipitation
70% ethanol wash
o remove contaminants precipitated by
absolute (e.g. salts)
Dissolve with TE (or other aqueous soln)
o Buffer solution with EDTA to prevent
activity of DNases by chelating divalent
cations

Nucleic acid yield and purity
Agarose gel electrophoresis
Absorbance
Fluorescent dyes that intercalate with DNA
Kits that allow you to add dyes to see amount
of fluorescence to determine quantity and
percent ot DNA added

Factors affecting Agarose Gel Electrophoresis
Molecular size of DNA
Concentration of DNA
Concentration of agarose gel
Conformation of DNA
o Loose conformation is retarded
o Presence of intercalating agents that
may alter characteristics of DNA
including shape
o Voltage
! In relation to amount of ions in
your gel.
! Higher voltage leads to higher
mobility but causes more
heating.
! If too slow, diffusion might
occur and lead to bad bands
o Type of agarose
o Electrophoresis buffers

*There are specific concentrations of agarose that
would yield best resolution. Typically, we use 1%
agarose because it is in the range of 500-1000 bp
*It is hard to resolve large and small DNAs on the
same gel.

Separation by size
PFGE (Pulse Field Gel Electrophoresis)
o Zig-zag path to increase effective path
Agarose
Acrylamide
o You cant view in UV if theres still a
glass plate. Check in UV, if not enough,
but back in glass plate

Conformation of DNA
Brightest band is usually supercoiled DNA
In order of increasing mobility: nicked, linear,
covalently bonded, supercoiled
Run in PCR to check size
It is sufficient because it is a check if plasmid
extraction is successful. If there is a band then
plasmid extraction is a success.

DNA visualization
Most common dye is Ethidium Bromide
o It intercalates with DNA in the
hydrophobic region and is therefore
considered as a mutagen
o Fluoresces when intercalated with DNA
because it is in hydrophobic region
and thus quenching is lessened
Gel red has a similar mode of action
Other dyes like acridine orange and SYBR
green, Actinomycin D

Determining DNA concentration
AGE run can be used to determine
concentration of DNA per band using a
special kind of marker
Same number of moles per band because they
are derived from one plasmid (lambda Hind III)
Some machines like GNOC can quantify
brightness
o Select band and tell you size of band
Higher mass = higher brightness because it
has more DNA;calculation based on % of mass

DNA purity
Nucleic acids, absorption peak at ~260 nm
A260/A280 ~ 1.8 for dsDNA and ~2.0 for ssRNA
o Ratios lower than 1.7 usually indicate
significant protein contamination
A260/A230 ratio of DNA and RNA should be
roughly equal to its A260/A280 ratio (greater than
or equal to 1.8)
o Lower ratios may indicate
contamination by organic compounds
(e.g. phenol, alcohol, or carbohydrates)
It measures light that is transmitted so you can
backtrack what light was absorbed.

Transformati on
Make your cell take up your plasmid
Uptake of exogenous genetic material
Methods:
o Electroporation
! Electrical shock makes cell
membranes permeable to DNA
o CaCl/Heat shock
! Chemical-component cells uptake
DNA

Competence
Some bacteria are naturally competent, this is
not the case for E. coli.
Artificial
o Chemical competence
o Electrical competence
Electroporation has a higher chance of killing
Heat Shock transformation
CaCl2 positive charges of Ca
2+
shields negative
charges of DNA phosphates, no longer ionic
1. Incubate on ice
Slows fluid cell membrane
2. Heat-shock
Increases permeability of membranes
3. Nutrient broth incubation
Allows beta-lactamase expression
Even if a cell has antibiotic resistance gene,
putting antibiotic right away would kill it.
Wait for 1 hour to ensure production of
antibiotic resistance gene

Selection and Screening
Selection such as antibiotic resistance
Screening: makes others grow but your
desired cells can be selectively chosen

Assessment of transformation
LB bacteria probably wont have fluorescence
because transformed cells are out competing
one another to even produce GFP
Dont over incubate because beta-lactamase
can seep out and enable growth of non-
resistant bacteria forming satellite colonies
Positive control tells you if cells are alive
Negative control tells you if amp works

Transformation efficiency
Expressed as number of CFU/ug of plasmid
In E. coli, the theoretical limit = 1 x 10
11
cfu/ug
Factors
o Plasmid size (inversely proportional)
o Conformation of DNA (supercoiled
DNA with highest efficiency)
o Amount of DNA
o Purity of DNA
o Source of DNA (Is it methylated)
o Growth of genotype of cells (consider
OD of cellsusually at 0.4)
o Method of transformation

Fluorescent Proteins
They are proteins based visual markers in
the study of biological processes
Localization and regulation of gene
expressions
Cell movement and cell fate during
development
Screenable marker to identify organisms

Heterologous protein expression
Synthesis of foreign proteins in a host
organism following transformation of that
organism by a vector carrying genes from a
different organism.

Expression systems
Bacteria, esp. E. coli
Yeast cells, insect cells, mammalian cells, cell
free (wheat germ extract, E. coli extract)
There are reasons why E. coli is preferred
o Fast growth
o Simple media
o Highest yield
*But proper folding is not attained for proteins
derived from higher organisms (glycosylation)

Expression Vectors
May differ depending on expression systems
Usually inducible. IPTG induction-lac operon is
inherent. Vectors that have a T7 promoter. T7
polymerase is inducible inside the cell
Arabinose induction-ara operon
His tag
Enterokinase site can be used to cut His tag
*yeast vectors require methanol

Ara operon
Arabinose acts as an effector. Binding of
arabinose cause downstream expression of
genes. Essentially, you just change what genes
are expressed

E. coli strains used for expression
BL21-protease-deficient; inducible high level
of expression uses T7 RNA polymerase
BL21(DE3)-lacI repression of T7 RNA
polymerase; tighter regulation
o IPTG binds to repressor on the gene
then now your polymerase

BL21(DE3) pLysS/pLysE-production of T7
lysozyme (inhibitor of T7 RNA pol transcription
o You have an inhibitor that removes
inherent T7 RNA pol transcription

Why use a starter culture?
Starter culture to select for living cells and
help even out inoculation.
In theory, same time to grow.

Extraction
Resuspend pellet
o proteins expressed in media
Sonicate 3x 30 sec/pulse
o Heat generated may denature proteins
60 secs on ice between pulses
Centrifuge to remove cell debris
Transfer clarified supernatant
*GFP makes it easy to check, look at fluorescence.
Possible that there is high expression but GFP
remains in the pellet.

Pol yacryl ami de Gel El ectrophoresi s

Acrylamide Polymerization
APS is the source of free radical that starts the
polymerization of acrylamide
TEMED catalyzes formation of free radical
from your APS
%T: % concentration of bis + acrylamide
%C: % of bisacrylamide or crosslinker
o Max %C is 5% because after that the
pore size becomes larger at higher %C
o 19:1 " 5%C

Different PAGE systems
Gradient PAGE
o Usually from pre-cast, because you
need a gradient mixer

Continuous vs. Discontinuous (Laemmli) PAGE
Stacking gel
o Zwitterionic glycine, less mobility
o Sandwich effect of protein
o pH 6.8
Resolving gel
o Glycinate form, greater mobility
o pH 8.8
SDS-PAGE
o Includes DDT and Beta-
Mecaptoethanol w/c are reducing
o Pseudonative PAGE: SDS but no
boiling and reducing agent. Run still
based in structure (This is in your
unboiled sample)
! Native PAGE is quite difficult
because there are many other
factors (+conformation, charge)
at play to get discrete bands

2 dimensional PAGE
Isoelectric focusing
SDS-PAGE

Treatment Buffer
Glycerol
Bromophenol blue~ 11 kDa

Protein visualization
Staining solution
o Fixation: water: acetic acid: methanol
o Coomassie Brilliant Blue R-250
! Slightly reddish at acidic pH
o Coomassie binds to proteins
Destaining solution
o I-same water:acetic acid: methoanol
ratio as staining solution
! Methanol is a fixative that
prevents dispersion
o II-lower concentration of methanol
! You can keep it in destain II for
a very long time.
o If you want to check protein activity, do
it before putting destaining solution
due to presence of acid

Protein visualization
Coomassie vs. Silver staining
o Increase detection of bands but you
actually need a lot of solutions
o High background is likely

Molecular Weight markers
Know what marker you used and buffer you
used. Thus, different mobilities in different
buffers
Aprotinin and Insulin are below limit of
resolution in our runs.
Precision Plus standards (Bio-Rad)

Protein Purification
Chromatography
o Size exclusion chromatography
o Ion exchange chromatography
o Affinity chromatography
o Hydrophobic interaction chrom
Decreases amount of proteins you have to
deal with because you elute out other proteins

Size exclusion chromatography
Larger proteins do not enter gel matrix and
they just flow out.
Smaller molecules elute at a later time
because they interact with matrix
Some proteins may have the same size

Ion exchange chromatography
Based on charge. Separate acidic/basic
Elute with salt gradients.

Immobilized Metal Affinity Chromatography
Co
2+
was used. Bound to linker, Sepharose
Binding of Co
2+
is lower than Ni
2+
creates
lower yield but higher purity
Elute out with imidazole or pH

Fast Protein Liquid Chromatography
Automated chromatography system
System consists of high precision pumps,
control unit, column, detection system, and
fraction collector
Vs. HPLC
o FPLC can withstand up to 5MPa only

Purification
Flowthrough
o Proteins that dont bind to resin
Wash
o Excess or non-specific binding
Elution
o Protein w/ His tag
Strip
o Remove imidazole using MES buffer
o Remove Co
2+
using EDTA
o Replenish afterwards
Why equilibrate resin before use
o Maintain binding pH
o Has a level of salt that prevent the non-
specific binding, which could occur.

Purification of GFP
Increases size from 25 kDa to 29 kDa due to
His tag and plasmid components
Unboiled samples, you generally wont get
size that you are looking for because some
structures are retained
Fraction 1 may have equilibration buffer

Protein quantification
Bradford assay
o Colorimetric assay for measuring total
protein concentration
o It involves the binding of Coomassie
Brilliant Blue G-250
1. Make solutions of a known proteins (Bovine
Serum Albumin)
2. Mix from each solution with Bradford reagent
(CBBG + Phosphoric acid + Ethanol)
Binds to basic residues
If higher basic, detect a lot more protein
therefore overestimate
3. Measure absorbance at 600 nm
Biuret assay
o Relies on reduction of copper (II) ions
to copper (I), the latter forma complex
with the nitrogens of the peptide bond
in an alkaline solution
o Absorption at 540 nm is directly
proportional to protein concentration
o Bind to peptide bond
Lowry Assay
o Uses Folin Ciocalteu reagent
(phosphomolybdic/phosphotungstic
acid)
o Cuprous ions (Cu
+
) reduction of Folin
Ciocalteu reagent produces blue color
that can be read at 650-750 nm. More
aromatic residues also detected
BCA assays
o React with reduced cuprous cation
o Intense purple-colored reaction
product reacts from the chelation of 2
molecules of BCA with one Cu
+
ions
o BCA/copper complex is water-soluble
and exhibits strong
o Linear absorbance at 562 nm with
increasing protein concentration
o Better because more specific