Flashcards in Test II Deck (34):
Give an outline of another method for isolation of DNA, which will give better purity and quality of genomic DNA?
-Lysis buffer: allows the release of the cells contents, including the DNA
-Resuspend in water
94 C=denaturing, 30 sec
44 C= annealing, 15-30 sec
72 C= Extending, 2 min
Cycle length= 30 rounds (depends on how much you want)
Tm-primer melting temp
Ta=annealing temp, usually 5 C less that Tm
What % of agarose and what buffers are typically used for gel electrophoresis of genomic DNA and PCR products?
0.7-1% agarose (depends on size)
Buffer: TAE, TBE
1.5-2 % agarose
Buffer: super buffer, TAE, TBE
Why should you purify your DNA insert before cloning?
To remove unincorporated dNTPs and primers.
-Purify the DNA to improve the efficiency
-The cleaner the DNA, the better
With the ligation reaction, what are we trying to achieve?
Ligation=the act of joining. Enzymatic reaction that joins two biomolecules with a covalent bond
Ligase makes a continuous phosphate backbone. It puts the PCR product of DNA into the vector
Whats the purpose of shaking your culture?
To make the culture aerobic. Adds air to the colonies
What is the function of X-gal of the LB+ medium?
-The vector contains the lacZ gene that codes for both subunits of the b-galactosidase enzyme. The enzyme is able to act of the substrate X-gal which is a modified galactose sugar.
-When X-gal is metabolised it is converted from colourless to blue
What is the function of IPTG on the LB+ medium?
-Inactivates the lacZ repressor, therefore inducing transcription of the lac operon.
Why is heat shocking the cells at 42 C used in bacterial transformation?
Open pores in the cell membrane. Moves plasmid from outside the cell to inside
Calculate the transformation efficiency (in cfu/µg of DNA) assuming 200 cfu were obtained using 0.001 ng of plasmid DNA`
0.001/1000= 1 x10^-6 µg
200/1 x10^-6 = 2 x10^8 cfu/µg (no. of colony forming units)
How many bands are in your plasmid preparations?
1 band, shows DNA is pure- no contamination as there are no other bands
If multiple bands:
Nicked open circle
Why is the genomic bacterial DNA not co-extracted or precipitated during the isolation of plasmid DNA?
gDNA is heavier and bigger than plasmid DNA/ gDNA also attracts proteins. The plasmids stay in solution
-When you spin it down, the heavy stuff (gDNA) goes to the bottom while the plasmid stays in solution.
Why do we use Pst1 and Nco1?
-They are compatible and can be used in the same buffer
-Not all restriction enzymes are compatible in the same buffer, they may not be able to be used in recombination
-Could of potentially used Eco1 cause it cuts in both places but it is known to have multiple cut sites
-In theory we could use any of the restriction enzymes but they need to work together
-Pst1 and Nco1 are on different sides so we can chop it out
What are the cut sites for Nco1 and Pst1?
Nco1= 37 bp
Pst1= 88 bp
Why do you think its necessary to add two different antibiotics to the broth?
Shows that the cell has both the pLys S plasmid and the GDHA plasmid being expressed. Together they should allow resistance to both antibiotics in the broth and their growth is selected for.
To make sure both plasmids stay in the bacteria. Providing resistance to ampicillin and chloraphenicol.
Why did you add IPTG to the broth after 4 hours of incubation?
IPTG inactivates the lac Z repressor therefore inducing transcription of the lac operon.
-Want the cells to get to a certain level of growth and then want to induce the protein GDHA.
-IPTG overrides the expression to produce to the protein of interest. Causes the increase expression of T7 polymerase
-It is added after 4 hours so the cells have reached a certain number. Want the cells to be in the exponential phase
-If we add it too soon, won't get enough of the protein
Why are there both blue and white colonies?
-LacZ has been disrupted producing an inactive enzyme due to the insert. This produces white colonies
-There is no insert and the enzyme is produced, results in blue colonies.
Transformation is not 100% efficient
Which coloured colonies are most likely to have the insert and why?
The insert is cloned into the lacZ gene sequence. This disrupts the gene producing an inactive lacZ enzyme
What is the approx. size of the amplified insert?
Give the diagram of the pBluescript II SK(+) cloning vector, describe some theoretical primers for amplifying the cloned insert
T7 Forward Primer and GDHA reverse primer. The T7 forward primer is in the vector while the GDHA primer is in the actual sequence
-Need one primer in the insert and one in the vector to ensure the insert is in the right orientation
-We have the insert but we don't know what orientation it is.
-Picking specific primers we can confirm the orientation or the PCR product
What are the function of :
The His tags
= T7 polymerase binds to the T7 promoter. Its a recognition site on the plasmid. The protein binds and initiates transcription of the gene of interest and the histidine residues. IPTG induces T7 polymerase, binds to promoter.
=His tags can be used to bind to the expressed protein. Any protein of interest with the His tag will stick to the Ni2+ column. Ni2+ binds to the His tags
=Multiple cloning sites
Designed with many different cut sites for restriction enzymes. Allows very specific manipulation using restriction enzymes. Allows manipulation of vector and insert
-where vector is inserted.
=Stops T7 RNA polymerase
What is special about the E.col BL21 (DE3)pLysS cell used for gene expression?
-pLys is a plasmid that produces the protein T7 lysozyme. It blocks T7 RNA polymerase. T7 polymerase makes the protein of interest from our plasmid
-Encodes chloramphenicol resistance
-Mutations in proteases. Less proteases present that destroys exogenous proteins
-Modifications to cell wall and membrane. Membrane is modified to allow cell to more easily lyse. Makes cells more prone to lyses with cold and heat shock.
In what structural form is the expressed gdhaA gene synthesised in the E.coli host cell?
-Conditions inside the cell are reducing so protein is in a linear form
-Normally proteins need chemical conditions for folding e.g. oxidation.
Given the size of gdhA gene of ~1362 bp, what is the theoretical size of the expressed protein?
Does this agree with the MW of your expressed protein?
No it doesn't.
-modifications (change weight)
-Histidine tags (adds weight)
The actual is small than the theoretical.
-The pLysS plasmid carried by the BL21(DE3)pLysS strain produces T7 lysozyme to reduce basal level expression of the gene of interest.
-pLysS confers resistance to
-Bacterial cells which have been treated for uptake and production of plasmid DNA.
-Can be prepared by washing with salt solutions and heat shocking or using organic solvents or electroporation.
Is the process where DNA/vector is introduced in bacterial cells
-It can then be replicated and the DNA expressed
-The DNA can then be isolated for downstream applications
-Artificial transformation is when competent cells are produced capable of exogenous DNA uptake.
-Requires chemically competent cells
-Uses divalent cations (e.g. CA2+) to increase permeability of the bacterial cell wall
-Increases likelihood of DNA acquisition.
-Includes heat shock step to increase DNA uptake
-Electrical field to the competent cells in suspension
-Introduces holes in the membrane
-The compromised cell allows exogenous DNA to pass through into bacterium.
-Gets plasmids into the cell using electric shock
-High efficiency of transformation but does kill some cells.
pGEM T easy vector
-Provides with cloned insert gdhA in DH5⍺ cells
-Used to isolate the gdhA gene for ligation into a cloning vector
-Also used for plasmid isolation and digestion
-T tailed vector, modified to have sticky ends, has T overhangs
-The PCR product has Adenines at the ends
-DNA ligase will put the PCR product into the vector due to complementary pairing
pBluescript II SK+ cloning vector
To ligate gdhA and transform into DH5⍺ cells
pIVEX2.4d transformed into E.coli BL21 (DE3) pLysS cells
Protein Expression Vector
-Contains His-tags, provided with cloned insert gdhA in BL21 cells also contains pLysS plasmid
-Used for proteins expression and purification
-Produces the protein T7 lysozyme, blocks T7 RNA polymerase. T7 polymerase makes the protein of interest in our plasmid
What was used to:
(a) Align sequences
(a) Cluster omega
Then did BLAST to see protein.