Supplementary Practical

Question 1

What is the purpose of the supplementary practical

Answer 1

Evaluating expression systems in ecoli effect on levels of exp of the transporter protein and allow targeting to specific cell compartments in ecoli via sds page

Question 2

What are the 4 expression systems used

Answer 2

A - plasmid with insert and pet21a vector with a T7 promoter regulation by iptg (upregulates lacZ)
B- plasmid with insert but with a periplasmic tag (signal peptide) directing it to periplasm
C- gene inserted into a non expression vector puc18 = no gene txn
D- ecoli with plasmid pet21a with no novel gene

Question 3

What were the 6 samples and the controls of bacterial expression systems

Answer 3

A cytoplasm
A periplasm 
B cytoplasm 
B periplasm 
C total 
D total

Question 4

How were samples removed from E. coli after exponential growth with iptg and cloning of vector in

Answer 4

Bacteria pelleted
Lysed via sonication
Centrifugation separated the periplasm and cytoplasm and the protein contents

Question 5

Why is it easier to purify proteins from periplasm

Answer 5

Less protein translocate there

Question 6

Before we do sds, how did we determine how much protein we should load to gel

Answer 6

Using a standard curve of BSA (bovine serum albumin)
And Bradford reagent

Determined Optical density (y) of them next to their known conc (x)

Given optical density for our protein samples. Determines conc via excel

Question 7

Because bsa was diluted to x10. What did we have to do to determine conc of our protein sample

Answer 7

X 10 after excel analysis

Question 8

What does sds do go protein

Answer 8

Causes a negative net charge by interacting with aa

Allows movement to anode in polyacrylamide gel

Question 9

What is added to sds to break protein disulfide bonds

Answer 9

DTT and heat(stops folding)

Question 10

What causes colour change in the sds psge

Answer 10

The Bradford reagent (reacts with protein basic residues)

Question 11

What is added to samples before going into polyacrylamide gel

Answer 11

Bromophenol blue and loading buffer (keeps in tank via glycerol, edta, dtt,sds,tris)

Question 12

What is added to the gel to visualise proteins

Answer 12

Instant blue via reacting with basic residues

Question 13

Why was it necessary to use the bsa standard against optical density

Answer 13

To determine conc of protein we have. Don’t want to load too much (contamination of wells) or too little on the gel

Question 14

What do you do to determine size of protein after sds

Answer 14

Measure mw ladder distance Rf value against its log 10 kda (x)

Then use this on excel to determine size of proteins via their Rf

Question 15

How do you work out Rf value

Answer 15

Distance moved by protein / distance moved by dye front (clumps at bottom)

Question 16

Which expression systems yields the most protein

Answer 16

Sample B periplasm (one with periplasmic peptide tag)

Question 17

A and B samples were induced by iptg how

Answer 17

Iptg binds to the lac repressor on lac operator. Changes it’s conformity to release it for expression of the novel gene via T7 promoter allowing T7 polymerase to work (inside exp vector pET21a induced by lac operator)

Question 18

Why would multiple bands show

Answer 18

Because no purification techniques

Question 19

What is the coding region called where insert has been added in A and B which is induced by iptg

Answer 19

pET21a

Question 20

Why would sample B periplasm have largest protein amount

Answer 20

Induced inside expression vector pET21a, and also has a periplasmic peptide signal tag on the novel gene. This facilitates easier recovery of protein because directed to periplasm

Question 21

What is mobility directly proportional to

Answer 21

Log10 mass of protein

Question 22

What happens to the novel gene before insertion into a vector then transformation

Answer 22

PCR amp

Then cut by ecor1 and inserted into vector pET21a or other

Question 23

How are bacteria selected for which take up plasmid

Answer 23

Amp resistance

And white blue selection (a and b are white)

Question 24

How is plasmid recovered to be then transferred into a bacteria with T7 polymerase

Answer 24

Miniprep

Question 25

Which phase of bacterial growth is iptg added

Answer 25

Exponential phase

Question 26

What are the 2 gels in sds page

Answer 26

Upper stacking gel (with wells)

Main gel (8.8ph)

Question 27

What is the % difference in polyacrylamide between upper stacking and main gel

Answer 27

Upper stacking gel has larger pores as lower percentage allows all proteins to enter

Question 28

Which component of loading buffer keeps ph constant

Answer 28

Tris

Question 29

Why is edta added to loading buffer

Answer 29

Chelates mg and ca

Question 30

What is added to polyacrylamide gel to visualise proteins

Answer 30

Instant blue via reaction with basic amino acids