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