Biochem 651: Exam 1

Front 1

Running a PCR requires what components?

Back 1

- Buffer
- Mg2+
- DNA template
- Primers (forward and reverse)
- dNTP mix (ATP, GTP, CTP, TTP)
- Taq polymerase (enzyme)

Front 2

How does PCR contrast with DNA replication?

Back 2

- Primers are DNA
- Only one enzyme is involved: taq polymerase

Front 3

What is the cycle of temperatures (approximately) in PCR?

Back 3

1. 94-95*C (denature DNA)
2. 50-65*C (anneal primers)
3. 72*C (extension/polymerization)

Front 4

What happens during PCR?

Back 4

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

Front 5

How do you ensure that the primers will anneal to the template strands?

Back 5

Make sure primers are in large excess

Front 6

How many target copies are generated after 30 PCR cycles?

Back 6

1,000,000,000 (one billion)

Front 7

At the end of 30 cycles what is most of the DNA?

Back 7

Target portion of the template strand

Front 8

What is a primer?

Back 8

A primer is a small nucleotide segment that is complementary to the replication template

Front 9

What must a primer have for it to be elongated during polymerization?

Back 9

A free 3' hydroxyl group to which the polymerase can add nucleotides

Front 10

Where do you add restriction enzymes to on primers? Why?

Back 10

The 5' end of primers so that the 3' hydroxyl group is still free for polymerization

Front 11

What are the characteristics of a primer?

Back 11

- 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)

Front 12

The forward primer is complimentary to what?

Back 12

3' end of the bottom strand

Front 13

The reverse primer is complimentary to what?

Back 13

3' end of the top strand

Front 14

How can PCR primers be used for cloning?

Back 14

If restriction sites are on the ends of the primers they can be digested and inserted into a vector plasmid.

Front 15

What does a restriction enzyme do?

Back 15

Cuts dsDNA (double-stranded DNA) at a specific sequence known as the restriction site

Front 16

What do most restriction enzymes recognize?

Back 16

A palindromic 6 base sequence

Front 17

What do you need to make sure of when adding restriction sites?

Back 17

Make sure the restriction enzyme does not cut anywhere else in your gene

Front 18

What makes up a "clone"

Back 18

(digested) Vector + (digested) PCR product with restriction sites

Front 19

When designing primers what do you need to consider?

Back 19

Make sure the start codons of the PCR product and the vector are in frame

Front 20

What is the DNA start codon?

Back 20

ATG

Front 21

Why don't you need to worry about the 3' end of the gene?

Back 21

The gene's stop codon will end translation

Front 22

If you want to use the vector's stop codon what do you need to do?

Back 22

Delete or change the gene's stop codon

Front 23

What must be the approximate content of the bases in a primer?

Back 23

~50% GC

Front 24

What should you avoid when designing PCR primers?

Back 24

Hairpins and dimers

Front 25

What should the Tm values be for the primers?

Back 25

Very similar; 50-60*C

Front 26

What do PCR and replication both require?

Back 26

DNA template, primers, nucleotides

Front 27

How do the PCR primers and replication primers differ?

Back 27

PCR primers are made of DNA; replication primers are made of RNA

Front 28

How are template strands denatured in PCR vs replication?

Back 28

PCR: heat
Replication: helicase

Front 29

How many enzymes does PCR require?

Back 29

One - Taq polymerase

Front 30

What can PCR be used for?

Back 30

- Amplify a specific sequence
- Introduce mutations
- Forensic analysis
- Prenatal diagnosis

Front 31

How can mutations be introduced via PCR?

Back 31

- Design primers with desired mutation
- Use error-prone polymerase to generate random mutations

Front 32

Why is Mg2+ required for PCR?

Back 32

It acts as a cofactor for Taq Polymerase

Front 33

Why is Taq buffer required for PCR?

Back 33

Sets up a conducive environment for the Taq polymerase to work

Front 34

What is a master mix?

Back 34

Contains all of the components that do not vary between reactions

Front 35

Why is using a master mix a good idea?

Back 35

- Reduces pipette error
- Increases reproducibility
- Saves time

Front 36

How are Tm values calculated?

Back 36

- G and C bases = 4*C
- A and T bases = 2*C

Front 37

What reaction does carbonic anhydrase catalyze?

Back 37

CO2 + H2O <--> HCO3- + H+

Front 38

What kind of enzyme is human carbonic anhydrase?

Back 38

alpha Zn-metalloenzyme

Front 39

What does "carbonic anhydrase is ubiquitous to all cell types" mean?

Back 39

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

Front 40

What is the importance of CAII?

Back 40

- Regulation of respiration and gas exchange
- Regulation of acid-base equilibria
- Transport of ions and fluids
- Development of bone and mineralization

Front 41

What reactions related to carbonic anhydrase go on in a cell?

Back 41

- 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+)

Front 42

What reactions related to carbonic anhydrase go on in the lungs?

Back 42

Bicarbonate is converted back to CO2 (6 HCO3- + 6 H+ --> 6 CO2 + 6 H2O)

Front 43

Why is HCAII a model system?

Back 43

- Stable
- Soluble
- Monomeric
- Relatively low molecular weight (30 kD)
- Many structures solved
- Biophysically characterized

Front 44

What are the three possible roles for Zn in the function of HCAII? Which are relevant to HCAII?

Back 44

- 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)

Front 45

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?

Back 45

- Zn has a stable +2 redox state
- CO2 --> H2CO3 is not a redox reaction (it's an addition/hydrolysis)

Front 46

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?

Back 46

- While Zn is structurally important for many folds, HCAII is stable in its app form
- Apoenzyme means without Zn

Front 47

Why is this particular function of metalloenzymes relevant to HCAII: they are positively charged and can act as Lewis acids?

Back 47

Zn stabilizes the OH- ion that performs the nucleophilic attack on the CO2 substrate

Front 48

How is Zn2+ situated in the HCAII enzyme?

Back 48

It is coordinated by three neutral histidines (H94, H96, and H119)

Front 49

What is the function of the Zn2+ situated in the HCAII enzyme?

Back 49

It has a high affinity for binding a negative hydroxyl ion

Front 50

After Zn2+ binds an OH- ion, what is it activated to do?

Back 50

Zn bound OH- is activated to perform a nucleophilic attack on the CO2 (making it Zn bound HCO3-)

Front 51

What kind of mechanism occurs for the HCAII enzyme?

Back 51

Ping-pong mechanism (2 steps)

Front 52

What occurs during the first step of the ping-pong mechanism?

Back 52

- 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-

Front 53

What occurs during the second step of the ping-pong mechanism?

Back 53

- 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

Front 54

What is the rate-limiting step of the ping-pong mechanism?

Back 54

The removal of the proton by the environment/solvent (B-)

Front 55

How effective is the HCAII enzyme?

Back 55

It is extremely efficient with an extremely high turn-over rate (kcat ~ 10^6 per sec)

Front 56

What is the catalytic rate constant (kcat) for HCAII?

Back 56

kcat ~ 10^6 per sec

Front 57

What does the efficiency of the HCAII enzyme approach?

Back 57

The limit of diffusional control
(kcat/Km ~ 10^8 1/M*sec)

Front 58

What property of the HCAII enzyme is useful for activity assays?

Back 58

- It has esterase activity
- p-Nitrophenol derivative becomes colored (absorbs in the visible range) once hydrolyzed

Front 59

Specifically how can the esterase activity of HCAII be used for activity assays?

Back 59

- 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

Front 60

What is the makeup of the active site of HCAII?

Back 60

~1/2 hydrophobic, ~1/2 hydrophilic

Front 61

What can be found on the protein data bank?

Back 61

Macromolecular structure data, including coordinates and electron density maps

Front 62

What does resolution refer to?

Back 62

The minimum distance between structural features that can be distinguished in an electron density map

Front 63

What are low, medium, high, and atomic values for resolution?

Back 63

- Low = 3 Ȧ
- Medium = 2.7 - 2 Ȧ
- High = 1.5 Ȧ
- Atomic = <1 Ȧ

Front 64

What does "R-work" refer to?

Back 64

A measure of how well the model matches the data (%)

Front 65

What does "R-free" refer to?

Back 65

An independent statistic that checks for over interpretation of the data

Front 66

Electrophoresis is what kind of technique?

Back 66

Separation

Front 67

How is electrophoresis useful for analysis?

Back 67

- Helps determine what is in an unknown sample
- Helps determine how much of a substance you have
- Helps determine how pure a sample is

Front 68

What key physical properties can be exploited for separation?

Back 68

- Size
- Shape
- Charge

Front 69

What is the purpose of the gel in electrophoresis?

Back 69

It acts as a molecular sieve

Front 70

What provides the driving force for a separation by electrophoresis?

Back 70

Electric field (E)

Front 71

What do charged molecules experience in an electric field (during electrophoresis)? What is the relevant equation?

Back 71

Force (F)

F=qE

Front 72

What equation relates the electric field, the force, and the charge?

Back 72

F=qE

F= force, q = charge, E = electric field

Front 73

If there was no friction what would happen during electrophoresis?

Back 73

The molecules would keep accelerating and never reach a steady state velocity

Front 74

What provides the necessary friction for electrophoresis?

Back 74

The gel (molecular sieve)

Front 75

What equation can be used to determine the velocity?

Back 75

v = qE/f

v=velocity,q = charge, E = electric field, f = frictional coefficient

Front 76

Would a sphere or an oblong shape of the same volume move faster in an electric field? Why?

Back 76

The sphere would move faster because it has a smaller frictional coefficient

Front 77

What characteristics make proteins much more complicated than simple spheres and oblong shapes?

Back 77

- Complex shapes
- Flexibility
- Unevenly distributed charges
- Counter ions are dragged in direction opposite protein

Front 78

In gel electrophoresis, how are proteins separated?

Back 78

By molecular weight

Front 79

Why is separating proteins with gel electrophoresis by molecular weight problematic?

Back 79

There is no common mass:charge ratio among proteins

Front 80

How can the problem of not having a common mass:charge ratio for proteins be overcome?

Back 80

Use a reagent that will introduce a constant mass:charge ratio (sodium dodecyl sulfate, SDS)

Front 81

What is the significance of SDS?

Back 81

- 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

Front 82

What is the structure of SDS (sodium dodecyl sulfate)?

Back 82

- Polar head group (negative, with Na+ counter ion)
- Hydrophobic tail group (12 carbons)

Front 83

What is the ratio that SDS binds proteins?

Back 83

1.4g SDS / g protein
(~1 SDS molecule / 2 AA's)

Front 84

When SDS is added to a protein what are the implications?

Back 84

- 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

Front 85

When SDS is used for polyacrylamide gel electrophoresis it is also commonly called what?

Back 85

Denaturing gel electrophoresis (because it denatures the folded shape of the protein)

Front 86

Why is DNA separation by electrophoresis so much less complicated than for proteins?

Back 86

- DNA has a constant shape (double helix)
- There is a constant charge/size ratio (-1 for every phosphate; -2 for every base-pair)

Front 87

How exact is DNA separation?

Back 87

Very good - can separate based on 1 base pair difference!

Front 88

How can the precision of DNA separation be utilized?

Back 88

DNA sequencing

Front 89

Why is RNA a bit more difficult to separate with electrophoresis than DNA?

Back 89

- It is single-stranded
- It often forms secondary structures which require denaturing

Front 90

How can RNA be denatured for electrophoresis?

Back 90

65*C and formaldehyde

Front 91

What are the two gels for electrophoresis?

Back 91

- Agarose
- Polyacrylamide

Front 92

When is an agarose gel used for electrophoresis?

Back 92

For high molecular weight macromolecules (e.g. nucleic acids)

Front 93

When is a polyacrylamide gel used for electrophoresis?

Back 93

For low molecular weight macromolecules (e.g. protein)

Front 94

What are the characteristics of agarose?

Back 94

- Linear, non-crosslinked polymer
- Used for high molecular weight macromolecules (e.g. nucleic acids)

Front 95

Why does cooled agarose need to be melted at a higher temperature than originally?

Back 95

After it is cooled to "set" the polymer chains become intertwined

Front 96

At what weight/volume ratio is agarose typically used at?

Back 96

0.7-3%

Front 97

How is polyacrylamide polymerized?

Back 97

Via a radical reaction

Front 98

At what weight/volume ratio is acrylamide typically used at? bisacrylamide?

Back 98

Acrylamide ~8-20%
Bisacrylamide ~3%

Front 99

How far does xylene cyanol move in agarose gel electrophoresis?

Back 99

The distance that 4000bp DNA fragments move

Front 100

How far does bromphenol blue move in agarose gel electrophoresis?

Back 100

The distance that 750-500bp DNA fragments move

Front 101

What must be the relationship between the ionic strength of the gel and the buffer? Why?

Back 101

They must be of equal ionic strength in order for proper separation of DNA

Front 102

What was the running buffer and solution used for the gel in the lab we made agarose gels?

Back 102

TAE = Tris-acetate-EDTA

Front 103

Should the wells be at the cathode or anode?

Back 103

Cathode

Front 104

In which direction does the DNA run?

Back 104

Runs to red (towards the anode)

Front 105

Why is EtBr added to the agarose gel?

Back 105

It absorbs UV light at 302nm and fluoresces at 590nm (this allows us to visualize the DNA in the gel)

Front 106

What are the three buffers used to purify DNA?

Back 106

- PB - binding buffer
- PE - washing buffer
- EB - elution buffer (contains the purified DNA)

Front 107

For determining DNA concentration, what ratio is important?

Back 107

260/280 ratio; a value of 2 would mean 100% pure DNA

Front 108

How does EtBr interact with the DNA?

Back 108

It fits between the bases - this causes the DNA to fluoresce at 590nm

Front 109

What are the three methods to visualize protein in gels?

Back 109

- Coomassie Brilliant Blue (least sensitive)
- Silver Staining
- Western Blotting (most sensitive)

Front 110

How does Coomassie brilliant blue interact with proteins in gels? What is the detection limit?

Back 110

- Associates with positively charged and aromatic amino acids
- Detection limit: ~25-50ng/band

Front 111

How does Silver Staining interact with proteins in gels? What is the detection limit?

Back 111

- Ag+ ions associate with various protein side chains
- Detection limit: ~0.5-1ng/band

Front 112

How does Western Blotting interact with proteins in gels? What is the detection limit?

Back 112

- Uses antibodies to generate, amplify signal
- Very specific and sensitive
- Detection limit: often picograms or less

Front 113

Why do plasmids make great vectors?

Back 113

- 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

Front 114

What are three salient features of a cloning vector?

Back 114

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)

Front 115

What is a MCS?

Back 115

Multiple Cloning Site
- Contains DNA sequences that can be digested with restriction enzymes

Front 116

What factors do you consider when choosing your cloning vector?

Back 116

- 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)

Front 117

What does electroporation of a cell do?

Back 117

A large electric field makes the bacterial cell wall permeable so circular DNA can leak in

Front 118

What does it mean for a bacterial cell to be "competent"?

Back 118

It is capable of accepting plasmids (electroporated)

Front 119

Is transformation (the uptake of a plasmid) an efficient process?

Back 119

No - it is very inefficient; only about 1 in every 10^5 cells may take up the DNA

Front 120

What is a way to check for which cells have successfully taken up the plasmic you want?

Back 120

Utilize the antibiotic resistance gene

Front 121

Do all of the antibiotic resistant colonies on your plate contain the desired clone product?

Back 121

No; they could also be re-ligated vector or the vector ligated to a different insert

Front 122

What are the four methods of controlling / screening for colonies that do not contain the desired insert?

Back 122

- Blue-White Screening
- Vector Re-ligation Control
- Restriction Digest Screen
- PCR Screen

Front 123

What does the blue-white screening technique distinguish between?

Back 123

Re-ligated plasmid and plasmid + insert

Front 124

What do the blue colonies represent from the blue-white screening technique? Why are they blue?

Back 124

- These colonies are re-ligated plasmids (no insert)
- The vector has the intact LacZ gene so beta-galactidose is produced

Front 125

What are the limitations of the blue-white screen?

Back 125

It will not tell you if your insert is in the gene; just whether the vector contains ANY insert

Front 126

What do the white colonies represent from the blue-white screening technique? Why are they white?

Back 126

- The white colonies represent plasmids with inserts in them
- The LacZ gene is interrupted so no beta-galactidose is produced

Front 127

What is necessary for the blue-white screening technique?

Back 127

- 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

Front 128

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?

Back 128

- 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

Front 129

What must occur to do a restriction digest screen?

Back 129

Plasmid DNA must be purified from E. coli

Front 130

How does the restriction digest screen work?

Back 130

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)

Front 131

Does the PCR Screen require DNA purification from E.coli?

Back 131

No (restriction digest screen does)

Front 132

How does the PCR screen work?

Back 132

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)

Front 133

What are the steps of Site-Directed Mutagenesis?

Back 133

1. Design primers
2. PCR
3. Digest parental plasmid
4. Transformation, growth

Front 134

For site-directed mutagenesis, how do the primers differ?

Back 134

The primers are complementary to the strand except for the place you want to introduce a mutation.

Front 135

What happens during the PCR step of site-directed mutagenesis?

Back 135

- 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

Front 136

During step three (digest parental plasmid) of site-directed mutagenesis, what happens?

Back 136

- DpnI is a restriction enzyme that only cuts methylated DNA
- It digests the template DNA and leaves the mutated products

Front 137

What is unique about the restriction enzyme DpnI? How can this be used for site-directed mutagenesis?

Back 137

- DpnI only cuts methylated DNA
- Template DNA is methylated, but the PCR products are not
- DpnI digests the template strands

Front 138

What happens during the transformation and growth stage of site-directed mutagenesis?

Back 138

- E. coli is transformed with the double-stranded (ds) PCR product
- The E. coli repair the nicked DNA and replicate the mutant

Front 139

What ingredients are necessary for a ligation?

Back 139

- Ligase (T4 DNA Ligase)
- Digested insert
- Digested vector
- ATP
- Mg2+
- Reducing buffer

Front 140

What does T4 DNA Ligase do?

Back 140

Catalyzes the phosphodiester bond between a 5' phosphate and a 3' OH

Front 141

How do you activate ligase?

Back 141

Incubate at room temperature for 30 minutes

Front 142

How do you inactivate ligase?

Back 142

Heat reaction at 65*C for 10 minutes

Front 143

What does transformation do?

Back 143

It's a process to introduce foreign DNA into a cell

Front 144

What are the two methods of transformation? What are the basic pros and cons of each?

Back 144

- Electroporation - more efficient but very slow
- Chemical Transformation - lower efficiency but faster

Front 145

How is chemical transformation done?

Back 145

- 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

Front 146

Why during the chemical transformation process were the cells heated for 50 seconds?

Back 146

To make the membrane more permeable

Front 147

Why was 500 μL of Ligase buffer added and the sample incubated for 45 minutes at 37*C?

Back 147

It helped the cells to recover from the trauma of being transformed

Front 148

What grows on the kanamycin plates?

Back 148

- E. coli with vector transformed
- If the vector is incomplete (w/o a plasmid) it will not grow

Front 149

Besides vector + insert (goal clone), what other forms of plasmid can grow on the LB + Kanamycin plates?

Back 149

- Recombinant vector
- Undigested vector
- Only one cut - can religate

Front 150

Why did we transform our clones into DHBα cells first (in lab four)?

Back 150

This is a cell line with a high copy number; i.e., high level DNA introduced

Front 151

What does recombinant protein expression take advantage of?

Back 151

Target cells' native machinery to make a protein of interest

Front 152

What is the process of recombinant protein expression?

Back 152

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

Front 153

When introducing a gene for recombinant protein expression, what are two necessary characteristics of the gene?

Back 153

- Should be replicable (ori)
- Gene should be under control of a strong promoter

Front 154

When you turn on a promoter, what happens?

Back 154

The cell transcribes mRNA

Front 155

What promoter does our pET-28b vector contain?

Back 155

T7 promoter

Front 156

When we transform our pET-28b vector into E.coli what is the problem with transcription?

Back 156

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

Front 157

How do we get the E.coli we are transforming to express our clone?

Back 157

- 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

Front 158

How do you get E.coli to express the T7 RNA polymerase?

Back 158

IPTG induction

Front 159

After you add IPTG to E.coli to induce expression of T7 RNA polymerase, what do you do?

Back 159

A time course via a gel (SDS-PAGE) to determine the time of maximum protein expression

Front 160

What are the advantages of a bacterial expression system (e.g., transforming E.coli with a clone)?

Back 160

- Relatively fast (1-2 weeks including cloning)
- Inexpensive
- High expression levels
- System is amenable to large scale preparations

Front 161

What are the disadvantages of a bacterial expression system (e.g., transforming E.coli with a clone)?

Back 161

- 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

Front 162

For eukaryotic cell expression systems, how do you get your gene into a eukaryotic cell?

Back 162

Use viruses

Front 163

What is the process of inserting your gene into a eukaryotic cell for expression?

Back 163

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

Front 164

Why during eukaryotic cell expression systems do we not purify the protein directly from the original cells we added the viral genome to?

Back 164

These cells may be exhausted/weak so you want to use new healthy cells to express the protein

Front 165

For insect cell expression systems, what is used to express the protein of interest?

Back 165

Baculovirus in insect cells

Front 166

The gene of interest during an insect cell expression must be under what control?

Back 166

The control of a baculovirus-specific strong promoter

Front 167

Once the gene of interest is recombined with the baculovirus, what do you do?

Back 167

Infect insect cells - during the course of its natural life cycle the virus will produce your protein of interest

Front 168

What does the baculovirus promoter regulate?

Back 168

Protein induction

Front 169

What are the advantages of a insect cell expression system (using baculovirus)?

Back 169

- Reasonably high protein expression levels
- Supports many post-translational modifications (PTMs)
- Proteins are likely to fold properly

Front 170

What are the advantages of a insect cell expression system (using baculovirus)?

Back 170

- 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

Front 171

How do eukaryotic cells differ from bacterial cells?

Back 171

- More expensive
- More difficult to maintain

Front 172

When using a tissue culture expression system, what must you do?

Back 172

- Cells are kept in culture
- Cells must be passaged every few days so they don't overgrow the plate and die

Front 173

How do you passage cells, as is required for tissue culture expression systems?

Back 173

Cells are removed from the original plate, counted and plated at the correct density on fresh plates

Front 174

When you are ready to express protein from a tissue culture, what do you do?

Back 174

Grow up many cells; it can take a week or so to get enough cells for expression

Front 175

What are the two ways to express proteins in mammalian cells?

Back 175

1. Virally
2. Transfection

Front 176

How are proteins expressed virally in mammalian cells?

Back 176

- 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

Front 177

Why are adenoviruses used for mammalian cell protein expression systems?

Back 177

Adenoviruses can infect many different mammalian cells (skin, muscle, brain, bone, nerve, liver cells, as well as embryonic stem cells)

Front 178

How can mammalian cells incorporate the gene of interest virally?

Back 178

Using an adenovirus containing your gene of interest

Front 179

What does the viral promoter regulate in a mammalian cell expression system?

Back 179

Regulates protein expression

Front 180

What are the advantages of the viral method to mammalian cell protein expression?

Back 180

- High levels of PTMs (post-translational modifications)
- Often able to obtain functional mammalian proteins
- Virus can be used in different cell types

Front 181

What are the disadvantages of the viral method to mammalian cell protein expression?

Back 181

- 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

Front 182

What happens during transfection mammalian cell expression systems?

Back 182

- Nucleic acid polymer can be introduced directly into the cells (non-virally)
- The cell regulates protein expression

Front 183

How do viral and transfection methods differ in regulation?

Back 183

- Viral - viral promoter regulates protein expression
- Transfection - cell regulates protein expression

Front 184

What are the advantages of transfection as a mammalian cell protein expression system?

Back 184

- 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

Front 185

What are the disadvantages of transfection as a mammalian cell protein expression system?

Back 185

- 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

Front 186

What are three cell-free expression systems?

Back 186

- Rabbit Reticulocyte Lysate
- E. coli Extract
- Wheat Germ Extract

Front 187

Describe the Rabbit Reticulocyte Lysate expression system.

Back 187

- Cell-free
- Lysed immature RBC that have been treated with micrococcal nuclease to degrade endogenous mRNA
- You add the RNA template

Front 188

Describe the E. coli extract expression system.

Back 188

- Cell-free
- E. coli extract that contains T7 polymerase
- You supply circular DNA with your gene cloned downstream of T7 promoter

Front 189

Describe the Wheat Germ Extract expression system.

Back 189

- Cell-free
- Micrococcal nuclease treated extract from wheat germ containing the components necessary for translation
- You add the RNA template

Front 190

What are the advantages of cell-free expression systems?

Back 190

- 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

Front 191

What are the disadvantages of cell-free expression systems?

Back 191

- Very low level of expression - nano grams
- Very expensive
- Little post-translational modifications (PTMs) - you can add reagents to get some though

Front 192

What is a mini-prep used for?

Back 192

Method to separate plasmid DNA from genomic DNA

Front 193

How can you separate plasmid DNA from genomic DNA?

Back 193

Using a mini-prep (ex: QIAGEN)

Front 194

What kind of column is used for separating plasmid DNA from genomic DNA (in a mini-prep)? Why?

Back 194

Column with silica-gel membrane - selectively binds DNA and separates nucleic acids with specific size parameters

Front 195

What are the 5 buffers/solutions that are used during the mini-prep to separate plasmid DNA from genomic DNA?

Back 195

1. P1 - Resuspension
2. P2 - Alkaline Lysis
3. N3 - Neutralization
4. PE - Ethanol Wash
5. EB - Elution Buffer

Front 196

What does the P1 solution (first step) do in the mini-prep to separate plasmid DNA from genomic DNA?

Back 196

- Resuspension of pellet containing RNA, genomic DNA, plasmic, and proteins
- Contains RNase A to remove RNA

Front 197

What does the P2 solution (second step) do in the mini-prep to separate plasmid DNA from genomic DNA?

Back 197

- Alkaline lysis
- SDS lyses cell membranes
- NaOH denatures DNA and proteins (time sensitive)

Front 198

What does the N3 solution (third step) do in the mini-prep to separate plasmid DNA from genomic DNA?

Back 198

- 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

Front 199

What does the PE solution (fourth step) do in the mini-prep to separate plasmid DNA from genomic DNA?

Back 199

- Wash
- Ethanol removes salts
- Plasmid remains attached to column

Front 200

What does the EB solution (fifth/last step) do in the mini-prep to separate plasmid DNA from genomic DNA?

Back 200

- Elution buffer
- Tris pH 8.5
- High pH and low salt makes plasmid wash off of column

Front 201

What were the two methods we could have used to screen our clones?

Back 201

- PCR screen
- Restriction digest screen

Front 202

Which screen was used to screen our clones?

Back 202

Restriction digest screen

Front 203

What happens during the restriction digest screen that allows us to differentiate between our desired clones and empty vectors?

Back 203

- 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

Front 204

What is the Rosetta DE3 E.coli strain designed for?

Back 204

- High transformation efficiency
- Over-expression of proteins
- Enhanced tRNA pool for codons found in eukaryotic protein sequences

Front 205

Before recombinant technology, where were proteins obtained from?

Back 205

Naturally abundant sources, such as hemoglobin, myoglobin, white egg lysozyme

Front 206

What kind of proteins were hard to study before recombinant technology?

Back 206

- Proteins with low abundances (needed an abundant source)
- Human proteins
- Proteins that are hard to purify
- Proteins that are unstable when removed from tissue

Front 207

What has recombinant technology allowed us to do?

Back 207

- 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

Front 208

Related to proteins, what can recombinant technology do?

Back 208

- 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)

Front 209

Why is protein over-expression not always a good thing?

Back 209

Massive amounts of foreign protein can be really toxic to the host cell, complicating cloning and actual expression

Front 210

The system we discussed is a controlled system based on what two key elements?

Back 210

- 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

Front 211

What are the characteristics of the bacteriophage T7 RNA polymerase?

Back 211

- 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)

Front 212

How efficient is the bacteriophage T7 RNA polymerase?

Back 212

- 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

Front 213

Why must the T7 RNA polymerase be controlled?

Back 213

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

Front 214

How is the T7 RNA polymerase controlled when inserted in E.coli?

Back 214

Lac operon

Front 215

What is a regulon?

Back 215

Group of genes that are regulated by the same protein

Front 216

What is an operon?

Back 216

Regulon and its regulatory elements (promoter, operator, and activator site)

Front 217

What is the structure of the Lac operon?

Back 217

- Promoter of LacI
- LacI gene (produces Lac repressor)
- Terminator for LacI
- Activator site
- Promoter containing operator
- LacZ, LacY, LacA and their Terminator

Front 218

What happens to the Lac Operon if there is no lactose present?

Back 218

- 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

Front 219

What happens to the Lac Operon if there is lactose present with a high concentration of glucose?

Back 219

- 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

Front 220

What happens to the Lac Operon if there is lactose present with a low concentration of glucose?

Back 220

- 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

Front 221

So if there is no lactose essentially what happens to expression of LacZ, LacY, and LacA?

Back 221

Expression is OFF

Front 222

So if there is lactose and glucose levels are high, essentially what happens to expression of LacZ, LacY, and LacA?

Back 222

Expression is LOW

Front 223

So if there is lactose and glucose levels are low, essentially what happens to expression of LacZ, LacY, and LacA?

Back 223

Expression is HIGH

Front 224

What do each of the Lac genes code for?

Back 224

- 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

Front 225

What does Lactose get hydrolyzed into?

Back 225

- Glucose
- Galactose

Front 226

What happens when the Lac Operon is activated?

Back 226

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

Front 227

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?

Back 227

- Low expression
- Some leaky expression

Front 228

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?

Back 228

- Any leaky expression may kill or affect the cells before they can grow
- Cloning will be hard

Front 229

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?

Back 229

- 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)

Front 230

It is best to have the target gene under the control of what?

Back 230

T7 Polymerase Promoter

Front 231

It is best to have T7 RNA polymerase under the control of what?

Back 231

Lac operon

Front 232

What are the two types of E.coli strains used for recombinant expression of protein?

Back 232

- Cloning strain
- Expression strain

Front 233

What is the cloning strain optimized for?

Back 233

- DNA preparation that does not contain the T7 RNA polymerase gene
- Ex: DH5α

Front 234

What is the expression strain optimized for?

Back 234

- Making proteins that expresses the T7 RNA pol under the lac operon
Ex: Rosetta Blue DE3 strain

Front 235

How is the E.coli genotype manipulated to make it optimized for cloning?

Back 235

- 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)

Front 236

How is the E.coli genotype manipulated to make it optimized for expression?

Back 236

- 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

Front 237

How can protein expression be induced under the lac operon?

Back 237

- Lactose
- IPTG

Front 238

Why is lactose unideal for induction of the lac operon?

Back 238

It is metabolized by cells and consumed over time

Front 239

Why is IPTG ideal for induction of the lac operon?

Back 239

- 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

Front 240

Following IPTG induction, what does T7 RNA polymerase do?

Back 240

Binds to T7 promoter in pET vector

Front 241

What does a higher absorption mean?

Back 241

More scatter of light = more cells