Genetic engineering

Question 0

What factor should be considered when choosing a host for protein synthesis

Answer 0

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

Question 1

Why may you need to change the codon useage?

Answer 1

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

Question 2

Disadvantages of Ecoli vector

Answer 2

Requires cDNA (no introns)

Lacks much post-translational processing

Possible protein stability/solubility/toxicity issues

Question 3

Examples of promoters

Answer 3

tac
T7 both these are regulated by the Lac operating system
ara

Question 4

The lac operator and lac repressor

Answer 4

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

Question 5

RBS

Answer 5

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

Question 6

What does a signal sequence look like

Answer 6

Basic region-hydrophobic region-cleavage Region

Question 7

Fusion protein/peptide and there affinity columns

Answer 7

Glutathione-S-transferase. glutathione

Maltose binding protein. Amylose from starch

Hexa-histidine tag. Metals (Ni/Co2+)

StrepTag II. StrepTactin (elute with desthiobiotin)

Question 8

How can fusion tags be removed from protein

Answer 8

By incorporating a peptide sequence that codes a suitable site for a protease cleavage enzyme. At the gene cloning stage

Question 9

Maltose binding site fusions

Answer 9

Use a protease cleavage site between the MBP and target protein, do the target protein can be separated from the MBP

Question 10

Strains containing the T7 RNA polymerase genomic insertion are referred to as what strains?

Answer 10

DE3 strains

Question 11

What expression system would you use to produce a protein for functional studies?

Answer 11

Mammalian

Question 12

Mammalian expression systems

Answer 12

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

Question 13

How to get forge in DNA into a eukaryotic cells cultures in vitro?

Answer 13

Mikroinjection

Lipofection

Viral transfection

Question 14

Tet/on Tet/off system

Answer 14

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

Question 15

Advantages of fluorescent protein in cellular imaging

Answer 15

Genetically encoded
Live cell imaging
Intrinsic fluorophore
Multiple colours 
Can be used as tag for other proteins of interest

Question 16

Disadvantages of fluorescent proteins

Answer 16

Low brightness
Complex photo physics such as dark States
Larger size
pH sensitivity

Question 17

How to check weather an elated protein is pure

Answer 17

SDS-PAGE to check molecular mass and any other contaminating proteins

Question 18

State a compound used to elute the protein from the column and the mode of action of this compound in bringing about protein eltution

Answer 18

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

Question 19

What is the name of the type of chromatography used to to purify a protein with a His tag

Answer 19

Immobilised metal affinity chromatography/Ni-NTA

Question 20

How does the resin that is used in affinity chromatography column allow protein purification

Answer 20

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

Question 21

What can DNA foot-printing reveal

Answer 21

The nucleotide sequence of the binding site of a protein

Question 22

What reagents are needed to perform a DNA-foot print analysis experiment

Answer 22

Radio labeled gene ( could be a regulatory sequence)

Protein ( that binds to the gene of interest)

DNase1

Question 23

Co-immunoprecipitation

Answer 23

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

Question 24

State the rules that you need to follow for a successful quick change mutagenesis experiment

Answer 24

At least 25nt long (25-40)
Central mutations 
Ideally G/C at 3' ends of primer
High Tm >75 to 78
Complementary primers
No need for 5' phosphorylation step as there is no ligation step