Concepts 2 Flashcards
(78 cards)
1
Q
What are plasmids?
A
small circular DNA molecules that replicate independently of the host chromosomes
2
Q
What features do plasmids constructed in the lab normally have?
A
- Usually have a predesigned cloning site and an amp resistance gene
3
Q
How are plasmids mass produced and isolated?
A
Plasmids are transformed into bacteria and may be purified and isolated
4
Q
How are plasmids edited?
A
Enzymes are used to insert desired pieces of foreign DNA into plasmids
5
Q
What type of plasmids are we using and why are we using it?
A
We are using a plasmid called a shuttle vector because they can be propagated in either bacteria or yeast
6
Q
Describe how shuttle vectors are used. What are they grown in? What markers do they contain?
A
- Plasmids are propagated in bacteria because they grow quickly and maintain multiple copies of the plasmids
- Shuttle vectors have origins of replication and selectable markers for propagation in bacteria and yeast
7
Q
What functional elements are found in our yeast plasmids?
A
URA3, Beta – lactamase gene, pBR322 ori, Yeast 2um origin, GAL1 promoter, C-Terminal tags
8
Q
What is the purpose of URA3 in our plasmids?
A
allows yeast to grow in the absence of uracil after they have been transformed with plasmids
9
Q
What is the purpose of the beta-lactamase gene?
A
amp resistance
10
Q
What is the purpose of the GAL-1 promoter in our plasmids?
A
upstream of gene of interest, allows for manipulation of expression
11
Q
What is the purpose of C-temrinal tags?
A
downstream of gene of interest, allows for purification and isolation in western blotting
12
Q
What physical properties allow for plasmid purification?
A
- Plasmids are much smaller than bacterial chromosomes
- Plasmids are supercoiled in their native forms, which allows them to renature quickly after being denatured
13
Q
What do bacterial cells with plasmids contain?
A
- Many different, well-folded proteins
- 1-2 copies of large, circular bacterial DNA complexed with proteins
- Multiple copies of small plasmids
14
Q
What does purification of plasmids from bacterial cells involve?
A
- Cells are first treated with alkaline lysis buffer containing base and a detergent (gently mix the contents by inverting the tube 4-6 times)
- Neutralization: Extract is neutralized to allow DNA molecules to renature
- Purification of ZYPPY silica resin
- Plasmid Elution
15
Q
What happens during the lysis step of plasmid purification? (Proteins, chromosomes, plasmids, solutions)
A
- Proteins denature irreversibly
- Chromosomal DNA denatures – will have difficulty renaturing because its length and many proteins complexed to it
- Plasmids denature, but strands stay together because of supercoiling
- Solution changes from cloudy to clear when the cells are lysed
• Too much agitation can shear chromosomal DNA
16
Q
What occurs during the neutralization step of plasmid isolation?
A
- Plasmids renature and are suspended in the supernatant following centrifugation
- Proteins and chromosomal DNA form an aggregate irreversibly forming a precipitate that can be collected by centrifugation
- When purifying plasmids use a micropipette to carefully remove the supernatant
17
Q
What is the purpose of the Zyppy endowash?
A
Endowash contains guanidine hydrochloride and isopropanol. It removes contaminating proteins that are bound to resin.
18
Q
What is the plasmid elution step of plasmid extraction?
A
- Transfer column to a clean labelled microcentrifuge tube
- Add TE buffer directly to column and allow to sit, then spin for 30 seconds.
19
Q
What is BLOSUM62 matrix? What do BLOSUM62 scores mean?
A
- evaluates how much an amino acid change impacts protein structure
- Positive and negative scores suggest amino acid changes that have been selected FOR (positive) or AGAINST (negative) during evolution
- Magnitude of the score selects the strength of selection
- Score of zero suggests that a particular substitution can be explained by chance alone
- Cysteine and proline cannot be effectively substituted for because they are special
20
Q
What is BLASTP?
A
Basic Local Alignment Search Tool, a computer algorithm for finding homologous sequences in databases
- BLASTP uses word matches as a starting point and extends them in both directions, looking for additional similarity
- As BLASTP extends the alignment out from the match, it calculates a running score – extension stops when the score drops below a threshold value
21
Q
How was the gene coding my met6 protein cloned into the plasmid?
A
- In the DNA there are introns
- An intron from another species can’t be processed by S. cerevisiae
- Processed mRNA is required
- RNA is extracted from our organism; cDNA is made using reverse transcriptase and PCR amplified from the start codon to the stop codon
22
Q
What are restriction endonucleases?
A
enzymes that cleave double stranded DNA at defined positions
23
Q
What is restriction mapping?
A
site-specific restriction endonucleases are used to identify DNA molecules
24
Q
What is the role of REs in bacteria?
A
• Restriction endonucleases are part of a bacterium’s defense against invaders
25
Describe restriction modification systems in bacteria.
- Restriction-modification systems allow the bacterium to distinguish self from non-self DNA
- Restriction: bacterial endonucleases cleave both strands of foreign DNA of specific recognition sites
- Modification: bacteria protect their own DNA by adding a methyl group to the recognition sites in their own DNA
26
What type of REs are used in molecular biology?
- Type II restriction enzymes are widely used in molecular biology: enzymes cleave, but do not modify their specific recognition sites
- REs with 6-nucleotide recognition sites (6-cutters) are widely used in molecular biology
27
How frequently do common lab (6 cutters) cut DNA? What are other aspects of their function?
- Sites would randomly be expected every 1/4096 nucleotides (1/46)
- Actual sizes vary widely with an average of ~4000 bp in length
- Recognition sites are often palindromes
- REs are often dimeric proteins
28
What are sticky ends and what are they useful for?
- sticky ends: 4 unpaired hydrogen bonds on nitrogenous bases hanging off the 5’ end
- Sticky ends are useful in generating recombinant DNA molecules
29
Describe EcoR1.
- EcoRI recognition site is a palindrome with an axis of symmetry
- EcoRI dimer binds sequence and catalyzes double strand cleavage
30
How did we perform restriction mapping?
- Predict bands with NEBCutter2.0
- Take 7 ul of the correct plasmid and add to correct tube (tube 1)
- Add 2ul of Cut Smart buffer and 2ul of restriction enzymes to tube 1
- Add 4ul of water to the uncut tube (Tube 2)
- Quick spin in microcentrifuge
- Place tubes in 37 degree incubator
31
What is yeast transformation?
uptake of foreign DNA by a cell changes its phenotype
32
Describe the yeast cell wall. What must cross this barrier during transformation?
- During transformation, DNA must cross the formidable yeast cell wall
- Fuzziness is characteristic of polysaccharides
- Cell wall is an extensively cross-linked network of proteins and polysaccharides
33
What is the transformation procedure in general?
Cells are treated with chemicals and submitted to a mild heat shock
34
What are the three chemicals necessary to transform yeast?
Polyethylene glycol, Lithium acetate, Single-stranded DNA salmon sperm
35
What is the purpose of polyethylene glycol in transformation?
Possible effects on membrane structure – may help DNA adhere to the cell wall (mechanical)
36
What is the purpose of lithium acetate in transformation?
Monovalent cations generally enhance uptake of DNA (electrical)
37
What is the purpose of the salmon sperm in transformation?
- Saturates non-specific binding sites for DNA in cell wall
- May provide protection from nucleases
- DNA has been boiled and quick chilled to make it single stranded
- Essentially acts as a chaperone
38
How are transformed cells selected for?
Transformed cells are isolated on selective media with no uracil in it
(Complementation of ura3 mutation in our strain and in plasmids)
39
What gel was the RE digest run on?
1% agarose gel
40
What was in the master mix for our transformation?
(200uL of Polyethylene Glycol + 50uL of Lithium Acetate + 2uL of B-mercaptoethanol (reduces disulfide bonds)
41
Describe the process of replica plating.
- The replica of the plate will be made on a piece of sterile velveteen (transferred with gentle tapping)
- The colonies are then transferred from the velveteen to a YC-ura plate, YC-met (galactose), and YC-met (glucose)
- The gal-1 promoter is induced by galactose and repressed by glucose so growth of the cells would be expected on the galactose plate and not on the glucose plate
42
What is yeast's preferred carbon source?
glucose
43
How are glucose and galactose different?
o Glucose and galactose are isomers and only differ in the orientation of a hydroxyl group at the C4 position
44
What happens when galactose replaces glucose as a carbon source?
- Transcription patterns change when galactose replaces glucose
- The glucose transporter in the membrane does not recognize the galactose and does not transport it into the cell, consequently cellular energy levels begin to decline
- Cells increase the expression of proteins that:
- Transport galactose in the cell (Gal2p)
- Convert galactose into glucose-1-P (Gal1p, Gal7p and Gal10p)
(This can enter glycolysis)
45
How are cells able to promote the transition to galactose processing genes?
Cells are able to coordinate the transcription of these genes because the promoters of all 4 genes have binding sites for the same transcriptional activator: Gal 4
46
What is the transcriptional activator for Gal1,10, and 7?
Gal4p transcriptional activator
47
Do other TFs bind GAl-1?
yes
48
What are the domains of the gal 4 protein
Dimerization Domains: Hydrophobic residues on one face of each helix bind the two subunits together
DNA binding domains: each contains a Zn finger that coordinates two zinc atoms (stabilized by cysteine and contains basic amino acids that bind to DNA)
• Binds 17bp sequence in promoters of multiple genes
• UAS = upstream activating sequence
49
Describe how the gal promoter is regulated.
- The GAL1 promoter is subject to both positive and negative regulation
- Positive and negative regulatory proteins bind to cis-elements in the GAL1 promoter
- Gal4 protein binds upstream activating sequence (UAS)
- Repressor proteins bind and repress expression when glucose is available
- Downstream is the MET coding sequence
50
How does galactose impact Gal4p function?
- Galactose relives Gal4p repression by a complex mechanism:
- In the absence of galactose, Gal80p inhibits Gal4p dimer
- Gal80p no longer binds Gal4p in the presence of galactose – activated Gal4p recruits transcriptional machinery
51
Describe transcription regulated by the Gal-1 promoter in the presence of differenct carbon sources.
- Glucose represses transcription
- In the presence of glucose inhibitory proteins will be bound to the repressor and Gal80p will bind Gal4p resulting in transcription being turned off
- Raffinose relives glucose repression – removes inhibitor proteins and some transcription occurs
- Galactose activates transcription 1000x the basal rate
- Inhibitor proteins are not bound to the repressor and Gal80p is not bound to Gal4p
52
How are proteins extracted from our yeast cells without being degreaded?
Our procedure depends on the rapid and efficient extraction of yeast proteins by denaturing the cell wall
53
What happens during denaturation of our protein?
- Proteins are stabilized by thousands of bonds
- Vast majority are non-covalent: ionic, polar, hydrogen, van der Waals
- Covalent disulfide bonds link cysteines
- All of these bonds are broken during the denaturation process
54
What are the denaturing conditions that proteins are extracted under?
- Heat – heat alone would break all bonds but would leave protein with consistency of a fried egg
- Denaturing detergent (SDS) – works slowly at room temperature
- Sulfhydryl reagent (2- aka B-mercaptoethanol) – reduces disulfide bonds
55
What is SDS? Describe structure.
- Sodium Dodecyl sulfate is a denaturing detergent with multiple roles in extract preparation
56
Describe structure of SDS
- Detergents are amphipathic molecules that render hydrophobic molecules soluble in aqueous solutions
- Long side chain binds hydrophobic regions in proteins and cell membranes
- Hydrophilic head group binds water molecules
57
What does SDS do?
- SDS binding imparts a uniform charge to mass ratios to all proteins in a sample
- binds and solubizes unfolded proteins: 1gram protein binds 1.4 grams SDS (1 SDS/2amoino acids)
58
How are SDS gels visualized?
- Stain sds gels with a protein stain
| - When you run a western blot don’t stain the gel before because it interferes with the ability to transfer proteins
59
What are antibodies?
- Antibodies are proteins produced by lymphocytes in vertebrate immune systems
- Antibodies bind tightly and specifically to their target molecules
60
What are antigens?
- antigens are foreign substances that stimulate the immune system to product antibodies
- Bacteria, viruses, allergens and other large molecules are antigens
61
What are epitopes?
the portions of the antigen recognized by the immune system – most antigens have multiple epitopes
62
Describe the structure of antibodies.
- They are bifunctional molecules
- Fab fragments bind antigen (there are two Fab domains)
- Fc fragments are used by the immune system to remove antigen-antibody complexes
63
Where exactly do antigens bind?
- Antigens bind hyper variable regions at the tips of Fab fragments
- Antigen binding has been compared to a lock and key fit
64
What determines processing of antibody antigen complexes?
A limited number of Fc regions determine how other cells will process antibody antigen complexes
65
Describe the antibodies used in molecular biology.
have a gamma heavy chain (IgG immunoglobins)
66
What are polyclonal antibodies?
- Polyclonal antibodies are semi-purified fractions derived from animal serum (antiserum)
- Polyclonal antibodies may recognize multiple epitopes on the same antigen
67
What are limitations of polyclonal antibodies?
- A limited amount of serum can be obtained from an animal
| - It is often useful to have antibodies with a defined specificity
68
What are monoclonal antibodies and what are they produced by?
- Monoclonal antibodies recognize a single well-defined epitope
- Produced by cultured hybridoma cells
69
What are hybridoma cells?
- Hybridoma cells are formed by fusing antibody secreting lymphocytes from an animal with myeloma cells
- Hybridoma cells can be maintained indefinitely in tissue culture
- Hybridoma cells secrete large amounts of monoclonal antibody that can be harvested from the culture medium
70
Give an outline of the monoclonal antibody harvesting process.
- Spleen cells are extracted from a mouse challenged with an antigen
- The spleen cells are fused with myeloma cells to form hybridoma cells
- Scientists select from hybridomas producing the antibody of interest and culture them, then harvest monoclonal antibodies
71
What are the two types of antibodies used in a western blot?
- Primary antibodies – often a mouse monoclonal antibody for an epitope of interest
- Secondary antibodies - - animals are injected with Fc fragments from a different species
- polyclonal antibodies are common
- enzyme or a chromochrome is often covalently attached to the secondary antibody
- signal is amplified
72
What is used to visualize the signal in our western blots?
Horseradish peroxidase (HRP) produces a colored reaction product
73
What do epitope tags do?
- Epitope tags add antibody binding sites to proteins
| - Proteins are expressed from plasmids that encode epitopes in the same reading frame with the cloned sequence
74
Problem: How can proteins encoded by pBG1805 and pYES2.1 plasmids be visualized on the same gel?
Solution: Use an alternative strategy to identify proteins encoded by pBG1805, base on the IgG binding domain of S. aureus protein A
- Protein A is a transmembrane protein with multiple binding sites for the Fc portion of IgGs
- Protein A helps bacterium to evade the hosts immune system during infections
- Protein A binds specifically to rabbit IgGs which we will use as the secondary antibody in our experiment
- PYES2.1 has specific V5 antibody
75
Describe what is expected to be recognized in pBG1805 and pYES2.1 encoded proteins.
- pYES2.1 encoded proteins - Mouse monoclonal antibody detects V5 antigen, Secondary antibody is HRP-conjugated rabbit anti-mouse IgG polyclonal antibody
- pBG1805 encoded proteins – won’t be bound by the mouse monoclonal antibody, but will be bound by the HRP-conjugated rabbit anti-mouse IgG polyclonal antibody
76
What is HRP?
- HRP-catalyzed reaction amplifies the signal and generates a visible product
- Horseradish peroxidase – attached to the secondary antibody and catalyzes reaction that produces a blue colored product
77
What steps are involved in westernblots?
- A multi-layer “sandwich” of filter papers and sponges encloses the gel and the PVDF membrane within a transfer cassette
- The cassette with its “sandwich” is inserted into the transfer apparatus
- Black plate of the sandwich is oriented toward black side of the cassette holder
- The electric current transfers proteins from the gel to the membrane
- When the transfer is done the membrane is removed from the cassette and a series of washes and incubation steps are performed
78
Describe the washing steps of western blot?
Blocking step – membranes are incubated with 5% non-fat milk
• PVDF membranes will bind proteins non-specifically. Milk contains high concentrations of casein proteins which binds and saturate these low affinity non-specific sites on the membranes
Primary antibody binding step
• Primary antibody is a mouse monoclonal antibody that recognizes the V5 epitope
• Drain the primary antibody from the membrane and wash the membrane several times in TBS buffer before proceeding to the next step
Secondary antibody is a rabbit polyclonal antibody that:
• Recognizes the constant region of the mouse IgGs
• Binds to S. aureus Protein A
Detection step
• Blot is incubated with substrates for HRP
• Blot is incubated until reaction products appear – time may vary based upon the level of expression of the yeast cells and the concentration of protein in our extracts
• Blue-black bands indicate the position of epitope-tagged proteins
Molecular weight standards have different colors and can therefore be used to accurately estimate the MW of your protein
