L5 Flashcards
true or false, pUC vector allows propagation of molecule as ssDNA strand
true
how to synthesize new genes in vitro
- determine desired amino acid sequence
- translate into desired DNA sequence
- synthesize overlapping oligonucleotides
- anneal together and ligate
- clone assembled oligos into plasmid using sticky ends
- sequence to confirm
what methods are used to manipulate DNA sequences
- cloning
- PCR
- sequencing
- oligonucleotide sythesis
- site directed mutagenesis
how do you express high value proteins in fermentation
- need high level promoter (to make lots of mRNA)
- inducible expression- temperature shift, chemical induction (ethanol/methanol)
- optimise translation- codon usage of host organism
- optimise stability
- optimise solubility
- post translational modification
How is DNA introduced in bacteria
electroporation or Ca2+ treatment
How is DNA introduced in yeast
Li+ treatment or Electroporation
How is DNA introduced in animal cells
Ca ppt of DNA
lipid encapsuation
microinjection
How is DNA introduced in plant cells
PEG
electroporation
biolistics
A.tumefaciens
true or false, if ori is present DNA replicated (all)
true
if ori is absent and C/O is present what happens
DNA integrates by homologous recombination (10^-3 to 10^-4)
what happens when ori is absent and there is a NHEJ
DNA integrates at random sites (10-3 to 10-4)
true or false, results from with ori, C/O and NHEJ are stable transformants
true
what does kanamycin do
inactivates aminoglycoside antibiotics (kanamycin, neomycin, gentamycin)
true or false, kanamycin resistance is the most common selectable marker for eukaryote cells
true
true or false, E.coli kanamycin resistance gene from Tn903: inactivates all of the mentioned antibiotics via phosphorylation
true
where is genticin used
animal cells and yeast
where is kanamycin used
plants
what does genticin G418 inhibit
synthesis of cytoplasmic ribosomes
what does kanamycin ihibit
chloroplast ribosomes
true or false, eukaryote signals are used to express the kan gene
true
in animals what is the promoter and the protein formed for kan gene expression
SV40 late promoter
SV40 poly A
in plants what is the promoter and the protein formed for kan gene expression
CaMV 35S prom
CaMV poly A
where are the signals derived from for kan gene expression
dsDNA of viruses, active in wide host range, direct high level of constitutive expression of transcript (can be turned on or off)
what is a reporter gene
gene used to monitor level of expression from promoters
- no background activity in host cells
what are common reporter genes
lac
gus
luc
gfp
what is lac used for
bacteria, yeast, animals
what is gus used for
plants
what is luc used for
yeast, animals, plants
what is gfp used for
yeast, animals , plants
true or false, lac and gus are standard reporter genes
true
true or false, luc is the most sensitive reporter gene
true
true or false, gfp is a non destructive reporter gene
true
true or false, yeast is the best model eukaryote for gene transfer
true
why is yeast the best model for eukaryote gene transfer
has set of vectors for many purposes
- integration
- high copy plasmid
- low copy plasmid
what are the commercial applications of yeast
hepatitis B vaccine
fermentation
What are the two kinds of integration events
1.) random:
occur anywhere on any chromosome ( by DNA repair systems called NHEJ: non homologous end joining, no DNA homology needed)
- allows gene addition but not gene replacement
- only dominant genes produces a phenotype
- variable expression
2.) targeted:
occur at sites of sequence identity (by homologous recombination system –> control of integration site)
- allows gene replacement as well as addition
- both dominant and recessive genes produce phenotypes, including null alleles)
true or false, targeted integration is the most powerful system
true
true or false, targeted integration is predominant in yeast
true
how do you do target integration
via homologous recombination to replace ORF in yeast
how do species without targeting integration reduce gene expression
by introducing dsRNA , inductions of a sequence specific nuclease that specifically degrades that target RNA, results in dominant negative transgene
