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61 Cards in this Set
- Front
- Back
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Which gene is studied in this lab?
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The Zap 70 protein kinase
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Which domain of this gene is studied?
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The tandem SH2 domains
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What is zap 70 required for?
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The proper initiation and amplification of the activation signal induced by the stimulation of the T lymphocyte antigen receptor.
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What are SH2 domains known to mediate?
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Phosphotyrosine-dependent protein protein interactions
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How big is the SH2 domain fragment?
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780 bp
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Which plasmid is used in this exercise and how big is it?
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The pGEX-3T prokaryotic vector is 4979 bp.
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What type of protein is generated?
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A GST (Glutathione S transferase) fusion protein
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What enzyme site was created at both sites of the fragment?
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BamH1
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What type of insertion is the zap-70 derived fragment into the pGEX-3T vector?
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Non-directional
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What does the origin of replication allow the plasmid to do?
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To be replicated as a separate entity
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What does the origin of replication determine?
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The copy number
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What is the name of the origin of replication in the pGEX-3T vector?
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pBR322 Ori
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How many copies does pBR322 allow pGEX-3T to have?
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Only a few
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In the case where the origin of replication only allows the protein to have a few copies, what treatment is helpful for the cells?
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A protein synthesis inhibitor to stop cell division and allow the DNA polymerase to replicate only the plasmid, therefore increasing the number of plasmids per cell.
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What is an example of a protein synthesis inhibitor?
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chloramphenicol
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What allows the selection of transformed cells over bacteria that do not contain the plasmid?
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An antibiotic resistance gene
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How does the plasmid allow its host cell to grow in antibiotic containing plates?
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It has an ampr element that encodes beta-lactamase, an enzyme capable of hydrolysing ampicillin
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What allows the insertion of foreign DNA in close proximity to the promoter?
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A Multiple Cloning SIte (MCS)
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What type of sequence is the MCS?
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A series of unique restriction sites that are not found anywhere else on the plasmid.
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What are four things that pGEX-3T has that allow it to express fusion proteins in bacterial cells?
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The Ptac promoter, GST gene, lacI repressor, and stop codons
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What does the Ptac promoter allow?
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Allows cellular bacterial enzymes to bind to the plasmid and initiate translation of the GST fusion protein encoded in part by the plasmid and the inserted fragment.
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What does the GST gene allow?
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The purification and detection of the fusion protein.
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What is the substrate of GST and with what type of affinity does it bind?
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The substrate of GST is glutathione and it binds with high affinity.
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What is glutathione available as a conjugate with?
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Sepharose or agarose beads
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What is required to generate the correct fusion protein?
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The DNA fragment MUST be inserted in the same reading frame as the GST gene
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What does the product of the lacI repressor have the ability to do?
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Bind to the Ptac promoter and prevent the cellular RNA polymerase from interacting with the promoter.
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What can the lacI repressor feature prevent?
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The expression of a potentiall toxic gene product.
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What can the inhibition of the Ptac promoter by lacI repressor be lifted by?
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IPTG (Isopropyl-beta-D-thiogalactopyranoside
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What is IPTG and how does it work?
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It binds to the repressor with very high affinity, lifting the repression and promoting transcription of the inserted protein of interest in the plasmid in stead of the beta-galactosidase it normally regulates.
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What does the presence of stop codons allow?
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The termination of translation immediately following the inserted DNA fragment.
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Even though the plasmid has stop codons, what is recommended for the cDNA fragment to have?
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Its own stop codon, to avoid the addition of unwanted amino acids
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What is screening?
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Identification of bacterial colonies containing recombinant plasmids
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What does the choic of a screening strategy depend on?
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The nature of the plasmid as well as the number of clones for screening
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What are five types of screening methods?
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-Restriction analysis of small-scale preparations of plasmid DNA
-alpha-complementation -insertional inactivation -colony lify assay and hybridization -PCR |
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What type of vectors does alpha complementation need?
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Vectors like pUC vectors that carry the coding sequence of the NH2 terminal 146 amino acids of the E coli beta-galactosidase gene.
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What is the requirement of the host cell for alpha complementation?
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It needs to express the COOH terminal portion of the beta-galactosidase gene.
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Where is the DNA fragment introduced in alpha complementation?
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Via a polycloning site within the beta-galactosidase coding sequence.
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How are blue colonies formed and what does this mean?
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Together, the NH2 end of the beta-galactosidase gene from the plasmid, and the COOH end from the host cell complement each other, resulting in Lac+ bacteria. Lac+ bacteria form blue colonies in the presence of X-gal.
This means the gene was not disrupetd and therefore there is no insert. |
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How do white colonies form?
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If the fragment was inserted into the vector, it would disrupt the beta-galactosidase gene and would prevent alpha-complementation.
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How does the technique of insertional inactivation work?
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In a vector that carries two antibiotic resistance genes, the fragment is inserted into one of the genes. Therefore clones will be resistant to the gene and sensitive to the antibitotic of the gene that was disrupted.
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What is the colony lift assay and hybridization technique used for?
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Simultaneous screening of a large number of colonies
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How does this technique work?
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Colonies are duplicated by laying a sterile filter on them. The filter is then placed on a Whatman that is saturated with SDS and then an alkaline solution and then a neutralising solution. It is then washed and incubated with a radiolabelled oligo specific for the insert.
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What type of filter is commonly used?
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Nitrocellulose or nylon
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What does SDS do?
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It is a detergent that lyses cells
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What do alkaline solutions do?
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Denature DNA
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How and on what type of bonds do restriction endonucleases act?
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They can hydrolyse the phosphodiester bond of nucleic acids
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What type of DNA can restriction endonucleases recognize?
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Short sequences (4-8) bases of unmethylated DNA
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Where can restriction endonucleases cut?
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Within the target sequence, or outside the recognised sequence
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What type of ends can restriction endonucleases generate?
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Blunt ends (same position on both DNA strands)
Cohesive ends (Overhangs are generates) |
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What is the definition of a vector?
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Plasmid, virus, or other vehicle used to carry DNA.
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What is the definition of transformation for bacteria?
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The aquisition of new genetic material
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What is the definition of transformation for eucaryotes?
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The conversion of a normal cell to a cancer cell
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What is the definition of competence?
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Ability of a bacterial cell to pick up DNA from its environment
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What does the 5' termini of a DNA strand contain?
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A phosphate group
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What does the 3' termini of a DNA strand contain?
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A hydroxyl group
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What direction is DNA read?
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5' to 3'
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What is the + strand?
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THe sense strand
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What is the - strand?
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The antisense strand
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What is the natural function of restriction enzymes for the host cell?
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To protect it from bacteriophages
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What is the size of the fragment generated by using Kpn1 for the RIGHT insertion? Wrong?
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Right orientation- 324 bp
Wrong- 488 |
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What is the size of the fragment generated using Xho1 for the RIGHT orientation? Wrong?
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Right- 488 bp
Wrong- 352 bp |
