L2 Flashcards
what are BAC vectors based on which plasmid
F plasmids; which are used to clone large inserts of DNA
define vector
DNA molecule that assists gene transfer ( in bacteria, vectors are plasmids and viruses that carry and replicate foreign DNA in the host cell)
define Replicon
nucleic acid molecule capable of autonamous replication (including the origin)
what are the 3 types of replicons
plasmids, chromosomes, viruses
define chromosome
a DNA replicon that is essential for the growth of the cell under normal conditions
define plasmid
autonomously replicating, extrachromosomal, DNA molecule
define virus
a particle consisting of nucleic acid and protein that must infect a living cell to replicate and reproduce ; capable of horizontal transmission
what are plasmid vectors
- almost all based on pMB1 origin or replication
- all have selectable markers
- most have a lac alpha region and MCS
- most have SP6/T3/T7 bacteriophage promoters adjacent to MCS for making RNA transcripts of inserts
- used for cDNA library construction
- used for subcloning small fragments for RE mapping, sequencing, construction of probes, mutagenesis
- basis for constructing shuttle vectors and expression vectors
true or false, shuttle vectors allow genes to be moved between hosts
true
what do shuttle vectors consist of
typically consist of two plasmids combined to allow genes to be shuttled between e.coli and another host cell
name 3 types of shuttle vectors
- gram negative bacteria; rhizobium
- gram positive bacteria; B.subtilis
- integrating mammalian vectors
what are plasmid expression vectors
- have higher expression
- expression conditional (inducible)
eg. lac promoter induced by IPTG
eg. T7 promoter with temp. sensitiveT7 polymerase gene
what are the benefits of fusion protein
for higher stability, solubility sometimes, easy purification
what are the implications of fusion protein
proteins often insoluble, inclusion bodies that need resolubilising; post translational modifications are different from mammals
true or false, plasmid vectors are excellent for small fragments, but are inefficient for cloning large fragments
true….
what E.coli vectors can carry and replicate large DNA fragments; how large are their inserts
bacteriophage lambda vectors (9-23kb)
cosmid vectors (15-40kb)
yeast artifical chromosome (YACS) 100-300 kb
bacterial artfiicial chromosome 100-150 kb
true or false, BACs are the vector of choice for genomic libraries
true
why are BACs the vector of choice for genomic libraries
- good size of insert
- high stability
- use E.coli rather than yeast
- technically quite difficult to make
what is bacteriophage lambda
- best studied bacterial virus
- has 48 kb linear DNA genome with 12 bp cohesive termini
- 15 kb central region, non essential (9-23 kb inserts )
- highly efficient in vitro packaging system using cos sites
- highly efficient transfection of cells
- forms plaques rather than colonies
what are cosmid vectors
plasmid + cos site (40 kb inserts)
efficient packaging and transfection, replicates as plasmid but unstable
true or false, BACs are plasmids with replication origin from F plasmid of E.coli
true
true or false, BACs have low copy number (1-4 per cell), stable inheritance via genes for replication and partition
true
how large are the inserts of BACs
100-150 kb
insert size of bacteriohage lambda
9-23kb
insert size of cosmid vectors
15-40 kb
insert size of YACs
100-300 kb
what are YACs
- can be used to clone very large fragments
- completely artificial
- have telomerases
- have selectable marker
- can clone 1Mb
