Lecture 3+4 - Vectors/PCR Flashcards
What is a plasmid?
Plasmids are circular DNA molecules found in bacteria that are replicated by the host’s machinery independently of the genome.
How is a plasmid replicated independently of the genome?
Plasmids can be replicated by host’s machinery independently of the genome by a sequence on the plasmid called ori (from ColE1), for origin of replication. Extra: ColE1 is a plasmid found in bacteria. Its name derives from the fact that it carries a gene for colicin E1 (the cea gene)
Are plasmids present in varying quantities in E. coli?
Yes, some plasmids are present in E. coli at 200-500 copies per cell, while others are present in only 1-2 copies.
What are selectable markers?
Selectable markers are used to distinguish host cells that have taken up vectors from host cells that have not, the vector should carry a select- able marker gene (usually an antibiotic resistance gene or the gene for an enzyme absent from the host cell). Selectable markers are genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Usually, the selectable markers provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.).
What does a bacteria require to grow on a medium containing antibiotics?
Bacteria will grow on medium containing antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. (For ampicillin, kanamycin, tetracycline, chloramphenicol, etc.)
What is pBR322?
pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, Davis, it was named after the Mexican postdoctoral researchers who constructed it. The p stands for “plasmid,” and BR for “Bolivar” and “Rodriguez.”
Who created pBR322?
Herbert Boyer in 1977.
What does pBR322 contain in it structure?
pBR322 contains
– origin of replication (ori and rop) from ColE1
– two antibiotic resistance genes: ampicillin (AMPR) tetracycline (TETR)
– there are 6 key restrict sites inside the ampR gene. – the plasmid is 4361 base pairs in length
– unique restriction enzyme recognition sites in AMPR and/or TETR
– the circular sequence is numbered so 0 is the middle of the EcoRI site (EcoRI is used as a restriction enzyme. It creates 4 nucleotide sticky ends with 5’ end overhangs of AATT.)
What are pUC plasmids?
pUC19 is one of a series of plasmid cloning vectors created by Messing in 1982. It is a circular double stranded DNA and has 2686 base pairs.
pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on colour differences of colonies on growth media.
In pUC18, a piece of synthetic DNA has been inserted which contains recognition sites for a number of restriction enzymes. This multiple cloning site allows a considerable degree of flexibility in the choice of restriction enzyme. The plasmid has been engineered so that each of these sites is unique, i.e. it contains no other sites recognized by any of these enzymes.
What is α-complementation?
Alpha-complementation is a quick, easy, 1-step process of determining whether a transformed bacterial colony has plasmid+insert or not (The tetracycline resistance of pBR322 works, it is just time consuming).
Extra:
The key to alpha-complementation is the fact that the lac-Z gene product (B-galactosidase) is a tetramer, and each monomer is made of two parts - lacZ-alpha, and lacZ-omega.
If the alpha fragment was deleted, the omega fragment is non-functional; however, alpha fragment functionality can be restored in-trans via plasmid. Hence, then name alpha-complementation.
A strain of E. coli that has the deletion of the lac Z-alpha (lacZ DM15 works well as a genotype), and a plasmid with the lacZ-alpha fragment as the scorable marker (such as pBluescript or pCR2.1) is required.
If plain plasmid is successfully transformed into a cell, then the cell will express functional B-galactosidase. However, if the plasmid+insert is transformed into a cell, then it will express non-functional B-galactosidase (the lac Z-alpha will be disrupted with the insert gene product).
Plate the cells out onto selection media based on the selectable marker, IPTG (induces lac repressor to disengage), and X-gal (chromogenic substrate that yields blue product when cleaved by B-galactosidase) and the white colonies (non-functional B-galactosidase) are the ones with plasmid + insert; the blue ones have plain plasmid.
In alpha-completation what is the role of the lacZ gene?
The lacZ gene encodes an enzyme (β- galactosidase) that can cleave a colourless substrate (X-gal) resulting in galactose and 5-bromo-4-chloro- indoxyl derivate.
What happens to the indoxyl derivate in alpha complementation?
The indoxyl derivate spontaneously dimerises and oxidises to form an insoluble blue dye.
What is Blue/White selection?
The blue-white screen is a screening technique that allows for the detection of recombinant bacteria in vector-based molecular cloning experiments.
DNA of interest is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal.
Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies.
What colour will E. coli carrying the mutant lac Z gene (lacZΔM15) appear on ampicillin and X-gal containing plates?
E. Coli carrying the lacZΔM15 mutation and pUC18 will be blue on ampicillin and X-gal containing plates (α- complementation)
What will occur if a DNA fragment is inserted into the multiple cloning site of pUC18?
Insertion of a DNA fragment into the MCS will disrupt the opening reading frame of the α-peptide
