Genetic Engineering Flashcards
Give 2 ways in which an oligohistidine tag be added to a protein?
1) cloning the protein coding sequence in-frame in an expression vector with an oligohistidine tag coding region
2) adding the coding region by PCR before cloning into vector
What is the name of a feature that can be associated with a recombinant protein that allows purification?
Oligohistidine tag
What is the type of chromatography used to purify an oligohistidine tagged protein?
Affinity
Immobilised, Ni-NTA
How does the resin in affinity chromatography allow protein purification?
Resin bind ps diva lent metal ions
Histidines coordinate to free positions on metal ions
How can a protein be eluted from a chromatography column using a histidine tag?
Use imidazole
Has structure similar to histidine = competition
How would you check whether your protein is pure?
Use SDS-PAGE to check molecular mass and any contaminating proteins
What is a fluorescent molecule?
Absorbs light at shorter wavelengths and emit at longer wavelength
What is labeled with a fluorophore during immunofluorescence?
Antibody, by cross-linking
3 advantages of fluorescent proteins in cellular imaging
Used in live cells Multiple colours Genetically encoded Intrinsic fluorophore Can be used as tag for other proteins of interest
Disadvantages of fluorescent protein?
Low brightness
Complex photo physics such as dark states
Large size
PH sensitivity
You have purified the regulatory protein ‘X’, thought to bind to upstream regulatory region of ‘Y’ gene, the nt sequence of which is known.
What reagents do you need to perform a gel shift experiment to confirm this interaction?
Labelled ‘Y’ gene regulatory sequence
Purified ‘X’ protein
OR!!!
Cell fractions and antibody to ‘Y’
What does a DNA footprint analysis show?
Nt region where a protein binds to a regulatory gene
What reagents are required for DNA footprint analysis?
Radio labelled regulatory region
Your protein of binding
DNase I
Described the steps in immunoprecipitation using an antibody against a protein of interest
1) Treat living cels with cross linking agent, formaldehyde
- -> this will cross link the regulatory region with the protein
2) lyse the cells
3) sonicate DNA to shear into fragments
4) use antibody to co-ppt your protein bound to target sequence
5) remove protein using protease
6) sequence DNA (binding site will be common to all purified fragments)
State the fuels needed for successful Quikchange mutagenesis
At least 25nt long
Ideally G or C at 3’ ends
High Tm phosphorylation
Using NaOH raises pH, does this degradation RNA or DNA?
RNA
What’s the function of Bal31
Removes nucleotides from both 5’ and 3’ ends of a double stranded DNA molecule
What is the function of exonuclease
To remove nucleotides from the 3’ end of DNA molecules
What are isochizomers?
Restriction enzymes from different bacteria capable of recognising the same restriction site
What is the purpose of high stringency conditions in terms of nucleic acid hybrids?
To destabilise less stable nucleic acid hybrids
Include a low salt concentration and a temperature close to the Tm
Briefly describe the process of nick translation
DNase I used to introduce single stranded breaks into DNA at random sites
Exploits 5’-3’ Pol activity and 5’-3’ exonuclease of E. coli DNA Pol
1) Enzyme binds to nicks created by DNase I
2) removes nts and replaces with labelled nts
What’s the relationship between E. coli Klenow fragments and random hexanucleotide primers?
Random hexanucleotide primers are used in different labelling procedure called random primer method using the Klenow fragment of E.coli DNA polymerase
What do you need to do first before cloning DNA?
Purify it
1) grow cells in culture
2) spin cells down
3) lyse cells
4) apply to affinity column (usually silica)
What is the purpose of an oligodT column?
To separate mRNA from other RNAs
mRNAs possess polyA tail which binds to strings of T residues
What are the initial steps in cloning for purifying vector DNA?
What about for target DNA (genomic, cDNA, silico sourced)
Vector
1) digest circular plasmid with restriction enzymes
2) alkaline phosphatase treat to remove 5’ Ps
Target DNA
Either
— digest DNA with restriction enzyme
— PCR amplify fragment with carefully designed primers and digest
What steps follow in basic cloning after genome purification?
1) ligate digested Vector and target DNA
- –> gives mixture of vector and recombinant a
2) transform into E. coli
3) plate onto agar with antibiotic
4) only cells containing the plasmid will grow
5) get a clone through replication colonies
6) screen colonies to identify those with recombinant clone
7) colony PCR or plasmid isolated & restriction digest
Outline PCR
Add template, nts, polymerase, buffer and primers
95 — 55 — 72
Denature the DNA
Allow binding of primers
Allow thermos table polymerase to copy DNA between primers
Why should you avoid complementary primer sequences
Production of primer dimers
Get no replication of DNA as a result
Appear as thick band at bottom of agarose
Primer sequences don’t need to be perfectly complementary
But why should they be at the 3’ end
Why is that advantageous for the 5’ end?
Region of extension
Need to ensure specificity of annealing to correct target sequence
Can add different sequences for mutagenesis, recombination, manipulation, addition of Restriction enzyme
What is the purpose of the origin of replication
To allow autonomous replication from the genome
Why would you add restriction sites to a clone
To allow insertion into a vector, e.g. pET28
What does the reaction vessel for PCR contain?
0.2-0.5ml of polypropylene
How would you analyse the DNA fragments from a PCR reaction?
What kind of fragments would move fastest/slowest?
Agarose gel electrophoresis
Supercoil will most fastest bc compact
Introduce a nick produces open circular = significantly slower
Second strand break produces linear = quicker than open circular
What are the 3 types of ends produced by restriction enzymes
5’ overhang = EcoRI
Blunt end = Pvu II
3’ overhang = Kpn I
Some enzymes recognise different sites but generate the same sticky ends
Why is alkaline phosphatase used to treat the plasmid vector before inserting your DNA?
Removes 5’ phosphates to prevent self ligation
Mixture need inactivation first at 37 for 30 mins to prevent insert from being dephosphorylated as well
How are vector and insert DNA joined?
Ligase buffer containing ATP and DNA ligase
Cells repair missing phosphate in vector
Following a ligation reaction of your insert into a plasmid vector, what steps follow next?
1) Aliquot transformed into E. coli cells
2) treated with CaCl2 to disrupt cell walls
(Frozen stored at -80)
3) then thawed on ice adding 40-50ng DNA
4) heat shocked for 1-2 mins at 42 to take up DNA
What is another method apart from antibiotic resistance to show the presence of the recombinant plasmid?
LacZ blue white selection
White colonies indicate the presence of an insert in lacZa which disrupts beta galactosidase formation
What are the advantages and disadvantages of using a prokaryotic expression system?
Advantages
- large quantities
- numerous expression systems can be tested
- high throughput
Disadvantages
- poor functional expression of eukaryotic proteins
- problems with solubility of multi domain proteins
- little post translational modification
What are the advantages and disadvantages of eukaryotic expression systems?
Advantages
- good functional expression if eukaryotic proteins
- expressed proteins have native fold
- post translational modification (glycosylation)
Disadvantages
- small quantities
- limited number if expression systems
Give examples of prokaryotic and eukaryotic expression systems
Prokaryotic
Yeast cells - saccharomyces
Prokaryote - e.coli
Eukaryotic
Mammalian cells - HEK293
Insect cells - DES S2
Fungal cells - aspergillus
What are the advantages and disadvantages of using E. coli as an expression system?
Advantages
- simple and rapid culture
- easy to transform
- well characterised
- range of vectors/markers
Disadvantages
- requires cDNA (no introns)
- lacks much lost-translational processing
- possible protein stability/solubility/toxicity issues
Why do you need to optimise codon usage within an expression organism?
1) E.g. certain E. coli strains have additional tRNA genes to enhance expression
2) mutate critical codons to more commonly used codons
3) Re synthesise the complete gene to reflect codon usage
Some codons in heterologous genes may inhibit protein synthesis as they are rarely used by the expression host
3 examples of promoters that could be used in expression systems
Tac
Arabinose
T7
What role does IPTG play in T7 system
Causes induction of T7 RNA polymerase to act on the polymerase to product large amounts of recombinant protein
Cells enter stationary phase with no continued growth
Explain the process of autoinduction for glucose and lactose
Cells grow on glucose to high cell density Glucose depletes Cells use lactose Convert to allolactose Switches on gene expression
Why can secondary mRNA structures affect translation
Prevent efficient translation initiation and access to RBS or initiating ATG
How can we improve translation and subsequent product expression
Introduction of N terminal silent mutations into first 6 codons preventing mRNA secondary structure around the ATG
How could we increase transcription of a fungal enzyme galactose oxidase when expressed in E. coli?
N-terminal silent mutation enhances expression in VITRO by preventing mRNA secondary structure around ATG
Silent mutations in first 6 codons
‘GO’ construct in Bl21 Star (DE3)
Mutation in rne gene (rne131) = an RNAse responsible for mRNA degradation
What is Strep tag affinity purification?
Use of streptactin binding to protein of interest
Trp-ser-his-pro-gln-phe-glu-lys
What are the advantages of using yeast as an expression system?
Flexibility Some eukaryotic post translational modifications Maintenance of multiple plasmids Cheap and easy to grow Well characterised
However DOES NOT work with all proteins
Describe Pichia pastoris
Methylotrophic yeast AOX1 promoter produces up to 5% mRNA Yields 30% total cell protein Genetically similar to saccharomyces Low transformation efficiency Not identical glycosylation to mammals Potentially very high yields: 1) 2.5g/l soluble/secreted 2) 1.3g/l cytoplasmic 3) 1mg/l membrane
In terms of expression from a vector, what are the differences between saccharomyces and Pichia?
Saccharomyces = autonomous or integration into genome
Pichia = must integrate into genome to be expressed
(Either by single or double cross-over event)
What does real time PCR entail?
Measuring the amount of amplified DNA by measuring fluorescence emitted during each cycle rather than at a fixed endpoint
Simple and sensitive
How does SYBR green work and why would you use it in preference to ethidium bromide?
Bind to double stranded DNA and fluoresces
Ratio between ds and ss is much higher than EB
Fluoresces much brighter than ethidium bromide
SYBR green doesn’t bind ssDNA
What two probes can be used in real time PCR?
Briefly describe how they work
Hydrolysis based probes
— e.g taqman (5’-3’ exonuclease, digests end of probe to release reporter group)
Use reporter quencher system, when separated fluoresces
Hybridisation based probes
E.g. Beacons and fret probes
Base pair with DNA changing their 3D structure
FRET = fluorescence resonance energy transfer
Uses 3’ donor fluorophore and 5’ acceptor fluorophore
When brought in close proximity 5’ fluoresces (occurs only after hybridisation)
What is the cycle threshold in RTPCR?
When the PCR mixture fluorescence exceeds the threshold fluorescence
(Baseline produced with no template)
Use these values to creates melting curve to assess DNA quality
Plot change in fluorescence against temp
Give 3 applications of RTPCR
Quantification of infectious agents (HIV, HPV)
Analysis of gene expression at mRNA level
Genotyping
Why is traditional OCR only semi-quantitative?
Insensitivity of ethidium bromide
Name 4 methods for quantification of mRNA
Northern blotting
Ribonuclease protection assay
In situ hybridisation
Reverse transcription PCR
Why is RTPCR a good technique to use for quantification of mRNA?
Most sensitive of all techniques
Discriminates between closely related mRNAs
Technically simple
Only requires small amount of mRNA
How do you calculate the ratio of target transcript in experimental and control samples from a northern blot?
(Fold change in target transcript) / (fold change in reference transcript)
In RTPCR what criteria should ‘standards’ fulfil?
Same copy number in all cells
Expressed in all cells
Expression doesn’t change when conditions of cell growth are changed
Medium copy number = correct more accurate
Give some examples of commonly used standards in RTPCR
Glyceraldehyde-3-phosphate dehydrogenase mRNA Beta-actin mRNA MHCI mRNA Cyclophilin mRNA mRNAs for certain ribosomal proteins (RPLP0) 28S or 18S rRNA
What is the importance of controls in RTPCR?
Negative (no DNA) = checks reagents for contamination
No reverse transcriptase control = detects if signal from contaminating DNA
Positive = checks reagents/primers working (especially important for showing absence of gene)
On a graph of RTPCR, where should the threshold value be overlapping?
Should be in the linear part of the reaction
Should be high enough for showing reactions due to amplification and not noise
What would a standard curve graph show for series 10 fold dilutions in RTPCR?
CT values for dilutions against concentration
Linear graph
Excellent correlation coefficient, >0.99
What is the efficiency deltadeltaCT method?
Approximation method
Assumes:
1) minimal correction for standard gene
2) standard and target have similar efficiencies
2deltadeltaCT value assumes efficiencies at 100%
DeltaCT = target - ref Difference = deltaCT(control) - deltaCT(experimental)
Name 4 techniques used to detect protein
Immuno dot blots
Western blot analysis
Immuncytochemistry
Immunoprecipitation
What technique is used to detect protein DNA interactions?
Chromatin Immunoprecipitation
What technique is used to detect protein-protein interactions?
Co Immunoprecipitation
What’s the difference between monoclonal and polyclonal antibodies?
Poly = recognise different regions, have different affinities for protein mono = only one region (produced using clonal cell line)
What are 2 ways in which proteins can be labelled directly?
Give 2 pros and cons of this
Radioactive
Fluorescent (FITC green or rhodamine red)
Pros: convenient, simple
Cons: health risk, potential poor signal
What does indirectly antibody labelling entail?
Secondary antibody that recognises constant region of primary antibody
Secondary antibody linked to fluorophore or fluorescence releasing enzyme (e.g horseradish peroxidase)
Describe the process of chromatin Immunoprecipitation
Determines DNA sequences bound by specific proteins or modified histones
1) cross link DNA and proteins and isolate chromatin
2) sonicate or digest chromatin
3) immunoprecipitate, reverse cross link and purify DNA
4) PCR amplify target sequences
Name 4 ways in which you can analyse ChIP products and describe what they reveal
1) PCR - does the protein bind
2) qPCR - quantify level of protein binding
3) microarray - determine if protein binds to large no. Of different sequences and its relative affinity
4) ChIPSeq - determine all sequences bound by protein
Describe the 3 steps in immune cytochemistry
1) FIX cells with formaldehyde = crosslinks protein and preserves structures
2) PERMEABILISE cells using detergent
- e.g. Triton X-100 or methanol/acetone = puts holes in membrane
3) VISUALISE using secondary antibody with fluorescence of enzyme reaction
What is immunohistochemistry used for?
Describe the steps involved
Measures rate of protein synthesis/degradation
1) incubate cells with 35S Met for set time (pulse)
2) incubate cell with excess unlabelled Met (chase)
3) make protein extracts at several time points
4) immunoprecipitate with antibody
5) run on SDS-PAGE
6) quantify 35S label in protein
What is immunocyto- and immunohistochemistry used for?
Protein localisation and co-localisation
What proteins are used in Immunoprecipitation for recovery of poly and monoclonal antibodies?
How do they facilitate this process?
Protein A and G
Type G Streptococci
A from staphylococcus aureus
Both bind constant region of IgG
Both can be linked to agarose/magnetic beads/solid supports
What does in VITRO protein synthesis complement?
Co-Immunoprecipitation
1) take wheat germ or reticulocytes
2) lyse cells
3) spin to remove nuclei, mitochondria and plasmids
4) products can be used in GST pull-down experiments
What do GST pull down assays detect?
Detect protein-protein interactions in VITRO
1) express GST-fusion of protein in E. coli
2) purify via GST beads
3) in VITRO transcribe and translate prey protein
Name 3 essential features of vectors
Ability to :
1) replicate in host cell
2) be readily introduced into host cell
3) readily insert foreign DNA into vector
Why are plasmids not the vector of choice for cloning gene clusters or making genomic libraries?
Gene clusters from humans have larger genomes which exceed max. packaging limit
Name 3 features of bacterial host needed for efficient transformation
1) host deficient in natural restriction modification systems
e. g. E. coli B instead of K intrinsically have less protease activity (from deletion of hsdR gene)
2) required stable maintenance of transformed DNA = avoid rearrangements by using mutants in recombination genes (recA or recF)
3) disabled host for safety reason = auxotophic on metabolite only produced in lab to prevent growth if accidentally released
Give 4 features of natural plasmids
1-100kb
1-1000 copies per cell
Replicate independently of chromosome
Carry e.g. Antibiotic resistance
What are the essential features in a plasmid for use as a vector?
ORI (15-20 per cell)
Selectable marker
Suitable region for DNA insertion (restriction sites)
Suitable size
Give an example of a typical plasmid cloning vector
pUC19 2686 bp >500 copies per bacterial cell Possess lacZ gene Contains poly linker = multiple restriction sites
Name 2 ways for plasmid uptake into bacteria
Chemical transformation - CaCl2 and heat shock
Electroporation
What is the genome size for insertion into bacteria?
Why is this a problem for creating genomic libraries?
0 to 10kbp
Eukaryotic genes are much larger
Size limitation and poor transformation efficiency for making genomic library, construction tricky
Describe genome organisation in bacteriophage lambda
Genome 49kb
Linear double stranded DNA, present in phage particles
Single stranded complementary termini of 12nt called cohesive ends
What is the purpose of cohesive ends in the bacteriophage lambda genome?
Allows recircularisation which initiates replication
Site of joining is called a cos site, producing a circular concatemer
What are the 2 life cycle stages of bacteriophage lambda?
Lysis
DNA replication, phage product synthesis, phage release
Lysogeny
Integration of lambda into host genome
Briefly describe the Lytic pathway in lambda phage
What kind of replication
Rolling circle mechanism
Replication starts at ORI
Produces long catenane, cut at cos sites to produce whole genome fragments
Head and tail assembled separately
Describe the process of concatenation packaging into phage prehead
Concatenation cleaved at cos site packaging into pre head
(Phage encoded endonuclease)
Fixed length is always packaged (packaging constraint)
Name 4 different methods of distinguishing an ‘empty’ vector (no insert) from a recombinant molecule (insert present)
1) differential antibiotic resistance (plasmids such as pBR322)
2) lacZ complementation (plasmids and phage)
3) Spi selection (specific for lambda replacement vectors)
4) ci selection (insertion and replacement vector)
Describe the lacZ gene
Encodes beta-galactosidase
N terminal 146 AA portion inserted into cloning vector (promoter and alpha-peptide)
This region is inactive
Vector contains regulatory sequences to bind repressor = repressed
How and what visual changes occur during lacZ complementation to identify plasmid uptake into E. coli cells
Plasmid engineered to produce n terminus alpha peptide
Vector engineered to produce c terminus beta peptide
Add IPTG (lacZ inducer) and X-gal (chromogenic substrate)
Active beta gal by alpha complementation produced
Observe blue colonies
Poly linker cloning site of parent vector is situated within the middle of the lacZa gene. Does this affect its synthesis
What happens if foreign DNA is inserted into this site?
No
Disrupts alpha peptide production
Therefore no beta gal formed
White colonies or plaques
Chemical transformation of phage DNA into E. coli is inefficient
How is it inserted?
Using normal phage infectivity
Describe the packaging constraints of phage lamba
Cannot exceed 50kb
Only DNA molecules of 78-105% of wild type (48kb) can be packaged
gt10 = commonly used vector
contains unique EcoRI site within cl gene encoding Lytic gene repressor = inactivation means no lysogen formation = plaques are clear not turbid
How would you make phage lamba accept larger inserts?
Remove DNA for non-essential lytic growth
20kb of integration and excision
gt10 vector contains 43.8kb genome, therefore takes up 9kb
CHARON series vectors can take up 22kb
What is the lower packaging limit in phage?
Limit otherwise cannot be propagated
Cannot delete more than 25% wild type
What does the Klenow fragments of E.coli DNA polymerase lack compared to the wild type?
5’ to 3’ exonuclease activity
What does formamide do to the Tm of nucleic acid hybrids?
Lowers it
Give four facts regarding illumina sequencing
Based on reversible cycle-terminators technology
Is a massively parallel sequencing technology
Clinical amplification of DNA fragments occurs on a surface
Sequencing of bacterial chromosomes to detect chromosomal mutations is now routine
Cells from the liver express a different set of genes compared to cells from the brain. What techniques can be used to measure these differences?
Microarray analysis
Western blot
Northern blot
2D gel electrophoresis
What kind of vectors are usually used for cloning cDNA?
Lambda replacement vectors
What two purposes does production of a fusion protein have in bacteria
Improve stability of foreign protein
Assist purification of the foreign protein
does GFP require cofactors of substrates in order to produce fluorescence?
No
What does sanger sequencing require and how does it separate fragments?
Dideoxynucleotides
Radioactively or fluorescently labelled deoxynucleotides
Separates by size
Vectors containing specific promoters such as phage T6 or SP7 promoter can be used to do what?
Generate riboprobes
What does cassette mutagenesis involve?
Replacing a restriction fragment with a synthetic DNA region containing appropriate mutations to be studied
What does the Quikchange system for high efficiency mutagenesis use DpnI for?
Digest the original wild type template DNA
In gel shift analysis, what does the addition of an excess of unlabelled DNA sequence do?
Decrease the mobility of the radio labelled DNA-protein complex
DNaseI can be used for what?
Foot printing the protein binding sites of a promoter
Which is more stable, RNA/RNA or DNA/RNA hybrids
RNA/RNA
When using biotin as a non-radioactive label for probes, what reagents can be used to carry out detection of the probe
Avidin
Biotin-conjugated alkaline phosphatase
Describe creation of a replacement vector
To create a vector, central non essential region is substituted with stuffer fragment
Approx. same size as wild type
Stuffer contains restriction sites for cutting itself into small pieces to prevent its own reinsertion during cloning
Replaced during cloning, designed to carry larger DNA, up to 22kb
In VITRO packaging what happens?
Introduce recombinant concatemer into e. Coli by in VITRO packaging into phage particles
Will package any molecule so long as has cos sites separated by 37-50 kb
Steps to making a cosmic library
Propagate cosmic vector as a plasmid
Cleave with restriction enzymes, add foreign DNA and ligate to form concatemer
Why can a recombinant cosmic not initiate the Lytic cycle
Lacks gene required for lambda particle production
Instead produce colonies on ampicillin plates
Acts as plasmid in e.coli
Briefly describe bacteriophage M13
What is the advantage of these in terms of size of insert
Filamentous phage
Single stranded DNA, 6.4kb
No head particle, just gets longer
Problem with large inserts = prone to rearrangements
How does M13 phage infect e. Coli for delivery of insert DNA?
Usually used for phage display
Male specific phage
Host needs to contain f plasmid to produce f pilus
M13 binds to F pilus, internalised
Converted to dsDNA and purified like a plasmid
How do you clone using M13 to produce plaques?
Mix transformed e.coli with non-infected e. Coli
Add too agar
Infected cells release more phage = infect neighbours
Produce slow growing plaque of uninfected cells
M13 does not kill the host
With plaques of e. Coli and M13 containing an insert, what steps follow for isolation of this DNA?
Inoculate e. Coli culture with plaque of infected cells
Grow liquid culture
Pellet bacteria
Either:
1) isolate ds recombinant DNA using regular plasmid prep
2) isolate ss recombinant DNA from released phage in supernatant
Why would we need to use cloning by expression and/or functional assays?
When no DNA or AA sequence available but have clearly defined functional property/antibody to protein
Functional assay in e. Coli or mammalian cell
Make library in expression vector
Describe the process for gene cloning by expression in e.coli
Transfer plaques expressing fusion protein to nitrocellulose filter
Incubate filters with available antibody to desired protein
Antibody binds to plaque with protein
Secondary antibody could with dye-based detection
Western blot reveals location
Name three ways in which you could detect a protein expressed in e. Coli and identify which clones contain these
Antibody labelling
Isolation of clones by membrane hybridisation (autoradiography and radiolabelled probe)
Degenerate oligonucleotide hybridisation marking
How can we make use of degenerate oligonucleotides for hybridisation to identify a clone containing a protein
For a 6 amino acid protein partial sequence
1) Make oligonucleotide mixture
2) Comprises 8 different oligos containing all possible DNA sequences that can encode all six amino acids
3) one of the oligos will be fully complementary to gene
Or could make guessmer based on known preference of codon usage in organism
How could you isolate clones containing your protein using membrane hybridisation?
Nitrocellulose/nylon filter make copy of plate
Treat filter to denature DNA (NaOH, bake)
Hybridise with radio labelled probe (65)
Wash off unhybridised probe (65)
Perform autoradiography
Signal appears over colony/plaque complementary to probe
Name 3 requirements of genomic DNA a when preparing for insert
What size fragments are usually inserted into replacement and Cosmid vectors
Pure, high MW, free of nicks
Replacement = 20-25kb Cosmid = 40kb
Avoid complete digestion otherwise fragments are too small
What kind of enzymes can you use to partially digest genomic DNA
EcoRI (GAATTC) = once per 4096bp
BamHI (GGATCC) = once per 4096bp
Sau3A (*GATC) = once per 256bp
How would we use the DNA of a whole genome in separate phage to reconstruct the genome?
Align the sequences and look for the same sequences at the start and end and match up
Briefly describe the sanger/dideoxy method of sequencing
Enzymatic approach
Extend a primer strand against a template strand
4 reactions, each terminated by different dideoxy analogue
ddNTP lacks 3’OH
Strand elongation is not possible is 3’OH is missing
What 3 activities does E. Coli DNA polymerase possess?
5’-3’ exonuclease (repair)
5’-3’ DNA synthesis
3’-5’ exonuclease (proof-reading)
What 2 assays can be used to test for lambda growth?
Liquid culture assay
— add phage stock to bacteria, culture
— lysogenisation goes from milky to clear beer
Plaque assay
— add aliquot of mixture to ‘top agar’ and pour onto agar
— turbid plaques
What can peptide mass fingerprinting facilitate the identification of?
Gene that encodes a protein that has been separated when using 2D SDS-PAGE
How would you digest a genomic library for insertion into a vector
Partially digest
Frequently cutting restriction enzymes
E.g. Sau3A
Purify fragments within desired size range (20-25kb) by gel electrophoresis or sucrose gradient centrifugation
Once packaged into phage, each one contains distinct piece of genomic DNA
For insertion of target DNA into a cloning vector, what should the ligation reaction mixture contain?
Suitable buffer
Plasmid and insert DNA
DNA ligase
ATP, energy for formation of phosphodiester bond between nt strands
What are some non-radioactive labels used in nucleic acid hybridisation?
Biotin
— fluorescently labelled streptavidin
— alkaline phosphatase linked streptavidin
Digoxigen
— using primary anti- and secondary antibody
— direct binding to anti-DIG antibody
(Both fluorescently labelled)
What are some of the uses of nucleic acid hybridisation?
Analysis of gene expression/complex restriction maps
Screening gene libraries
Identifying mutations/mRNs size and conc
Identifying restriction fragments carrying target gene
Give some examples of uses of in situ hybridisation
Detecting:
1) infectious agents
2) mRNA temporal/spatial gene expression
3) gene expression in diagnostics
4) genetic defects/chromosome mapping
Give an example of a well known proto-oncogene and its analysis by in situ hybridisation and treatment regime
HER2 (ERBB2) - encoding 185kDa transmembrane tyrosine kinase receptor
Promotes cell division/survival, inhibits apoptosis
Herceptin - humanised monoclonal antibody has positive outcome for breast cancer treatment
Give four uses or site directed mutagenesis
Creating desired sequence changes
Investigating role of DNA/RNA
Understanding protein structure/function relationships
Creating new proteins
What were some of the problems with early site directed mutagenesis approaches? How were they solved?
Poor ligation (phosphorylate primer, improve conditions) Strand displacement (T4/7 pol) Mismatch repair (mutL/S Mutant recovery often less than 0.1%
What is the general principle of high efficiency mutagenesis approaches?
Once template has been used to copy mutant strand it is of no further use and can only reduce mutant recovery
What is the purpose of an amber stop codon
Read as a stop codon or glutamine in different e. Coli stains
What is the limited size of synthetic oligonucleotides that can be made for site directed mutagenesis?
About 60nt
What is the purpose of fusion tags?
Easy identification and purification of protein
Improved stability/folding
Added N or C terminal of in-frame
What needs to be removed when adding first a C terminal fusion tag and secondly an N terminal fusion tag
C = stop codon to create fusion with tag N = start codon so that fusion tag is not missed
2 ways to detect tagged protein
Recombinant protein expression
Coomassie stained gel
Western blot detection of 6His tag
Why may we want to remove a fusion tag in recombinant protein expression? How can this be done?
Purposes of crystallising the protein
Or when protein is large (e.g. maltose binding protein)
At gene cloning stage, incorporate site for protease cleavage enzyme
After purification, protein passed through column again and enzyme added to remove fusion tag
How would you add a promoter in recombinant protein expression?
Usually provided by the vector
Can put in during PCR
Name 2 ways in which protein expression can be switched on during recombinant protein expression
Tac
pBad
Lac
Describe how the tac promoter works in recombinant protein expression
Lac repressor binds to operator preventing RNA polymerase binding
Induced upon addition of IPTG or autoinduction
Describe how pBAD promoter works in recombinant protein expression
Arabinose or ramnose utilisation operon respond to increasing concentrations of their corresponding compound in growth medium
Describe show the lac promoter is used in recombinant protein expression
IPTG induction
Autoinduction
IPTG binds to repressor molecule allowing e. Coli RNA pol to transcribe T7 RNA polymerase
IPTG has also bound to repressor on other operator
T7 RNA pol can transcribe gene of interest
How may we improve the translation of proteins when designing a gene for recombinant protein expression?
Insert a signal sequence after the ATG to cause secretion into periplasm
Periplasm is an oxidising environment = favours formation of disulphide bonds
What kind of promoter causes constitutive activation for expression of RNA transcription?
CMV promoter
Name four types of expression and how they are activated
Constitutive - permanently active promoter e.g. CMV
Inducible - hormone/drug/metabolite-dependent binding
Transient - short period when no selection for vector in expression cell
Stable - permanent expression by the host cell
How are eukaryotic cells grown by tissue culture?
Sterile flask (25-300cm2)
Contain necessary nutrients and bicarbonate buffer
Incubators - 37 degrees, O2/N2/CO2
Mostly cancer cells - immortalised/rapid growth/amenable to vector introduction
Name 4 ways to get foreign cDNA into a eukaryotic cell cultured in vitro
Lipofection (DNA/lipid complex
Electroporation
Spontaneous uptake using Ca2+ complex or polycationic carrier
Successful only for small proportion of cells
Use viruses to deliver DNA
Give examples of viral and mammalian promoters that can be used within mammalian expression systems
Viral - SC40, CMV, MMTV
Mammalian - actin, heat shock protein
Name the components of a typical mammalian expression system and the use of each component
PCMV & polyA - drive mRNA transcription
PS40, NeoR & polyA - selection in stable transformed cells
AmpR & ORI - drive replication and allow antibiotic selection in e.coli
How does the tet-on system function in mammalian expression systems
Engineered transcription activator protein binds to tetracycline response element only in the presence of antibiotic (tetracycline-like doxycycline)
Describe the tet-off system in mammalian expression systems
Transactivator is modified to normally bind to promoter allowing transcription
Addition of antibiotic prevents Transactivator binding to promoter = transcription terminates
What is the function of the 2 plasmids required for tet-on/off systems in mammalian expression systems?
1) contains cDNA for protein of interest downstream of promoter
2) drives expression of Transactivator and contains selectable marker
Briefly describe the natural life cycle of baculoviruses spodoptera
1) viral particles with polyhedron coat protein digested
2) alkaline gut pH dissolves particle, capsids released
3) capsids taken up by epithelial cells
4) replicate using host cell machinery
5) bud off from infected cell using gp64
In which 2 ways can a recombinant baculoviruses genome be created?
Double cross-over recombination
Transposons-mediated recombination
For organisation of baculoviruses vectors, what components are required for replication in e. Coli and independently integration with baculoviruses DNA to create backed DNA
E.coli
— lacZ, f1region, AmpR, ORI
Integration
— Tn7L&R, GenR, pPolh, MCS, SV40 polyA
What are the reported yields for insect cell expression in both baculovirus sf9 cells and drosophila S2 cells
Baculovirus
Soluble: intracellular = 10-25mg/l, secreted 18-40 mg/l
Histamine receptor/substances P receptor: total protein = 0.1-3.9 ug/mg
Drosophila
Soluble (dopamine hydroxylase): 16mg/l secreted
Glucagon receptor: 12ug/mg total protein
Dopamine receptor: 200ng/mg total protein
How would using viruses other than baculoviruses in mammalian expression systems be advantageous?
Lentivirus - good for transfecting ‘difficult’ cells e.g. Primary cells
Adenovirus - gives potentially higher expression levels
Special containment needed even though not component in autonomous replication
Briefly describe lentivirus structure
RNA E.g. VSV Core shell, p24 Surrounded by membrane envelope overlying matrix protein Contains gp41 and gp120
Briefly describe the lentivirus life cycle in 293T cells
1) host transfected with virus and packaging mix of helper plasmids for replication and assembly
2) viral RNA transcribed, exported from nucleus
Recognised by psi sequence and new virus cores assemble
3) virus core binds to virally encoded envelope glycoproteins at inner plasma membrane
What components would a lentiviral expression plasmid need to contains? (Lenti-X)
3' and 5' LTRs Psi viral packaging protein recognition RRE, WPRE - RNA stability/nucleus export cPPT - increased infectivity PCMV - target transcription promoter PPGK - constitutive expression promoter of antibiotic marker
Name and briefly describe the 4 steps required for turning siRNA into a drug
1) sequence selection
- – Bioinformatics and in VITRO screening for cytotoxicity on off-target cells
2) synthesis and modifications
- – Chemical stabilisation - methylation may change specificity
3) packaging and delivery
- – Eliminates off target effects/deals with immune response/cytotoxicity/clearance from organism/accumulation in unwanted tissues
4) targeting
- – recognition of specific cells
How can you study differential gene expression
Measuring mRNA abundance
Looking at protein abundances (IEF and 2D separation)
What animal model is frequently used for studying gene expression
Nematode worms
C. Elegans
80% of animals on earth and these
899 cells
Briefly describe 3 experiments of showing that genes are not lost during differentiation
Adult frog and unfertilised egg - egg nucleus destroyed and injected with adult nucleus, normal embryo and tadpole form
Carrot section - proliferating cell mass - separate in liquid medium - single cell - organised clone of diving cells - young embryo - carrot
Cows
Epithelial cells from oviduct fused with unfertilised egg with removed meiosis spindle and associated chromosomes
Reconstructed zygote and embryo eventually form young calf
Give 2 examples of changes in gene expression caused by gene amplification
Very rare
2 chorion genes in drosophila
Structural protein for eggs shells
Amplified 16 or 64 fold
Xenopus egg rRNA genes
Needed in massive amounts
Excised to form circular plasmids to allow 3000x amplification
Name 4 methods for direct detection of transcripts via hybridisation
Northern blot
In situ hybridisation
Quantitative PCR
Microarrays
What kind of reporters could you use to detection of a protein?
Fluorescence (GFP)
Enzymatic (beta-gal, beta-glucuronidase, luciferase)
Briefly describe GFP
Natural product of aequorea victoria 238 aa long 11 strand beta barrel Central chromosohore No required cofactors of substrates 1hour time lag
For enzymatic reporters, where would each one typically be used and what changes would be detected
Beta galactosidase
Not in plants as beta-gal is naturally high
Blue colour
Beta glucuronidase
Blue colour
Luciferase
Photinus pyralis or renilla
Needs luciferin substrate
What is cassette mutagenesis used for?
Inserting DNA fragments of up to 100bp between restriction sites
What is PCR mutagenesis used for?
Removes the limitation of length from cassette mutagenesis
What is sticky feet mutagenesis used for?
Insertion of fragments above 100bp to several kbp
Is there a limit of how big a deletion can be made in deletion mutagenesis?
No
What is saturated mutagenesis used for?
Random mutagenesis throughout the sequences
Generates all possible mutations are a specific site in a narrow gene region
Briefly describe affinity chromatography
Specific binding of protein due to tag Load NTA resin with nickel divalent ions Load lysate with tagged protein Wash off any unspecific binding Eluted with imidazole having competitive binding with his tag Pure protein is eluted
Briefly describe streptag streptactin purification
Similar principle as hexa histidine purification
Collect cells and lyse
Centrifuge and purify supernatant with your recombinant tagged protein
Binds to streptactin via streptag (mimics biotin)
Competitive binding with desthiobiotin
Pure protein eluted
Briefly describe the tet on and tet off systems
Tet-on
Engineered transcription Activator protein binds tetracycline responsive element upstream of DNA only in presence of antibiotic
Tet-off
Modified Transactivator Constitutively binds to promoter to allow transcription
Antibiotic added prevents this = no transcription of gene
Briefly describe the steps in baculovirus as a vector
Gene of interest subcloned into transfer vector
Second vector contains parts of baculovirus particle
Both recombine in specially engineered e.coli cells to form bacmid DNA by recombination between tn7 sites of transfer and compatible bacmid site
Introduce bacmid DNA into sf9 or sf21 insect larval cells
What steps would you take to ensure successful transfection of your insert into the vector
Recover your bacmid DNA
Check for insert using PCR not electrophoresis bc too big
Transfect insect cells
Recover recombinant virus produce from strong polyhedrin promoter
SDS-PAGE and immunoblotting for time-post infection
Briefly describe the process of RNA interference
Binds to DICER (endonuclease) = fragments approx. 21nt
Short fragments bind to argonaut protein
Guide strand selected, other degraded
Forms RISC complex (RNA induced silencing complex)
siRNA directs RISC to mRNA binding through complementarity
Argonaut catalyses cleavage of mRNA which is degraded
Briefly describe the process of miRNA
Processed in nucleus by drosha/pasha complex
Binds to DICER
Then argonaut
The ‘seed’ guides RISC
Imprecise pairing with mRNA allows targeting of hundreds of endogenous mRNAs
RNA degradation of translation inhibition
Briefly describe quantitative PCR
Reverse transcribe mRNA in cDNA
PCR with fluorescently labelled primers of dye that binds dsDNA
Fluorescence emitted when product made
Rate of product appearance relates to concentration of mRNA
What is the even skipped gene (Eve) required for and in what organism
Drosophila
Essential gene
Defines body segment formation
Discrete stripes, well characterised
What causes eve expression?
Long upstream region 7.3kb
Controls:
1) switching on of expression
2) defining where eve is expressed (stripe 1-7)
How could we define the eve gene sequence?
Substitute eve ORF for a reporter ORF and see where the gene is expressed
Make random deletions to the regulatory region
Introduce altered genes into eggs
Does expression still occur?
What 3 ways could deletions be made to a regulatory region to define it?
Restriction enzyme digestion
Nucleases digestion
Site directed mutagenesis
Explain briefly the use of restriction enzyme digestion for analysing use of a gene
Make first set of deletions
Look for expression
Make smaller deletions in critical region
Describe briefly the use of nuclease digestion in elucidating gene use
Restriction digest
Exonuclease III digest 3’ termini of exposed DNA duplex
Ligate back together
Make bigger deletions for time course
No need to multiple restriction sites
Don,t depend on position of enzyme site
Can rapidly introduce variety of deletions
What 4 methods could be used for studying protein?
2 for what proteins binds
2 for where they bind
Gel mobility. Shift assay
Affinity chromatography
DNA footprint analysis
Chromatin Immunoprecipitation
Briefly describe gel mobility shift assay
Make ‘hot’ DNA fragment
Fractionated cell proteins by centrifugation
Mix fractions with “hot’ DNA
Find which cell proteins bind to ‘hot’ regulatory region
Use antibody to elucidate protein
Relies of DNA movement slower when bound to protein
DNA moves in proportion in size on gel
Describe the use of DNA affinity chromatography
Make DNA fragment of regulatory region Attach to solid matrix and line on column Add protein, only protein binds to DNA Eluted with salt Defines which proteins bind
Briefly describe DNA footprint analysis
Purify your protein Make 'hot' regulatory region Bind DNA and protein Add DNase I Protein protects binding site Run hot DNA fragments on gel with control
Briefly describe chromatin Immunoprecipitation
Treat living cells with formaldehyde to crosslink
Extract genomic DNA protein complex
Cleave in 300 bp fragments
Some will contain bound protein - purify with antibody
Remove protein using nuclease
Determine nucleotide sequence of bound proteins
How could you arrange the expression of the eve reporter in all cells of the drosophila embryo
Introduce plasmid by injection into G0 embryo
Transposons integrates into nuclei at embryo pole
Pole nuclei become pole cells (form germline)
Foreign genes now in germline
Transgenic organisms are the offspring of these modified organisms
How do you get a reporter gene construct to express in other higher organisms?
And how would you screen for the gene?
Pronuclear micro injection of 200-300 gene copies to the fertilised egg before fusion of the nuclei
fertilised eggs divide forming early embryo
Embryo implanted into uterus of surrogate mother developing as normal
Time consuming - wait for 3 weeks for birth
Insertion random, success rate low
How would you get a reporter gene construct to express in other higher organisms using embryonic stem cell transfection?
Transgene introduced into stem cells, grown to colonies Combine with early embryo Forms hybrid embryo Hybrid incubated in female mouse Screen offspring No need for intita, microinjection Screening doesn't involve live animal
How could you use gene transfer in plants?
Agrobacterium tumefaciens Mediated by natural plasmid called Ti Use T-DNA repeat for transfer of gene Gene of interest flanked by T-DNA repeat Bacterium infects plants Grow into crown gall and plant Antibiotics used to select for transgene recipients
How do you get plasmids into cells for expression of your protein?
Transfection:
1) liposomes - cationic detergents, high efficiency
2) calcium phosphate - low efficiency, unknown mechanism
Electroporation
(placed in electric field, pore forms, DNA diffuses in, high efficiency)
Bombardment
(fires 0.3-1.6um gold particles, coated with DNA/RNA, used for gene delivery)
What is another way to study the function of gene X or protein X other than altering its expression or looking for its expression in different cells etc.?
Prevents it correct expression
Knock out or knock in mutants
Replace functional gene with non functional gene using homologous recombination
Then infected stem cells, grow colonies, combine with early embryo = hybrid! incubated in female mouse! screen offspring
