Molec Biotech Exam 2

protein purification process

1. starting material + buffer to maintain stability

2. applying pressure/ mehc stress/ enzymes

protein stability

relates to temperature, pH, pressure, enzymes

breaking open cells

releases protein of interest + other cellular material

purification via centrifuge

1. centrifuge: pellet cont. insoluble material + debris.

2. collect supernatant: contains soluble protein fraction & insoluble material is removed.

3. it can now be used for precipitation or chromatography

methods to break cell

sonication, homogenization, high-speed blending, osmotic shock, beadbeater, french press, enzymes, mortar & pestle, frozen tissue

how to verify success of cell lysis

• compare samples before & after

• general protein assays

• enzyme activity assay (if pro is enzyme)

• western blotting/ HPCL- based assays/ ELISA

chromatography

column containing a stationary phase (resin) and a mobile base (buffer) to separate proteins accoording to how they interact with the resin

Ion exchange chromatography

separates proteins based on their net electrical charge. proteins have diff charges depending on the pH of their surrounding environment and their isoelectric point (PI)

types of IEX

• cation exchange

• anion exhcange

cation exchange

resin has negative groups that attract & bind positive proteins (basic pH binds, negatives pass thru)

anion exchange

resin has positive groups that attract negative proteins (acidic pH binds, positives pass thru)

IEX process

1. equilibrate column with buffer at specific pH & low salt conc

2. add protein mix

3. proteins with correct charge bind, those that dont just pass thru

4. wash column to remove contaminants

5. elution by gradually increasing salt conc or changing pH

6. salt ions compete with proteins to bind on column so proteins detach and elute

7. proteins with weaker interactions elute first, stronger ones need more salt

hydrophobic interaction chromatography

separates proteins based on their surface hydrophobicity

HIC process

1. column contains resin modified with hydrophobic ligands

2. protein sample preped in high salt buffer

3. high salt conc promotes hydrophobic interactions between proteins and resin

4. proteins that dont bind are washed away

5. salt conc. is decreased w salt gradient

6. as salt decreases, H-phobic interactions weaken so pros detach

7. less H-phobi elute first, more phobic needs lower salt conc,

temp in HIC

high temp strengthens hydrophobic interactions

how affinity chromatography works

separates biochemicals based on highly specific interactions between protein and another molec (Antigen, antibody , enzyme + subs, receptor + ligands)

why affinity chrom is desirable

provides a way to selectively bond one component from a complex mix & provides higher enrichment in a single step

elution strats for affinity chrom

• ionic strength, pH, temp, cahotropic agents, solvent change, denaturants, competitor ligands/analogs with more affinity than ligand

IMAC

separates proteins based on ability to bind to metal ions attached to resin

IMAC process

1. metal ions (Ni²+) attached to sollid support using chelating ligand

2. mix is passed through column

3. proteins with multiple His residues bind to immobilized nickel

4. unwanted proteins are washed away

5. His-tagged proteins are released by adding imidazole to elute

methods to isolate DNA

• organic isolation

• inorganic isolation

• silica binding

• chromatography

• density gradient centrifugation

organic isolation

purifies by removing contaminants using methods like high salt, low pH, mix of phenol + chloroform.

RNAse is added to avoid contamination

inorganic isolation

“salting out”.

using low pH and salt to selectively precipitate proteins. DNA is left in soln and precipitated using ethanol or isopropanol

silica binding

• binding occurs in presence of high salt & disrupted by elution with water

• the rxn of the silica’s negative charge due to ionic strength of buffer allows for DNA absorption

• disrupt the shell of hydration & positively charged ions from a salt bridge between silica and DNA.

• washed with high salt ethanol and eluted with low salt

chromatography DNA isolation

• negatively charged DNA can be purified by anion exchange columns.

• This increases the # of samples screened, isolation in limited amounts, isolation, isolation of challenging samples.

• helpful for fast quantitation by PCR & not done in clinical lab

density gradient centrifugation

• separates DNA based on its density using a gradient (commonly CaCl2) DNA is centrifuged at high speeds & molecs move through the gradient until they reach a region where the soln’s density matches their own.

• DNA forms distinct bonds that can be collected & separated from other cellular components
  
  

strategies to prevent RNA degradation

• DEPC

• Vanadyl rubonucleoside complexes

• protein inhibitors of RNAse

• inactivate RNAse with GITC

• gloves + cleaning surfaces

• using RNAse free reagents

• proper storage

• keep samples cold

how to determine if DNA/RNA quality is good

• spectroscopy

• agarose gel electrophoresis

• capillary electrophoresis

• fluorometry

spectrophotometry (nano drop)

measures absorbande at diff wavelengths to estimate nucleic acid conc. and purity

• 260nm = DNA & RNA

• 280nm = Proteins

• 230nm = contaminants

• A260/A280: DNA (1.8), RNA (2.0)

• A260/A230: 2.0-2.2, lower = contaminants

agarose gel electrophoresis

separates DNA/RNA by size, allows evaluation of integrity by staining with fluorescent dye

• good quality = clear bands + minimal smears

• RNA should show 2 ribosomal bonds

• smearing/ loss of bands = degradation

capillary electrophoresis

provides more accurate assessment of RNA quality than GE due to sensitive detector + algorithms to evaluate components

• evaluates 28S & 18S

• high quality: intact bonds (2:1 ratio, less that 1.5:1 = degradation)

• calculates RNA integrity number (1-10)

fluorometry

uses fluorescent dyes that binds specifically to DNA or RNA.

• fluoresence increases when dye binds to nucleic acids

• requires a negative control + standard with known conc.

souther and northern blotting similarity

• use gel electrophoresis

• transfer nucleic acids to a membrane

• hybridize with a labeled probe that binds to complementary seq

• require washing to remove unbound probes

southern blotting

used to determine if specific DNA seqs are present in sample.

Can also be used for genetic mapping, ID testing, gene linkage, & detecting gene markers

southern blotting procedure

1. isolate genomic DNA & cut into smaller frags using REs

2. separate according to size with GE

3. treat DNA with alkaline soln to separate double strands (depurination might be needed)

4. DNA frags from gel moved onto nylon or nitrocellulose membrane

5. permanently attach DNA to membrane using heat or UV

6. add labelled single stranded DNA probe complementary to target seq

7. wash away unbound probes & detect labeled probes using autoradiography or chemiluminesence

southern blotting result

visible band indicating target DNA is present in a sample

Northern blotting

determines the presence, size, and amount of specific RNA molec.

Specially useful for measuring gene expression & comparing expression between diff tissues, cell types. or developmental stages

northern blotting procedure

1. isolate total RNA from cells or tissue

2. separate RNA by size using RNA gel under denaturing condition to avoid secondary structures

3. transfer separated RNA to a membrane

4. attach RNA to membrane

5. add labeled probe that binds to RNA seq of interest

6. wash away unbound probes and detect the labeled probe

northern blotting result

intensity of the bands provide info about the relative abundance of the RNA (position = size)

Type II restriction enzyme

recognize specific DNA seq and cut both DNA strands near those sites

• used for cloning and recombinant DNA construction

T4 ligase

joins DNA fragments by forming phosphodiester bonds between 5’ PO4 and 3’ OH ends

• used to insert DNA frags into plasmid vectors

Taq DNA Polymerase

synthesizes DNA from a primer using a template

• used because its heat stable and helps amplify DNA in PCR

Terminal Deoxynucleotidy transferase

adds nucleotides to the 3’ end of DNA without requiring a template

• used to add nucleotide tails or label DNA ends

T7 RNA Polymerase

synthesizes RNA from DNA template containing a T7 promoter

• used for in-vitro RNA transcription

Reverse Transcriptase

synthesizes complementary DNA from an RNA template

• used in cDNA synthesis & RT-PCR

T4 Polynucleotide Kinase

Transfers phosphate group from ATP to the 5’ -OH end of DNA or RNA

• used to prepare nucelic acids for ligation or end-labeling

Alkaline Phosphate

removes the phosphate group from 5’ ends of DNA or RNA

• used to prevent a cut plasmid vector from self-ligating during cloning

PCR

technique used to amplify the # of copies of a specific region of DNA generating millions-billions of copies of that DNA sample that can be used for cloning / sequencing/ assays

what can PCR be used for

amplifying defective genes to help diagnose illness, identify pathogens, and the amplified frags can be “genetic fingerprints”

qPCR

combined target amplification and detection, eliminating the need to detect products on a gel. Monitors fluoresence emitted during rx as indicator or amplicon production at each PCR cycle as opposed to end point detection.

qPCR uses

measuring initial quantity of DNA in sample, quantifying pathogen DNA, measuring viral DNA copy numbers, detecting & quantifying specific genetic sequences

RT-PCR

based on process of reverse transcription. It uses mRNA, total RNA, or viral RNA. it allows for the detection of rare or low copy mRNA seq by amplifying the complementary DNA generated in its first step

RT-PCR uses

considered gold standard for gene expression analysis and detection of RNA viruses

RT-qPCR

converts RNA into cDNA and then quantitatively measures the amplification in real time

RT-qPCR uses

measuring gene expression, comparing DNA transcript levels between exp and control samples, detecting and quantifying RNA virus (HIV, influenza, COVID) & studying treatment effects on gene activity

steps to insert PCR product into plasmid & amplify in cells

1. amplify gene of interest with PCR

2. prepare plasmid vector (linearization with REs, may need to add alkaline phosphatase to dephosphorilate 5’ ends)

3. ligate adding insert to vector with T4 ligase, ATP, and Mg 2+

4. transform bacterial cells and allow cells to recover in growth medium

5. select positive transformants to grow on selection meda (blue/white screening)

6. isolate plasmid DNA by cleaving sample with correct RE (AGE/SB/Colony PCR/ seq)

7. confirm orientation & seq using sanger sequencing with appropriate vectpor or insert primers

cloning vectors

• plasmid, lambda phage, cosmids, P1 phages, BAC, YAC, MAC

• they all have bacterial origin of replication, selectable marker genes, and multiple cloning sites

plasmid vector

from bacterial circular extrachromosomal DNA

• host: e. coli

• cap: 15kb

lambda phage

derived from bacteriophage lambda

• host: e.coli

• cap: 25kb

cosmids

combines elements of plasmids with featues from lambda phage

• host: e.coli

• cap: 35-45 kb

P1 phages

derived from bacteriophage P1 cloning systems

• host: e. coli

• cap: 100-300 kb

BAC

based on bacterial F plasmid

• host: e.coli

• cap: 350 kb

YAC

linear DNA based on yeast chromosomes

• host: s. cerevisiae

• cap: 100-2000kb

MAC

retroviral vector have elements allowing integrations of transgene into mammalian cell DNA

• host: mammalian cells

• capacity: >1000kb

blunt-end cloning techniques

easy & versatile cloning ( does not rquire cloning sites)

linearized vector with blunt ends is required and typically dephosphorylated. Less efficient than cohesive cloning

cohesive end cloning

DNA frags have short single-stranded overhangs called “Sticky ends” made with restriction enzymes. These pair temporarily through H-bonds and ligase seals them with phosphodiester bonds.

Generally more efficient due to easy pairing of ends.

Using 2 different REs can help control insert orientation

TA Cloning

1. amplify DNA insert with PCR using Taq

2. use plasmid vector with complimentary single-thymine overhangs

3. overhangs on PCR product base pair with T overhangs on the vector

4. T4 ligase joins insert and vector using phosphodiester bonds ( needs ATP + Mg 2+)

5. introduce recombinant plasmid into competent cells and screen colonies for insert

6. analyze plasmid to determine wether insert is present and in correct orientation with PCR or sequencing

TOPO TA cloning

1. amplify DNA insert with PCR using Taq

2. vector contains complementary T overhangs and pre-activated with covalently attached topoisomerase I

3. A & T overhangs anneal and topoisomerase I enzyme catalyzes joining of the insert to the vector ( helps avoid adding ligase or ATP)

4. introduce resulting recombinant plasmid into competent cells

5. select & screen by using the vector selectable marker and do colony screening to perform PCR or RE assays to verify orientation of insert

Transformation

altering cell through direct uptake & incorporation of genetic material from its surroundings. 2 methods: chemical or electroporation of competent cells

transformation chemical method

• log phase e. coli culture & centrifuge

• resuspent in CaCl2

• chill on ice & alliquot competent cells

• add amplified plasmid DNA & chill on ice

• heat shock in 42° H2O bath

• plate on LB + ampicillin

Transfection

deliberately introducing nucleic acids into mammalian cells using non-natural methods.

aided by cationic polymers like PEI, Calcium phosphate, or cationic lipids to coat DNA or through electroporation

Transfection chemical aided

1. cationic lipid transfection reagent interacts with DNA

2. DNA-cationic lipid complex forms

3. complex interacts with mammalian cell line

4. expression happens following successful transfection

Transduction

introducing DNA into another cell using a viral vector.

Done through infection of bacteria by bacteriophage or mammalian cells with retrovirus used to introduce recombinant DNA

Gateway cloning

uses site specific recombination based on the natural recombination system of bacteriophage lambda to trasnfer a gene of interest into different expression vectors.

• main advantage is that the gene only needs to be cloned once into an entry vector. it can then be transfered into multiple expression vectors designed for diff host cells.

Gateway cloning process

1. amplify gene of interest using PCR designed to contain attB sites

2. PCR product with attB sites is combined with donor vector containing attP sites → entry clone with attL sites

3. entry clone combined with destination vector that has attR sites. (LR clonase recombination between attL and attR)

4. destination vector determines which host can express the gene cuz it contains the appropriate reg elements

5. expression clone is introduced into the selected host cells, where the gene can be expressed to produce the desired protein

lac operon

controls genes involved in lactose metabolism in e. coli

• no inducer: lacl repressor binds to operator, blocking RNA poly from transcribing

• with inducer: allolactose binds to repressor, changing its shape so it releases operator so transcription occurs

• CAP-cAMP: dual regulation. low glucose = cAMP increases → binds CAP → lac activated; high glucose= cAMP decreases → CAP doesnt binds → RNA poly no assisstance so low transcription

trp operator

controls genes involved in tryptophan biosynthesis in e. coli

• low trp: repressor is inactive and cannot bind the operator so RNA trascribes for trp synthesis

• high trp: trp is co-repressor. binds to repressor and activates it. complex binds to operator and block transcription

• leader seq trpL: high = ribos translate leader pep easily cuz a lot of trp available, mrna forms hairpin and rna poly stops abruptly; low trp = ribos stall cuz no trp, mrna forms hairpin, rna poly continues transcript = enzyme for trp production are porduced.