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….
