Name fields that rDNA has a major impact with examples?
Medicine -
Agriculture-
What is the mail application of rDNA technology?
Production of proteins from a cloned gene
Give the basic procedure of production of proteins from cloned gene
obtain animal gene
insert into a vector
recombinant into bacteria
bacteria transcribe & translate animal gene
animal protein is produced
What are the 3 special signals an expression vector should have? Explain each.
promoter
-short sequence, where transcription begins, via delta subunit RNA pol. binds the promoter
terminator
- stops transcription here
- usually for stem loop structures
ribosome binding site
- nt seq. shortie
- recognized by ribosome
- initiation codon is a few nucleotides downstream
Why eukaryotic signals are not recognized by E. coli RNA pol? So what should be done to clone?
seq. s are diff
- place within the R & T under the control of bacterial expression signals
Explain as to why promoter is the most important & critical component of expression vector? 2 types?
Controls initial stages of expression - rna pol binds here
determine the rate at which RNA is synth.ed
determine the quantity of rProtein formed
- strong
- weak
Factors to consider when choosing a promoter for expression why?
should be a strong one - efficient
can be regulated - can control the amount of protein produced - or too much protein can become toxic
What are the 2 major types of regulation? explain each(regulator)
induction - expression is turned on in the presence of an inducer(usually the substrate)
repression - switched off in the presence of the regu. chemical
List several types of E.coli promoters and their inducers/repressors?
lac promoter - IPTG inducer, transcription of lac z gene
trp promoter - controls synth. of tryptophan
- repressed by tryptophan
- induced by 3 – β – indoleacrylic acid
tac promoter - hybrid b/w lac & trp
-induced by IPTG
lambda PL promoter - very strong promoter
- transcribe lambda protein
- repressed by cI protein
- heat sensitive
What is 3 expression signals combined to form? Draw a typical vector and where the foreign gene is inserted?
cassette
within R & T
What is a hybrid gene & how is it formed?
foreign gene fused to the initial part of an E.coli gene
What is a hybrid protein?
N-terminus consists of a few AAs of E.coli
What is the advantage of creating a hybrid gene-hybrid protein?
product is more stable
efficient translation
possible to engineer
- include a signal peptide in N-terminus-easy to export
- fus.proteins that can be recovered by affinity chromatography
