Genetic engineering Flashcards
What factor should be considered when choosing a host for protein synthesis
Do you want disulphide bonds, if so then protein should be secreted into the peri plasm, as cytoplasm is an oxidising environment.
mRNA stability
Codon useage
Why may you need to change the codon useage?
May be expressing protein not native to the species that it’s being expressed in, so will have different tRNA’s.
Same amino acids but different nucleotide sequence
Disadvantages of Ecoli vector
Requires cDNA (no introns)
Lacks much post-translational processing
Possible protein stability/solubility/toxicity issues
Examples of promoters
tac
T7 both these are regulated by the Lac operating system
ara
The lac operator and lac repressor
No expression: lac repressor is bound to the lac operator
Expression: Add ITPG changes conformation of the lac repressor and cause the lac repressor + inducer to move away. Allowing polymerase to bind to lac promoter transcribing T7 pol. Which in turn then transcribes the gene of interest
Both the T7 pol coding region and the gene of interest have the lac operator bound by the lac repressor when the gene isn’t being expressed.
Cells are allowed to grow before expression is turned on
RBS
Has secondary structure so could prevent access to the site or initiating ATG
Can be a problem when you expressing a protein which had a fusion parter at the C terminal
What does a signal sequence look like
Basic region-hydrophobic region-cleavage Region
Fusion protein/peptide and there affinity columns
Glutathione-S-transferase. glutathione
Maltose binding protein. Amylose from starch
Hexa-histidine tag. Metals (Ni/Co2+)
StrepTag II. StrepTactin (elute with desthiobiotin)
How can fusion tags be removed from protein
By incorporating a peptide sequence that codes a suitable site for a protease cleavage enzyme. At the gene cloning stage
Maltose binding site fusions
Use a protease cleavage site between the MBP and target protein, do the target protein can be separated from the MBP
Strains containing the T7 RNA polymerase genomic insertion are referred to as what strains?
DE3 strains
What expression system would you use to produce a protein for functional studies?
Mammalian
Mammalian expression systems
High level expression, but expensive to produce large quantities of cells
Constitutive or inducible promotors based on viral (CMV, SV40 MMTV) or mammalian (actin, heat shock proteins) promotors
How to get forge in DNA into a eukaryotic cells cultures in vitro?
Mikroinjection
Lipofection
Viral transfection
Tet/on Tet/off system
Tet-On: Engineered transcription activator protein (rtTA) binds to tetracycline responsive element (TRE) upstream of cDNA only in the presence of tetracycline-type antibiotic (doxycycline)
Tet vector must also be transfected into mammalian cells
Advantages of fluorescent protein in cellular imaging
Genetically encoded Live cell imaging Intrinsic fluorophore Multiple colours Can be used as tag for other proteins of interest
Disadvantages of fluorescent proteins
Low brightness
Complex photo physics such as dark States
Larger size
pH sensitivity
How to check weather an elated protein is pure
SDS-PAGE to check molecular mass and any other contaminating proteins
State a compound used to elute the protein from the column and the mode of action of this compound in bringing about protein eltution
Imidazole as it has a structure similar to histidine and can therefore compete to bind the Ni 2+
EDTA mode of action is to chelate the Ni 2+ removing it from the column and so the protein is also eluted
What is the name of the type of chromatography used to to purify a protein with a His tag
Immobilised metal affinity chromatography/Ni-NTA
How does the resin that is used in affinity chromatography column allow protein purification
The metal is able to bind divalent metal ions usually Ni 2 +
And then the histidines in the oligo his tag can co-ordinate with the free positrons of the metal
What can DNA foot-printing reveal
The nucleotide sequence of the binding site of a protein
What reagents are needed to perform a DNA-foot print analysis experiment
Radio labeled gene ( could be a regulatory sequence)
Protein ( that binds to the gene of interest)
DNase1
Co-immunoprecipitation
Treat living cells with a cross linking reagent (formaldehyde) to cross link protein with DNA sequence.
Lyse cells
Sonnicate the DNA to shear into fragments
Use antibody bound to beads against protein of interest to co-precipitate protein with gene.
Remove supernatant
Remove protein by reversing cross links/ digest protein using a protease
Sequence the DNA
The binding site will be common to all purified fragment
