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241 Cards in this Set

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Running a PCR requires what components?
- Buffer
- Mg2+
- DNA template
- Primers (forward and reverse)
- dNTP mix (ATP, GTP, CTP, TTP)
- Taq polymerase (enzyme)
How does PCR contrast with DNA replication?
- Primers are DNA
- Only one enzyme is involved: taq polymerase
What is the cycle of temperatures (approximately) in PCR?
1. 94-95*C (denature DNA)
2. 50-65*C (anneal primers)
3. 72*C (extension/polymerization)
What happens during PCR?
1. Template DNA is denatured (94-95*C)
2. Primers are annealed (50-60*C)
3. Taq polymerase extends the primers (past the target region) (72*C)
4. Denature template and new pieces and REPEAT
How do you ensure that the primers will anneal to the template strands?
Make sure primers are in large excess
How many target copies are generated after 30 PCR cycles?
1,000,000,000 (one billion)
At the end of 30 cycles what is most of the DNA?
Target portion of the template strand
What is a primer?
A primer is a small nucleotide segment that is complementary to the replication template
What must a primer have for it to be elongated during polymerization?
A free 3' hydroxyl group to which the polymerase can add nucleotides
Where do you add restriction enzymes to on primers? Why?
The 5' end of primers so that the 3' hydroxyl group is still free for polymerization
What are the characteristics of a primer?
- 15-30 base pair DNA fragments
- Complimentary to the 3' end of both the top and bottom strands of the template (in other words the first 15-30 bases of the 5' ends of the forward and reverse strands)
The forward primer is complimentary to what?
3' end of the bottom strand
The reverse primer is complimentary to what?
3' end of the top strand
How can PCR primers be used for cloning?
If restriction sites are on the ends of the primers they can be digested and inserted into a vector plasmid.
What does a restriction enzyme do?
Cuts dsDNA (double-stranded DNA) at a specific sequence known as the restriction site
What do most restriction enzymes recognize?
A palindromic 6 base sequence
What do you need to make sure of when adding restriction sites?
Make sure the restriction enzyme does not cut anywhere else in your gene
What makes up a "clone"
(digested) Vector + (digested) PCR product with restriction sites
When designing primers what do you need to consider?
Make sure the start codons of the PCR product and the vector are in frame
What is the DNA start codon?
ATG
Why don't you need to worry about the 3' end of the gene?
The gene's stop codon will end translation
If you want to use the vector's stop codon what do you need to do?
Delete or change the gene's stop codon
What must be the approximate content of the bases in a primer?
~50% GC
What should you avoid when designing PCR primers?
Hairpins and dimers
What should the Tm values be for the primers?
Very similar; 50-60*C
What do PCR and replication both require?
DNA template, primers, nucleotides
How do the PCR primers and replication primers differ?
PCR primers are made of DNA; replication primers are made of RNA
How are template strands denatured in PCR vs replication?
PCR: heat
Replication: helicase
How many enzymes does PCR require?
One - Taq polymerase
What can PCR be used for?
- Amplify a specific sequence
- Introduce mutations
- Forensic analysis
- Prenatal diagnosis
How can mutations be introduced via PCR?
- Design primers with desired mutation
- Use error-prone polymerase to generate random mutations
Why is Mg2+ required for PCR?
It acts as a cofactor for Taq Polymerase
Why is Taq buffer required for PCR?
Sets up a conducive environment for the Taq polymerase to work
What is a master mix?
Contains all of the components that do not vary between reactions
Why is using a master mix a good idea?
- Reduces pipette error
- Increases reproducibility
- Saves time
How are Tm values calculated?
- G and C bases = 4*C
- A and T bases = 2*C
What reaction does carbonic anhydrase catalyze?
CO2 + H2O <--> HCO3- + H+
What kind of enzyme is human carbonic anhydrase?
alpha Zn-metalloenzyme
What does "carbonic anhydrase is ubiquitous to all cell types" mean?
It is found in all cell types because CO2 is the terminal product of oxidative metabolism and there needs to be a way to remove it from all cells
What is the importance of CAII?
- Regulation of respiration and gas exchange
- Regulation of acid-base equilibria
- Transport of ions and fluids
- Development of bone and mineralization
What reactions related to carbonic anhydrase go on in a cell?
- Respiration generates 6 molecules of CO2 (C6H12O6 + 6 O2 --> 6 CO2 + 6 H2O)
- CO2 is converted to bicarbonate (6 CO2 + 6 H2O --> 6 HCO3- + 6 H+)
What reactions related to carbonic anhydrase go on in the lungs?
Bicarbonate is converted back to CO2 (6 HCO3- + 6 H+ --> 6 CO2 + 6 H2O)
Why is HCAII a model system?
- Stable
- Soluble
- Monomeric
- Relatively low molecular weight (30 kD)
- Many structures solved
- Biophysically characterized
What are the three possible roles for Zn in the function of HCAII? Which are relevant to HCAII?
- Metalloenzymes often have multiple stable oxidation states and can participate in re-dox reactions (NO)
- Metalloenzymes can bind to 4+ ligands and act as multi dentate cross-linking agents, stabilizing a fold (NO)
- Metalloenzymes are positively charged and can act as Lewis acids (YES)
Why is this particular function of metalloenzymes NOT relevant to HCAII: they often have multiple stable oxidation states and can participate in re-dox reactions?
- Zn has a stable +2 redox state
- CO2 --> H2CO3 is not a redox reaction (it's an addition/hydrolysis)
Why is this particular function of metalloenzymes NOT relevant to HCAII: they can bind to 4+ ligands and act as multi dentate cross-linking agents, stabilizing a fold?
- While Zn is structurally important for many folds, HCAII is stable in its app form
- Apoenzyme means without Zn
Why is this particular function of metalloenzymes relevant to HCAII: they are positively charged and can act as Lewis acids?
Zn stabilizes the OH- ion that performs the nucleophilic attack on the CO2 substrate
How is Zn2+ situated in the HCAII enzyme?
It is coordinated by three neutral histidines (H94, H96, and H119)
What is the function of the Zn2+ situated in the HCAII enzyme?
It has a high affinity for binding a negative hydroxyl ion
After Zn2+ binds an OH- ion, what is it activated to do?
Zn bound OH- is activated to perform a nucleophilic attack on the CO2 (making it Zn bound HCO3-)
What kind of mechanism occurs for the HCAII enzyme?
Ping-pong mechanism (2 steps)
What occurs during the first step of the ping-pong mechanism?
- The Zn bound hydroxyl group is converted to Zn bound bicarbonate
CO2 + E-Zn-OH+ --> E-Zn-HCO3+
- It is then displaced by an H2O molecule
E-Zn-HCO3+ + H2O --> E-Zn-H2O(2+) + HCO3-
What occurs during the second step of the ping-pong mechanism?
- A proton is passed from the Zn bound water to His-64
E-Zn-H2O(2+) + His64 --> E-Zn-OH+ + His64-H+
- Next the proton is taken away from His64-H+ by a base from the environment
His64-H+ + B- --> His64 + BH
What is the rate-limiting step of the ping-pong mechanism?
The removal of the proton by the environment/solvent (B-)
How effective is the HCAII enzyme?
It is extremely efficient with an extremely high turn-over rate (kcat ~ 10^6 per sec)
What is the catalytic rate constant (kcat) for HCAII?
kcat ~ 10^6 per sec
What does the efficiency of the HCAII enzyme approach?
The limit of diffusional control
(kcat/Km ~ 10^8 1/M*sec)
What property of the HCAII enzyme is useful for activity assays?
- It has esterase activity
- p-Nitrophenol derivative becomes colored (absorbs in the visible range) once hydrolyzed
Specifically how can the esterase activity of HCAII be used for activity assays?
- para-nitrophenal acetate (PNPA) can be hydrolyzed to para-nitrophenol
- para-nitrophenol can be deprotonated and the formation of that product is measured due to its ability to absorb in the visible range
What is the makeup of the active site of HCAII?
~1/2 hydrophobic, ~1/2 hydrophilic
What can be found on the protein data bank?
Macromolecular structure data, including coordinates and electron density maps
What does resolution refer to?
The minimum distance between structural features that can be distinguished in an electron density map
What are low, medium, high, and atomic values for resolution?
- Low = 3 Ȧ
- Medium = 2.7 - 2 Ȧ
- High = 1.5 Ȧ
- Atomic = <1 Ȧ
What does "R-work" refer to?
A measure of how well the model matches the data (%)
What does "R-free" refer to?
An independent statistic that checks for over interpretation of the data
Electrophoresis is what kind of technique?
Separation
How is electrophoresis useful for analysis?
- Helps determine what is in an unknown sample
- Helps determine how much of a substance you have
- Helps determine how pure a sample is
What key physical properties can be exploited for separation?
- Size
- Shape
- Charge
What is the purpose of the gel in electrophoresis?
It acts as a molecular sieve
What provides the driving force for a separation by electrophoresis?
Electric field (E)
What do charged molecules experience in an electric field (during electrophoresis)? What is the relevant equation?
Force (F)

F=qE
What equation relates the electric field, the force, and the charge?
F=qE

F= force, q = charge, E = electric field
If there was no friction what would happen during electrophoresis?
The molecules would keep accelerating and never reach a steady state velocity
What provides the necessary friction for electrophoresis?
The gel (molecular sieve)
What equation can be used to determine the velocity?
v = qE/f

v=velocity,q = charge, E = electric field, f = frictional coefficient
Would a sphere or an oblong shape of the same volume move faster in an electric field? Why?
The sphere would move faster because it has a smaller frictional coefficient
What characteristics make proteins much more complicated than simple spheres and oblong shapes?
- Complex shapes
- Flexibility
- Unevenly distributed charges
- Counter ions are dragged in direction opposite protein
In gel electrophoresis, how are proteins separated?
By molecular weight
Why is separating proteins with gel electrophoresis by molecular weight problematic?
There is no common mass:charge ratio among proteins
How can the problem of not having a common mass:charge ratio for proteins be overcome?
Use a reagent that will introduce a constant mass:charge ratio (sodium dodecyl sulfate, SDS)
What is the significance of SDS?
- Sodium dodecyl sulfate - a detergent used to introduce a mass:charge ratio for proteins being separated via gel electrophoresis
- Also denatures the protein so it loses its folded shape
What is the structure of SDS (sodium dodecyl sulfate)?
- Polar head group (negative, with Na+ counter ion)
- Hydrophobic tail group (12 carbons)
What is the ratio that SDS binds proteins?
1.4g SDS / g protein
(~1 SDS molecule / 2 AA's)
When SDS is added to a protein what are the implications?
- The protein gets decorated by - charges according to mass; these charges are >>> than charges on native protein
- Protein is denatured so it loses its folded shape
When SDS is used for polyacrylamide gel electrophoresis it is also commonly called what?
Denaturing gel electrophoresis (because it denatures the folded shape of the protein)
Why is DNA separation by electrophoresis so much less complicated than for proteins?
- DNA has a constant shape (double helix)
- There is a constant charge/size ratio (-1 for every phosphate; -2 for every base-pair)
How exact is DNA separation?
Very good - can separate based on 1 base pair difference!
How can the precision of DNA separation be utilized?
DNA sequencing
Why is RNA a bit more difficult to separate with electrophoresis than DNA?
- It is single-stranded
- It often forms secondary structures which require denaturing
How can RNA be denatured for electrophoresis?
65*C and formaldehyde
What are the two gels for electrophoresis?
- Agarose
- Polyacrylamide
When is an agarose gel used for electrophoresis?
For high molecular weight macromolecules (e.g. nucleic acids)
When is a polyacrylamide gel used for electrophoresis?
For low molecular weight macromolecules (e.g. protein)
What are the characteristics of agarose?
- Linear, non-crosslinked polymer
- Used for high molecular weight macromolecules (e.g. nucleic acids)
Why does cooled agarose need to be melted at a higher temperature than originally?
After it is cooled to "set" the polymer chains become intertwined
At what weight/volume ratio is agarose typically used at?
0.7-3%
How is polyacrylamide polymerized?
Via a radical reaction
At what weight/volume ratio is acrylamide typically used at? bisacrylamide?
Acrylamide ~8-20%
Bisacrylamide ~3%
How far does xylene cyanol move in agarose gel electrophoresis?
The distance that 4000bp DNA fragments move
How far does bromphenol blue move in agarose gel electrophoresis?
The distance that 750-500bp DNA fragments move
What must be the relationship between the ionic strength of the gel and the buffer? Why?
They must be of equal ionic strength in order for proper separation of DNA
What was the running buffer and solution used for the gel in the lab we made agarose gels?
TAE = Tris-acetate-EDTA
Should the wells be at the cathode or anode?
Cathode
In which direction does the DNA run?
Runs to red (towards the anode)
Why is EtBr added to the agarose gel?
It absorbs UV light at 302nm and fluoresces at 590nm (this allows us to visualize the DNA in the gel)
What are the three buffers used to purify DNA?
- PB - binding buffer
- PE - washing buffer
- EB - elution buffer (contains the purified DNA)
For determining DNA concentration, what ratio is important?
260/280 ratio; a value of 2 would mean 100% pure DNA
How does EtBr interact with the DNA?
It fits between the bases - this causes the DNA to fluoresce at 590nm
What are the three methods to visualize protein in gels?
- Coomassie Brilliant Blue (least sensitive)
- Silver Staining
- Western Blotting (most sensitive)
How does Coomassie brilliant blue interact with proteins in gels? What is the detection limit?
- Associates with positively charged and aromatic amino acids
- Detection limit: ~25-50ng/band
How does Silver Staining interact with proteins in gels? What is the detection limit?
- Ag+ ions associate with various protein side chains
- Detection limit: ~0.5-1ng/band
How does Western Blotting interact with proteins in gels? What is the detection limit?
- Uses antibodies to generate, amplify signal
- Very specific and sensitive
- Detection limit: often picograms or less
Why do plasmids make great vectors?
- Circular DNA chains
- Occur naturally in bacteria
- Confer a selective advantage
- Can be "horizontally transmitted" from one bacterial cell to another
- Cloning vectors lack genes for cell-to-cell transmission
What are three salient features of a cloning vector?
1. Origin or replication (ori)
2. Selection marker (commonly an antibiotic resistance gene)
3. A region to clone (and ultimately express) your gene of interest; a multiple cloning site, MCS)
What is a MCS?
Multiple Cloning Site
- Contains DNA sequences that can be digested with restriction enzymes
What factors do you consider when choosing your cloning vector?
- Size: smaller vectors are usually easier to work with
- What selection markers do you want?
- What features do you want in the MCS (restriction enzyme sites, protein tags)
What does electroporation of a cell do?
A large electric field makes the bacterial cell wall permeable so circular DNA can leak in
What does it mean for a bacterial cell to be "competent"?
It is capable of accepting plasmids (electroporated)
Is transformation (the uptake of a plasmid) an efficient process?
No - it is very inefficient; only about 1 in every 10^5 cells may take up the DNA
What is a way to check for which cells have successfully taken up the plasmic you want?
Utilize the antibiotic resistance gene
Do all of the antibiotic resistant colonies on your plate contain the desired clone product?
No; they could also be re-ligated vector or the vector ligated to a different insert
What are the four methods of controlling / screening for colonies that do not contain the desired insert?
- Blue-White Screening
- Vector Re-ligation Control
- Restriction Digest Screen
- PCR Screen
What does the blue-white screening technique distinguish between?
Re-ligated plasmid and plasmid + insert
What do the blue colonies represent from the blue-white screening technique? Why are they blue?
- These colonies are re-ligated plasmids (no insert)
- The vector has the intact LacZ gene so beta-galactidose is produced
What are the limitations of the blue-white screen?
It will not tell you if your insert is in the gene; just whether the vector contains ANY insert
What do the white colonies represent from the blue-white screening technique? Why are they white?
- The white colonies represent plasmids with inserts in them
- The LacZ gene is interrupted so no beta-galactidose is produced
What is necessary for the blue-white screening technique?
- The multiple cloning site (MCS) must be within the LacZ gene
- The LacZ gene encodes for beta-galactidose which breaks down a chemical in the plates to produce a blue color
- If an insert is in the plasmid it will disrupt the LacZ gene and prevent the blue color from forming
If you are cloning into a vector that does not have the LacZ gene, what could you do to estimate how much of your vector re-ligates?
- Run a reaction without any insert so that there is only digested plasmid
- The vector will re-ligate to itself
- When this is plated you will see how many colonies form
- This will be like a baseline amount of colonies to compare the plasmid + insert plate to
What must occur to do a restriction digest screen?
Plasmid DNA must be purified from E. coli
How does the restriction digest screen work?
1. Pick colonies
2. Grow cultures
3. Isolate DNA (must be purified from E.coli)
4. Two samples - 1. desired clone, 2. empty vector
5. Restriction digest (vector will have one cut site, desired clone will have two cut sites because one is in insert)
6. Put on agarose gel (1. will have two shorter pieces, 2. will have one longer piece)
Does the PCR Screen require DNA purification from E.coli?
No (restriction digest screen does)
How does the PCR screen work?
1. Pick colonies
2. Two samples (1. desired clone, 2. empty vector)
3. PCR reaction with primers for insert
4. Agarose gel of products (only the desired clone will create copies of the insert leading to a single heavy band, the empty vector will have no bands)
What are the steps of Site-Directed Mutagenesis?
1. Design primers
2. PCR
3. Digest parental plasmid
4. Transformation, growth
For site-directed mutagenesis, how do the primers differ?
The primers are complementary to the strand except for the place you want to introduce a mutation.
What happens during the PCR step of site-directed mutagenesis?
- Primers with desired mutation are added with a high-fidelity polymerase
- The resulting PCR products have incorporated the primers so they contain the desired mutation
- These products are nicked
During step three (digest parental plasmid) of site-directed mutagenesis, what happens?
- DpnI is a restriction enzyme that only cuts methylated DNA
- It digests the template DNA and leaves the mutated products
What is unique about the restriction enzyme DpnI? How can this be used for site-directed mutagenesis?
- DpnI only cuts methylated DNA
- Template DNA is methylated, but the PCR products are not
- DpnI digests the template strands
What happens during the transformation and growth stage of site-directed mutagenesis?
- E. coli is transformed with the double-stranded (ds) PCR product
- The E. coli repair the nicked DNA and replicate the mutant
What ingredients are necessary for a ligation?
- Ligase (T4 DNA Ligase)
- Digested insert
- Digested vector
- ATP
- Mg2+
- Reducing buffer
What does T4 DNA Ligase do?
Catalyzes the phosphodiester bond between a 5' phosphate and a 3' OH
How do you activate ligase?
Incubate at room temperature for 30 minutes
How do you inactivate ligase?
Heat reaction at 65*C for 10 minutes
What does transformation do?
It's a process to introduce foreign DNA into a cell
What are the two methods of transformation? What are the basic pros and cons of each?
- Electroporation - more efficient but very slow
- Chemical Transformation - lower efficiency but faster
How is chemical transformation done?
- Bacterial cells are bathed in CaCl2 solution
- Ca2+ forms a barrier between repulsive - phospholipid heads of cell membrane and - DNA backbone
- Cl- can permeate cells through membrane pores; the difference in [Cl-] causes a water influx that helps with DNA uptake
Why during the chemical transformation process were the cells heated for 50 seconds?
To make the membrane more permeable
Why was 500 μL of Ligase buffer added and the sample incubated for 45 minutes at 37*C?
It helped the cells to recover from the trauma of being transformed
What grows on the kanamycin plates?
- E. coli with vector transformed
- If the vector is incomplete (w/o a plasmid) it will not grow
Besides vector + insert (goal clone), what other forms of plasmid can grow on the LB + Kanamycin plates?
- Recombinant vector
- Undigested vector
- Only one cut - can religate
Why did we transform our clones into DHBα cells first (in lab four)?
This is a cell line with a high copy number; i.e., high level DNA introduced
What does recombinant protein expression take advantage of?
Target cells' native machinery to make a protein of interest
What is the process of recombinant protein expression?
1. Introduce a gene (gene should be replicable and under the control of a strong promoter)
2. Turn on the promoter (cell will transcribe the mRNA)
3. mRNA is translated into protein
When introducing a gene for recombinant protein expression, what are two necessary characteristics of the gene?
- Should be replicable (ori)
- Gene should be under control of a strong promoter
When you turn on a promoter, what happens?
The cell transcribes mRNA
What promoter does our pET-28b vector contain?
T7 promoter
When we transform our pET-28b vector into E.coli what is the problem with transcription?
E.coli RNA polymerase does not recognize the T7 promoter of the pET-28b vector because the T7 RNA polymerase is not native to E.coli
How do we get the E.coli we are transforming to express our clone?
- We must use an E.coli strain that has been modified to express the T7 RNA polymerase (the polymerase that will transcribe our clone)
- This occurs with IPTG induction
How do you get E.coli to express the T7 RNA polymerase?
IPTG induction
After you add IPTG to E.coli to induce expression of T7 RNA polymerase, what do you do?
A time course via a gel (SDS-PAGE) to determine the time of maximum protein expression
What are the advantages of a bacterial expression system (e.g., transforming E.coli with a clone)?
- Relatively fast (1-2 weeks including cloning)
- Inexpensive
- High expression levels
- System is amenable to large scale preparations
What are the disadvantages of a bacterial expression system (e.g., transforming E.coli with a clone)?
- Protein may be insoluble
- Protein may be mis-folded
- Minimal post-translational modifications (e.g., glycosylation, phosphorylation, and disulfide bonds)
- Expression of functional mammalian proteins may be difficult
For eukaryotic cell expression systems, how do you get your gene into a eukaryotic cell?
Use viruses
What is the process of inserting your gene into a eukaryotic cell for expression?
1. Isolate the viral genome
2. Insert your gene under the control of a strong viral promoter
3. Put recombinant viral genome into cells
4. Cells will produce a lot of recombinant virus
5. Infect new plates of cells with the recombinant virus
6. During the viral life cycle your protein will be produced
Why during eukaryotic cell expression systems do we not purify the protein directly from the original cells we added the viral genome to?
These cells may be exhausted/weak so you want to use new healthy cells to express the protein
For insect cell expression systems, what is used to express the protein of interest?
Baculovirus in insect cells
The gene of interest during an insect cell expression must be under what control?
The control of a baculovirus-specific strong promoter
Once the gene of interest is recombined with the baculovirus, what do you do?
Infect insect cells - during the course of its natural life cycle the virus will produce your protein of interest
What does the baculovirus promoter regulate?
Protein induction
What are the advantages of a insect cell expression system (using baculovirus)?
- Reasonably high protein expression levels
- Supports many post-translational modifications (PTMs)
- Proteins are likely to fold properly
What are the advantages of a insect cell expression system (using baculovirus)?
- Takes a long time (at least 2-4 weeks to get a recombinant virus)
- Expensive (tissue culture, transfection reagents, special cloning vectors)
- Complicated cloning
- Must keep passaging cell lines
How do eukaryotic cells differ from bacterial cells?
- More expensive
- More difficult to maintain
When using a tissue culture expression system, what must you do?
- Cells are kept in culture
- Cells must be passaged every few days so they don't overgrow the plate and die
How do you passage cells, as is required for tissue culture expression systems?
Cells are removed from the original plate, counted and plated at the correct density on fresh plates
When you are ready to express protein from a tissue culture, what do you do?
Grow up many cells; it can take a week or so to get enough cells for expression
What are the two ways to express proteins in mammalian cells?
1. Virally
2. Transfection
How are proteins expressed virally in mammalian cells?
- Cells can be infected with a recombinant Adenovirus containing your gene of interest
- Gene of interest is cloned down stream of a strong, constitutive, viral promoter
- Virus uses the cellular machinery to produce the protein
Why are adenoviruses used for mammalian cell protein expression systems?
Adenoviruses can infect many different mammalian cells (skin, muscle, brain, bone, nerve, liver cells, as well as embryonic stem cells)
How can mammalian cells incorporate the gene of interest virally?
Using an adenovirus containing your gene of interest
What does the viral promoter regulate in a mammalian cell expression system?
Regulates protein expression
What are the advantages of the viral method to mammalian cell protein expression?
- High levels of PTMs (post-translational modifications)
- Often able to obtain functional mammalian proteins
- Virus can be used in different cell types
What are the disadvantages of the viral method to mammalian cell protein expression?
- Lower level of protein expression than E.coli or baculovirus systems
- Takes a long time (2-3 weeks) to get recombinant virus
- Expensive (tissue culture, transfection reagents, special vector system)
- Complicated cloning and virus generation system
- Must keep passaging cell lines
What happens during transfection mammalian cell expression systems?
- Nucleic acid polymer can be introduced directly into the cells (non-virally)
- The cell regulates protein expression
How do viral and transfection methods differ in regulation?
- Viral - viral promoter regulates protein expression
- Transfection - cell regulates protein expression
What are the advantages of transfection as a mammalian cell protein expression system?
- High levels of post-translational modifications (PTMs)
- Often able to obtain functional mammalian proteins
- Fast - once nucleic acid is introduced cells make protein within a couple of days
What are the disadvantages of transfection as a mammalian cell protein expression system?
- Low level of protein expression
- Cells must be transfected each time you need protein
- Expensive (tissue culture, transfection reagents)
- Generally not used to over express and purify large amounts of protein
What are three cell-free expression systems?
- Rabbit Reticulocyte Lysate
- E. coli Extract
- Wheat Germ Extract
Describe the Rabbit Reticulocyte Lysate expression system.
- Cell-free
- Lysed immature RBC that have been treated with micrococcal nuclease to degrade endogenous mRNA
- You add the RNA template
Describe the E. coli extract expression system.
- Cell-free
- E. coli extract that contains T7 polymerase
- You supply circular DNA with your gene cloned downstream of T7 promoter
Describe the Wheat Germ Extract expression system.
- Cell-free
- Micrococcal nuclease treated extract from wheat germ containing the components necessary for translation
- You add the RNA template
What are the advantages of cell-free expression systems?
- Very fast (hours vs days or weeks)
- Easy labeling (e.g. isotopes)
- Little endogenous protein produced
- Flexible template DNA or RNA
- Can express toxic proteins
What are the disadvantages of cell-free expression systems?
- Very low level of expression - nano grams
- Very expensive
- Little post-translational modifications (PTMs) - you can add reagents to get some though
What is a mini-prep used for?
Method to separate plasmid DNA from genomic DNA
How can you separate plasmid DNA from genomic DNA?
Using a mini-prep (ex: QIAGEN)
What kind of column is used for separating plasmid DNA from genomic DNA (in a mini-prep)? Why?
Column with silica-gel membrane - selectively binds DNA and separates nucleic acids with specific size parameters
What are the 5 buffers/solutions that are used during the mini-prep to separate plasmid DNA from genomic DNA?
1. P1 - Resuspension
2. P2 - Alkaline Lysis
3. N3 - Neutralization
4. PE - Ethanol Wash
5. EB - Elution Buffer
What does the P1 solution (first step) do in the mini-prep to separate plasmid DNA from genomic DNA?
- Resuspension of pellet containing RNA, genomic DNA, plasmic, and proteins
- Contains RNase A to remove RNA
What does the P2 solution (second step) do in the mini-prep to separate plasmid DNA from genomic DNA?
- Alkaline lysis
- SDS lyses cell membranes
- NaOH denatures DNA and proteins (time sensitive)
What does the N3 solution (third step) do in the mini-prep to separate plasmid DNA from genomic DNA?
- Neutralization of NaOH with acidic potassium acetate
- High salt conc. precipitates genomic DNA and proteins
- Must remove supernatant and put on column
- High salt conc. allows renatured plasmid DNA to bind column resin
What does the PE solution (fourth step) do in the mini-prep to separate plasmid DNA from genomic DNA?
- Wash
- Ethanol removes salts
- Plasmid remains attached to column
What does the EB solution (fifth/last step) do in the mini-prep to separate plasmid DNA from genomic DNA?
- Elution buffer
- Tris pH 8.5
- High pH and low salt makes plasmid wash off of column
What were the two methods we could have used to screen our clones?
- PCR screen
- Restriction digest screen
Which screen was used to screen our clones?
Restriction digest screen
What happens during the restriction digest screen that allows us to differentiate between our desired clones and empty vectors?
- The desire clone will be digested by Pst1
- It will not travel as far as the empty vector because it is cut
- The empty vector will not be digested by Pst1 - it will remain supercoiled
- The empty vector will travel further in the gel because of this
What is the Rosetta DE3 E.coli strain designed for?
- High transformation efficiency
- Over-expression of proteins
- Enhanced tRNA pool for codons found in eukaryotic protein sequences
Before recombinant technology, where were proteins obtained from?
Naturally abundant sources, such as hemoglobin, myoglobin, white egg lysozyme
What kind of proteins were hard to study before recombinant technology?
- Proteins with low abundances (needed an abundant source)
- Human proteins
- Proteins that are hard to purify
- Proteins that are unstable when removed from tissue
What has recombinant technology allowed us to do?
- Ability to chemically synthesize oligonucleotides
- Ability to amplify genetic material (PCR)
- Ability to sequence DNA
- Ability to cut DNA (with restriction enzymes) and past it specifically into a vector
- Ability to introduce genetic material into organisms
Related to proteins, what can recombinant technology do?
- Over-express proteins in heterologous organisms
- Easily mutate
- Express only selected domains
- Create fusion proteins
- Add tags for detection (epitope tags) or purification (His tag)
- Produce completely synthetic sequences (de novo design)
Why is protein over-expression not always a good thing?
Massive amounts of foreign protein can be really toxic to the host cell, complicating cloning and actual expression
The system we discussed is a controlled system based on what two key elements?
- A viral (phage T7) RNA polymerase that produces extremely high amounts of mRNA
- The lac-operon, used as a control system to unleash this powerful expression potential, only when it is required
What are the characteristics of the bacteriophage T7 RNA polymerase?
- 99kD, single subunit RNA pol
- Highly selective for its own promoter (T7 Promoter needs the T7 RNA pol and vice versa)
- Promoter does not occur naturally in E.coli
- Highly efficient (5x faster than E.coli RNA Pol)
How efficient is the bacteriophage T7 RNA polymerase?
- Highly efficient (5x faster than E.coli RNA Pol)
- It can accumulate extremely high levels of mRNA saturating the translational machinery
- Target protein can accumulate to >50% of total cell protein in 3 hrs
Why must the T7 RNA polymerase be controlled?
It can produce massive amounts of protein but this can kill the host cell; need a control mechanism so that it turns on only when the culture has reached a high density
How is the T7 RNA polymerase controlled when inserted in E.coli?
Lac operon
What is a regulon?
Group of genes that are regulated by the same protein
What is an operon?
Regulon and its regulatory elements (promoter, operator, and activator site)
What is the structure of the Lac operon?
- Promoter of LacI
- LacI gene (produces Lac repressor)
- Terminator for LacI
- Activator site
- Promoter containing operator
- LacZ, LacY, LacA and their Terminator
What happens to the Lac Operon if there is no lactose present?
- Lac repressor is synthesized by LacI
- Lac repressor binds the operator region of the LacZ, LacY, and LacA promoter
- RNA pol does not bind
- NO mRNA is produced
What happens to the Lac Operon if there is lactose present with a high concentration of glucose?
- Lac repressor is synthesized by LacI
- Lac repressor binds to allolactose and it is released
- RNA pol can bind to promoter
- mRNA is produced at LOW levels
What happens to the Lac Operon if there is lactose present with a low concentration of glucose?
- cAMP levels are high
- CAP binds to cAMP
- CAP-cAMP complex binds to the activator site and activates transcription
- RNA pol binds with higher efficiency
- mRNA is produced at HIGH levels
So if there is no lactose essentially what happens to expression of LacZ, LacY, and LacA?
Expression is OFF
So if there is lactose and glucose levels are high, essentially what happens to expression of LacZ, LacY, and LacA?
Expression is LOW
So if there is lactose and glucose levels are low, essentially what happens to expression of LacZ, LacY, and LacA?
Expression is HIGH
What do each of the Lac genes code for?
- LacI = Lac Repressor
- LacZ = β-galactosidase (converts lactose with a 1,4-β bond to glucose and galactose (and a bit to allolactose with a 1,6-β bond))
- LacY = Lactose Permease (transports across membrane)
- LacA = β-galactoside transacetylase
What does Lactose get hydrolyzed into?
- Glucose
- Galactose
What happens when the Lac Operon is activated?
1. Lac permease uses the protein gradient to pump lactose into the cell
2. Lactose is hydrolyzed by the β-galactosidase to glucose and galactose (and some to allolactose)
3. Allolactose binds to Lac Repressor
4. Lac Repressor is released from the operator site
5. Transcription is activated and more β-galactosidase and Lac permease are produced
If the plasmid with the target gene was under the control of the Lac operon and used the E.coli RNA polymerase what are the implications?
- Low expression
- Some leaky expression
If the T7 Polymerase was under the control of the Lac Operon and the target gene under the T7 Promoter with both on the plasmid what are the implications?
- Any leaky expression may kill or affect the cells before they can grow
- Cloning will be hard
If the target gene is under the control of the T7 promoter on the plasmid and the T7 RNA Pol is under the control of the lac operon on the genome, what are the implications?
- This is the best option
- Cloning can be done in an E.coli strain that does not contain the T7 RNA pol gene (i.e. not DE3)
- For expression the plasmid is transferred to a DE3 strain (with the RNA pol gene in the genome)
It is best to have the target gene under the control of what?
T7 Polymerase Promoter
It is best to have T7 RNA polymerase under the control of what?
Lac operon
What are the two types of E.coli strains used for recombinant expression of protein?
- Cloning strain
- Expression strain
What is the cloning strain optimized for?
- DNA preparation that does not contain the T7 RNA polymerase gene
- Ex: DH5α
What is the expression strain optimized for?
- Making proteins that expresses the T7 RNA pol under the lac operon
Ex: Rosetta Blue DE3 strain
How is the E.coli genotype manipulated to make it optimized for cloning?
- F- = cell is unable to mate through conugation
- recA1 = cell is deficient in DNA repair, reduces unwanted recombination
- endA1 = inactivates an endonuclease that degrades DNA during mini-prep
- hsdR17 = eliminates endonuclease that digest unmethlyated DNA; important for PCR-derived DNA (which are unmethylated)
How is the E.coli genotype manipulated to make it optimized for expression?
- DE3 - incorporates λ prophage, expressing the T7 RNA pol under the control of the Lac operon
- LysS RARE - pLysS expresses T7 lysozyme which suppresses basal T7 RNA pol expression; contains tRNA genes for rare codons
- LacI9 - mutated LacI promoter; leads to overproduction of Lac repressor
How can protein expression be induced under the lac operon?
- Lactose
- IPTG
Why is lactose unideal for induction of the lac operon?
It is metabolized by cells and consumed over time
Why is IPTG ideal for induction of the lac operon?
- It is a lactose (activally allolactose) analog that is not hydrolyzable by the cell so levels remain constant
- This leads to a lot of expression
Following IPTG induction, what does T7 RNA polymerase do?
Binds to T7 promoter in pET vector
What does a higher absorption mean?
More scatter of light = more cells