Molecular cloning II Flashcards
What are the key steps involved in generating a cloning vector?
- Adding inserts into a vector
- Adding the vector to host cells with transformation
- Amplifying the vector
- Purification of the cloning vector for analysis
- Detecting inserts in vector
What is a restriction nuclease ?
A restriction enzyme, an enzyme produced mainly by certain bacteria, that cleaves DNA at specific nucleotide sequences.
What are the properties of restrictoin nucleases?
- DNA recognition sequence is usually 4-8 bp
- Different restriction nucleases recognize different sequences
- The enzymes are named after the organisms from which they were isolated, e.g. EcoR1
- The cuts may result in blunt or sticky-ends
- The sticky-ends may have overhanging 5’ or 3’ends
- The average distance between cutting sites is determined by how long the recognition sequence is and the probability of finding each nucleotide
Different types of restriction enzymes gnerate different ends: state the different ends that can be generated
- Blunt ends
- Sticky ends
Adding inserts to a vector-Restriction Enzymes cloning:
Describe ligation
A reaction mediated by ligases where the insert covalently links to the vector.
- Insert is released from the donor plasmid using restriction nucleases
- Recipeint plasmid cut open (digest)
- Ligation occurs to merge the insert with the recipient plasmid
Adding vectors to host organisms:
Describe chemical transformation
A simple procedure that uses calcium ions and temperature shift to open up the bacterial cell wall which then allows the constructs to enter the cytoplasm of the bacterial cell.
Adding vectors to host organisms:
Describe electroporation
Using an electric current to open up the cell wall of bacteria, yeast or plant cells which allows the contracts to move into the cytoplasm.
Detecting inserts in vectors in Bacteria
- Antibiotic resistance (against tetracycline)
- Reporter genes
- Blue/White colour screening→ Use restricion enzymes to target the β-gal gene (breaking it) so if the vector is present it can’t express β-gal so no blue colour in the colony
- CcdB
Describe β-galactosidases’ mechanism of action
- β-galactosidase, also called lactase, beta-gal or β-gal, is:
glycosidehydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides through the breaking of a glycosidic bond.
- An active enzyme may be detected using artificial chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, X-gal.
Draw a diagram to show the amplification and Isolation of cloning vectors
- Obtain the pellet containing chromosomal DNA, the plasmid and protein following lysis and centrifugation
- Renature the pellet
- Follwoing further centrifugation, the chromosomal DNA, protein and plasmid are left in the supernatant
- Chloroform/phenol treatment to separate the plasmid and protein
- Plasmid will be in the supernatant and protein in the pellet
- Supernatant (plasmid) treated with alcohol to enable it to be used for furhter analysis
Analysis of the cloning vectors-Electrophoresis (Agarose Gel)
Three main samples
- Uncut plasmid
- control- cut insert
- Cut vector
TRUE or FALSE: All cloning vectors can be expressed
FALSE
Not all cloning vectors can be expressed!
- Both the structural gene and promoter were cloned on the same segment of DNA →Gene expression
- Only the structural gene was cloned
A promoter is provided by the plasmid →Gene expression
No promoter present in the plasmid →NO Gene expression
What is an expression vector?
A specifically designed to place a cloned gene under control of a plasmid-borne promoter.
In practice, the gene under investigation is normally first cloned in a general cloning vector and then transferred to an expression vector.
