supplementary pracitcal

Question 1

chemical hazard

Answer 1

bacterial lysate
acrylamine in the gel is a neurotoxin and carcinogen

Question 2

SDS- PAGE separates proteins by

Answer 2

molecular weight

Question 3

what does SDS-PAGE stand for

Answer 3

sodium dodecyl sulphate - polyacrylamide gel electrophoresis

Question 4

insertion of a novel gene into different expression vectors can increase

Answer 4

expression of the protein

Question 5

how much of our samples do we need to add to SDS gel

Answer 5

can work out from BSA standard curve

Question 6

4 E.coli bacterial strains
stain A

Answer 6

A - expression plasmid with insert, expression regulated by IPTG

Question 7

periplasm

Answer 7

space between two membranes

Question 8

strain B

Answer 8

B - as for A but with a periplasmic tag (tag attached to N-terminal and translocated protein to periplasm)

Question 9

strain C

Answer 9

expression under bacterial control (no promoter in plasmid)

Question 10

IPTG

Answer 10

switches on the promoter region, increasing expression of protein

Question 11

stain D

Answer 11

no expressed protein

Question 12

6 samples to analyse

Answer 12

AC - strain A cytoplasm
AP - strain A periplasm
BC - strain B cytoplasm
BP - strain B periplasm
CT - total protein strain C
DT - total protein strain D

Question 13

how to get the samples from the bacteria

Answer 13

centrifugation
sonication - sound waves breaks and disrupts the bacteria

Question 14

why periplasm

Answer 14

not many proteins in this region so if we traffic our protein there itll be more easier to purify our protein and easy to identify it

Question 15

BSA

Answer 15

BSA is the universally accepted reference protein for total protein quantitation

Question 16

graph shows the optical density of known BSA concentrations

Answer 16

compare optical density of our proteins using bradford reagent to work out protein concentration

Question 17

10x dilution factor

Answer 17

so concentration needs to be multiplied by 10 to get results

Question 18

SDS-PAGE

Answer 18

separation of proteins in an acrylamide gel by molecular weight
proteins denatured by the presence of SDS that gives the protein a negative charge therefore move upward towards the anode (red)

Question 19

DTT breaks

Answer 19

disulphide bonds

Question 20

Rf =

Answer 20

distance moved by protein / distance moved by dye front

Question 21

plot Rf of y axis

Answer 21

plot log10 MW on X axis

Question 22

the beer lambert law
A = Ecl

Answer 22

A = absorbance
E = extinction coefficient
C = concentration
L = path length (cm)

Question 23

want 20 microliters so do 20/protien conc from BSA graph

Answer 23

5.24mg/ml = 5.24 ug/ul
need 20ug so 20/5.25 = 3.819ul (4 ul)

Question 24

T7 promoter is controlled by

Answer 24

IPTG

Question 25

novel gene is in what expression vector in strain A

Answer 25

pET21a

Question 26

novel gene is in what non- expressing vector in stain C

Answer 26

pUC18 (non expressing wont make our protein)

Question 27

pET21a

Answer 27

ampicillin resistance
lacl - lac repressor
has own promoter
multiple cloning site (Multiple cloning sites are a feature that allows for the insertion of foreign DNA without disrupting the rest of the plasmid)

Question 28

IPTG binds to lac repressor and pulls the repressor off the lac operator allowing it to work allowing full expression T7 and protein