Supplementary Practical Flashcards
What is the purpose of the supplementary practical
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
What are the 4 expression systems used
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
What were the 6 samples and the controls of bacterial expression systems
A cytoplasm A periplasm B cytoplasm B periplasm C total D total
How were samples removed from E. coli after exponential growth with iptg and cloning of vector in
Bacteria pelleted
Lysed via sonication
Centrifugation separated the periplasm and cytoplasm and the protein contents
Why is it easier to purify proteins from periplasm
Less protein translocate there
Before we do sds, how did we determine how much protein we should load to gel
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
Because bsa was diluted to x10. What did we have to do to determine conc of our protein sample
X 10 after excel analysis
What does sds do go protein
Causes a negative net charge by interacting with aa
Allows movement to anode in polyacrylamide gel
What is added to sds to break protein disulfide bonds
DTT and heat(stops folding)
What causes colour change in the sds psge
The Bradford reagent (reacts with protein basic residues)
What is added to samples before going into polyacrylamide gel
Bromophenol blue and loading buffer (keeps in tank via glycerol, edta, dtt,sds,tris)
What is added to the gel to visualise proteins
Instant blue via reacting with basic residues
Why was it necessary to use the bsa standard against optical density
To determine conc of protein we have. Don’t want to load too much (contamination of wells) or too little on the gel
What do you do to determine size of protein after sds
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
How do you work out Rf value
Distance moved by protein / distance moved by dye front (clumps at bottom)
Which expression systems yields the most protein
Sample B periplasm (one with periplasmic peptide tag)
A and B samples were induced by iptg how
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)
Why would multiple bands show
Because no purification techniques
What is the coding region called where insert has been added in A and B which is induced by iptg
pET21a
Why would sample B periplasm have largest protein amount
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
What is mobility directly proportional to
Log10 mass of protein
What happens to the novel gene before insertion into a vector then transformation
PCR amp
Then cut by ecor1 and inserted into vector pET21a or other
How are bacteria selected for which take up plasmid
Amp resistance
And white blue selection (a and b are white)
How is plasmid recovered to be then transferred into a bacteria with T7 polymerase
Miniprep
Which phase of bacterial growth is iptg added
Exponential phase
What are the 2 gels in sds page
Upper stacking gel (with wells)
Main gel (8.8ph)
What is the % difference in polyacrylamide between upper stacking and main gel
Upper stacking gel has larger pores as lower percentage allows all proteins to enter
Which component of loading buffer keeps ph constant
Tris
Why is edta added to loading buffer
Chelates mg and ca
What is added to polyacrylamide gel to visualise proteins
Instant blue via reaction with basic amino acids
