8/10/2019 MBB 130.1 lab notes

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MBB 130 1: Molecular Biophysics Lab LE 1

Methods and Processes

oPlasmid extraction

oTransformation

oProtein expression

oPurification

oDNA 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 selfo 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 unwindo 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

our el.

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! 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.

Transformation

Make your cell take up your plasmid

Uptake of exogenous genetic material

Methods:

o Electroporation

! Electrical shock makes cel

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

CaCl2positive charges of Ca2+shields negative

charges of DNA phosphates, no longer ionic

1. Incubate on ice

Slows fluid cell membrane

2. Heat-shock

Increases permeability of membranes

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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 11cfu/ug

Factors

o Plasmid size (inversely proportional)

o Conformation of DNA (supercoiled

DNA with highest efficiency)

o

Amount of DNAo 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, cel

free (wheat germ extract, E. coli extract)

There are reasons why E. coliis 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 leve

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 ulses

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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.

Polyacrylamide Gel Electrophoresis

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 solutiono I-same water:acetic acid: methoano

ratio as staining solution

! Methanol is a fixative that

prevents dispersion

o II-lower concentration of methanol

! You can keep it in destain II fo

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 todeal 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

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Ion exchange chromatography

Based on charge. Separate acidic/basic

Elute with salt gradients.

Immobilized Metal Affinity Chromatography

Co2+was used. Bound to linker, Sepharose

Binding of Co2+ is lower than Ni2+ 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

Flowthrougho 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 Co2+using EDTA

o Replenish afterwards

Why equilibrate resin before useo 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) ionsto 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