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Concepts 2 Flashcards

1
Q

What are plasmids?

A

small circular DNA molecules that replicate independently of the host chromosomes

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2
Q

What features do plasmids constructed in the lab normally have?

A
  • Usually have a predesigned cloning site and an amp resistance gene
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3
Q

How are plasmids mass produced and isolated?

A

Plasmids are transformed into bacteria and may be purified and isolated

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4
Q

How are plasmids edited?

A

Enzymes are used to insert desired pieces of foreign DNA into plasmids

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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

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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
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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

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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

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9
Q

What is the purpose of the beta-lactamase gene?

A

amp resistance

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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

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11
Q

What is the purpose of C-temrinal tags?

A

downstream of gene of interest, allows for purification and isolation in western blotting

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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

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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
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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
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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
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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
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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.

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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.

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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
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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
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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
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22
Q

What are restriction endonucleases?

A

enzymes that cleave double stranded DNA at defined positions

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23
Q

What is restriction mapping?

A

site-specific restriction endonucleases are used to identify DNA molecules

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24
Q

What is the role of REs in bacteria?

A

• Restriction endonucleases are part of a bacterium’s defense against invaders

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25
Q

Describe restriction modification systems in bacteria.

A
  • 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
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26
Q

What type of REs are used in molecular biology?

A
  • 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
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27
Q

How frequently do common lab (6 cutters) cut DNA? What are other aspects of their function?

A
  • 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
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28
Q

What are sticky ends and what are they useful for?

A
  • sticky ends: 4 unpaired hydrogen bonds on nitrogenous bases hanging off the 5’ end
  • Sticky ends are useful in generating recombinant DNA molecules
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29
Q

Describe EcoR1.

A
  • EcoRI recognition site is a palindrome with an axis of symmetry
  • EcoRI dimer binds sequence and catalyzes double strand cleavage
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30
Q

How did we perform restriction mapping?

A
  • 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
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31
Q

What is yeast transformation?

A

uptake of foreign DNA by a cell changes its phenotype

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32
Q

Describe the yeast cell wall. What must cross this barrier during transformation?

A
  • 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
Q

What is the transformation procedure in general?

A

Cells are treated with chemicals and submitted to a mild heat shock

34
Q

What are the three chemicals necessary to transform yeast?

A

Polyethylene glycol, Lithium acetate, Single-stranded DNA salmon sperm

35
Q

What is the purpose of polyethylene glycol in transformation?

A

Possible effects on membrane structure – may help DNA adhere to the cell wall (mechanical)

36
Q

What is the purpose of lithium acetate in transformation?

A

Monovalent cations generally enhance uptake of DNA (electrical)

37
Q

What is the purpose of the salmon sperm in transformation?

A
  • 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
Q

How are transformed cells selected for?

A

Transformed cells are isolated on selective media with no uracil in it
(Complementation of ura3 mutation in our strain and in plasmids)

39
Q

What gel was the RE digest run on?

A

1% agarose gel

40
Q

What was in the master mix for our transformation?

A

(200uL of Polyethylene Glycol + 50uL of Lithium Acetate + 2uL of B-mercaptoethanol (reduces disulfide bonds)

41
Q

Describe the process of replica plating.

A
  • 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
Q

What is yeast’s preferred carbon source?

A

glucose

43
Q

How are glucose and galactose different?

A

o Glucose and galactose are isomers and only differ in the orientation of a hydroxyl group at the C4 position

44
Q

What happens when galactose replaces glucose as a carbon source?

A
  • 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
Q

How are cells able to promote the transition to galactose processing genes?

A

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
Q

What is the transcriptional activator for Gal1,10, and 7?

A

Gal4p transcriptional activator

47
Q

Do other TFs bind GAl-1?

A

yes

48
Q

What are the domains of the gal 4 protein

A

 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
Q

Describe how the gal promoter is regulated.

A
  • 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
Q

How does galactose impact Gal4p function?

A
  • 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
Q

Describe transcription regulated by the Gal-1 promoter in the presence of differenct carbon sources.

A
  • 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
Q

How are proteins extracted from our yeast cells without being degreaded?

A

Our procedure depends on the rapid and efficient extraction of yeast proteins by denaturing the cell wall

53
Q

What happens during denaturation of our protein?

A
  • 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
Q

What are the denaturing conditions that proteins are extracted under?

A
  • 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
Q

What is SDS? Describe structure.

A
  • Sodium Dodecyl sulfate is a denaturing detergent with multiple roles in extract preparation
56
Q

Describe structure of SDS

A
  • 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
Q

What does SDS do?

A
  • 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
Q

How are SDS gels visualized?

A
  • 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
Q

What are antibodies?

A
  • Antibodies are proteins produced by lymphocytes in vertebrate immune systems
  • Antibodies bind tightly and specifically to their target molecules
60
Q

What are antigens?

A
  • antigens are foreign substances that stimulate the immune system to product antibodies
  • Bacteria, viruses, allergens and other large molecules are antigens
61
Q

What are epitopes?

A

the portions of the antigen recognized by the immune system – most antigens have multiple epitopes

62
Q

Describe the structure of antibodies.

A
  • 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
Q

Where exactly do antigens bind?

A
  • Antigens bind hyper variable regions at the tips of Fab fragments
  • Antigen binding has been compared to a lock and key fit
64
Q

What determines processing of antibody antigen complexes?

A

A limited number of Fc regions determine how other cells will process antibody antigen complexes

65
Q

Describe the antibodies used in molecular biology.

A

have a gamma heavy chain (IgG immunoglobins)

66
Q

What are polyclonal antibodies?

A
  • Polyclonal antibodies are semi-purified fractions derived from animal serum (antiserum)
  • Polyclonal antibodies may recognize multiple epitopes on the same antigen
67
Q

What are limitations of polyclonal antibodies?

A
  • A limited amount of serum can be obtained from an animal

- It is often useful to have antibodies with a defined specificity

68
Q

What are monoclonal antibodies and what are they produced by?

A
  • Monoclonal antibodies recognize a single well-defined epitope
  • Produced by cultured hybridoma cells
69
Q

What are hybridoma cells?

A
  • 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
Q

Give an outline of the monoclonal antibody harvesting process.

A
  • 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
Q

What are the two types of antibodies used in a western blot?

A
  • 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
Q

What is used to visualize the signal in our western blots?

A

Horseradish peroxidase (HRP) produces a colored reaction product

73
Q

What do epitope tags do?

A
  • 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
Q

Problem: How can proteins encoded by pBG1805 and pYES2.1 plasmids be visualized on the same gel?

A

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
Q

Describe what is expected to be recognized in pBG1805 and pYES2.1 encoded proteins.

A
  • 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
Q

What is HRP?

A
  • 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
Q

What steps are involved in westernblots?

A
  • 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
Q

Describe the washing steps of western blot?

A

 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

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