1
Q
Plasmid Elements
A
- promoter: RNAP binding
- operator: controls expression, molecule binding induces DNA shape change
- RBS: ribosome binding site
- ATG
- gene
- stop signal
- terminator
2
Q
Promoters
A
- inducible promoters such as lac operon, arabinose, or galactose used
- lac I binds to operator preventing RNAP binding
- lac I constituitively expressed except in presence of lactose
- lactose binds lac I allowing gene transcription of lac z/y/a
3
Q
Optimisation of Expression
A
- switch on expression during mid exponential phase
- reduce temperature with increased inducer conc.
4
Q
Autoinduction
A
- relies on consumption of a carbon source in the media
- removes repression allowing induction
- switch on expression of protein
5
Q
Plasmid T7 promoter
A
- regulates T7 polymerase expression
- expression of recombinant proteins can be toxic due to leaky expression
- separate cloning of gene from expression
- T7 promoter inactive in E. Coli lacking T7 polymerase
- no nonspecific activation
- engineered cells with T7 polymerase in genome
- control of different promoters for T7 polymerase like lac
6
Q
TA Cloning
A
- cloning PCR fragments using A overhangs
- non directional
- sequence to identify which clones are in correct orientation
- incubate PCR product with Taq
- generate A overhangs in product complementary to plasmid T overhangs
- topoisomerase cleaves vector
- PCR product attacks covalent bonds between vector and RE
7
Q
Traditional Cloning
A
- uses RE and ligase
- cut sites can’t be present in gene
- double digest of PCR and vector
- complementary overhangs
- ligate with T4
- often inefficient
8
Q
Ligation Independent Cloning
A
- no restriction digestion
- phosphatase treatment or ligation needed
- PCR insert must have 15bp of identical sequence with each end of linearised vector
- cleave vector
- T4 polymerase removes nucleotides leaving an overhang
- gene anneals using complementary sequences
- transform into E. Coli for screening
9
Q
Fusion Proteins
A
- express protein of interest as a fusion protein
- linked to a tag sequence cleaved later on via protease
- cut site engineered into gene
10
Q
Eukaryotic Expression Systems
A
- bacterial systems cheaper/easier but not suitable for all proteins due to a lack of folding mechanisms, inclusion bodies, incorrect modifications/targeting, lack of correct lipids
- yeast, insect, mammalian, cell free
- consider gene source, specific protein requirements, downstream applications
11
Q
Pichia Yeast
A
- methylotrophic: uses methanol as carbon source
- AOX protein allows this (controlled by strong promoter)
- alcohol oxidase produced only when methanol present so used for recombinant protein production
- grows to high density
- selection markers used for selection/screening
- not plasmid based : integrated into genome
- can have high expression with multicopy incorporation
- plasmid cut and transformed into cell
- integrated into endogenous AOX
- linearised AOX non functional but made functional via integration
12
Q
Saccharomyces Yeast
A
- homologous recombination
- amplify gene with forward and reverse primers with extensions to regions within vector sequence
- mix with linearised vector
- recombination with extensions
13
Q
Insect Cell Systems
A
- recombinant baculovirus and cultured insect cells
- bacmid DNA contains all virus genetic information
- insert donor plasmid into bacmid
- E. Coli containing recombinant bacmid
- bacmid + GOI contain all information to produce complete virus and protein
- initial transfection of insect cells released virus particles
- particles used to infect insect cells and target protein then harvested
14
Q
Mammalian Cell Systems
A
- constitutive or inducible promoters
- standard cloning/HR cloning/TOPO cloning
- tags for investigating protein protein interactions
- FRET tags for monitoring location
15
Q
Transfection of Mammalian Cells
A
- liposome mediated uptake of plasmid DNA
- electroporation and calcium phosphate
- inefficient
- transient or stable depending on is your want short of long term production
16
Q
Cell Free Expression
A
- mixture of all necessary components with added DNA
- express proteins not possible in other systems
