Recombinant DNA Practical

Question 0

What does recombinant DNA technology allow ?

Answer 0

Easy manipulation and amplification of DNA sequences

Question 1

What is recombinant DNA also known as ?

Answer 1

Genetic engineering or molecular biology(this is a broad term)

Question 2

What has recombinant DNA technology been exploited for ?

Answer 2

To isolate new genes
To produce drugs
Gene therapy- introducing healthy genes into disease affected tissues
Expressing drug targets for pharmacological drug screening

Question 3

What are restriction enzymes ?

Answer 3

Symmetrical proteins which recognise specific palindromic base sequences
The specific base sequence is known as the recognition site
They cleave a specific phosphodiester bond on each strand

Question 4

What naturally produces restriction enzymes ?

Answer 4

Bacteria so they can protect themselves against bacteriophage infection

Question 5

What is the restriction- modification system?

Answer 5

It is a system used by bacteria to protect themselves
They produce methylase enzymes to modify their DNA to prevent them being cleaved by their restriction enzymes
The methylase enzyme causes the backbone of the bases to rotate

Question 6

Why are restriction enzymes used in recombinant DNA technology ?

Answer 6

To cleave DNA so we can move/delete/insert fragments of DNA to and from a plasmid

Question 7

What are sticky ends/ cohesive ends ?

Answer 7

They are overhangs of bases produced by restriction enzymes
If the sticky ends are complementary then they can ligate back together using DNA ligase enzymes to reform the phosphodiester bond

Question 8

What are the 3 types of restriction enzymes ?

Answer 8

Type 1, 2 and 3
1- have specificity, modification and restriction subunit
2- recognises palindromic sequences in DNA and requires magnesium for cleavage
3- contains 2 subunits, m which recognises and modifies DNA and r which has nuclear actions

Question 9

How do the type 2 restriction enzymes work ?

Answer 9

It has a catalytic core which is activated when the enzyme undergoes conformational changes when it is activated by the recognition of its specific recognition site
It cuts the DNA producing 3’-OH and a 5’-P end

Question 10

What is a plasmid ?

Answer 10

Double stranded closed circular DNA molecule present in bacteria and yeast
Separate from chromosomal DNA

Question 11

What do plasmids contain and what does it enable ?

Answer 11

Origin of replication

This enables them to replicate themselves so they can be passed into daughter cells

Question 12

What is conjugation ?

Answer 12

Process in which bacteria can exchange plasmids with one another

Question 13

What is transformation ?

Answer 13

Process in which bacteria can be encouraged to take up plasmids from their environment

Question 14

Why do bacteria contain plasmids ?

Answer 14

Because these plasmids often contain genes which confer them with advantageous properties such as antibiotic resistance

Question 15

What advantageous properties can plasmids provide bacteria with and how do they work ?

Answer 15

Antibiotic resistance - genes encode proteins which can either metabolise the antibiotic or modify it ensuring it is inactive
Decrease entry or increase efflux of drugs- antibiotic resistance

Question 16

What is a constitutive promoter ?

Answer 16

It is a plasmid which is continually switched on

Therefore once it is present in the bacteria it transcribes and translates the protein encoded by the gene

Question 17

What is a stringent plasmid ?

Answer 17

Plasmids with low copy numbers within the cell

Question 18

What are relaxed plasmids ?

Answer 18

Plasmids with high copy numbers within cells

• these are more useful in recombinant DNA technology
• these have been deliberately engineered to contain multiple cloning sites to allow insertion of the gene of interest

Question 19

What processes can be used to add sticky ends to a gene of interest so it can be inserted into a plasmids ?

Answer 19

PCR or ligation of adaptors

Question 20

Why is using 2 restriction enzymes better when inserting DNA into a plasmid ?

Answer 20

Because the enzyme cuts the plasmid at 2 different points so the ends are not complementary so it prevents them rejoining

Question 21

What is added after the restriction enzyme if only one restriction enzyme is used ?

Answer 21

To prevent complementary sticky ends reforming a phosphatase enzyme is added after the restriction enzyme
It catalyses the removal of the 5’ phosphate group which is necessary for DNA ligase to ligase the DNA back together

Question 22

What does it mean by a bacterial cell being competent ?

Answer 22

It means the bacterial cell wall must be weakened/punctured - this enables it to take up the plasmid
Encouraged by heat-shocking the cells at 42 degrees with the plasmid and then chilling on ice

Question 23

Not all the bacterial cells will take up the plasmid so how do you known which ones have ?

Answer 23

The ones which have taken up the plasmid will contain the gene of interest and therefore they will confer antibiotic resistance whereas those they didn’t take up the plasmid won’t confer resistance
- so the cells are grown in a petri dish containing ampicillin and the cells which grow will be the ones that have taken up the plasmid

Question 24

Why is it relatively easy to produce large amounts of engineered plasmid constructs ?

Answer 24

Because when bacteria divide they pass on their plasmids

Question 25

What substance is often used in chemical transformation and why ?

Answer 25

Calcium chloride | It affects the cell wall and may also bind the DNA to the cell wall

Question 26

What are the benefits of preparing large amounts of engineered plasmids from e.coli ?

Answer 26

- can be used transfer prep into other cells such as mammalian cells so the gene of interest is expressed and so it can be used to analyse drugs - if the gene of interest is placed downstream of the inducible promoter then the bacteria can be induced so the gene of interest can be transcribed and translated

Question 27

Why is agarose gel electrophoresis used in plasmid analysis ?

Answer 27

To test if the gene of interest has been successfully taken up into the plasmid by checking the size of the construct - in our experiment we used a 3000bp plasmid and a 1000bp gene of interest so if the plasmid had taken up the gene of interest then electrophoresis would have shown 2 strips , 1 at 3000bp and one at 1000bp

Question 28

When using gel electrophoresis how is the DNA visualised ?

Answer 28

It is stained with ethidium bromide which intercalated between the bases of DNA and fluoresces under uv light of 260-300nm

Question 29

Why is it bad to run a circular plasmid on agarose gel and what happens when the plasmid is removed from e.coli ?

Answer 29

Circular DNA is supercoiled DNA which has been supercoiled by DNA gyrase- this configuration runs through the gel very rapidly Once plasmid is removed it unwinds as DNA strands become nicked just to even just one phosphodiester bond breaking - nicked DNA runs much slower than supercoiled DNA

Question 30

What is the run speed of linear DNA ?

Answer 30

It is I between supercoiled and nicked

Question 31

What fragments are further down the gel plate and why ?

Answer 31

Smaller fragments are because they are able to run through the gel more rapidly because they get less tangled within the pores

Question 32

What is not the relationship between DNA size and rate of migration ?

Answer 32

It is not a direct inverse relationship

Question 33

What happens if you increase the concentration of agarose gel ?

Answer 33

Causes a decrease in the pore size which increases resolution Using a higher concentration is better for distinguishing bonds that are closer together

Question 34

What is the difference between cutting the plasmid with 1 or 2 restriction enzymes ?

Answer 34

Using 1 will linearise the plasmid which allows comparison based on size with no bps lost but cutting with 2 restriction enzymes means the plasmid will be slightly smaller because some of the bps will have been cut out

Question 35

Where is the multiple cloning site in pBluescript plasmid ?

Answer 35

Placed right in the middle of DNA which codes for lacZ-alpha gene

Question 36

What does the lacZ-alpha gene produce ?

Answer 36

Beta galactosidase enzyme | Under control of an inducible promoter so it is only produced when the inducers IPTG is present

Question 37

What does the production of beta galactosidase do ?

Answer 37

It rescues the activity of an inactive enzyme and hydrolyses disaccharides

Question 38

In blue/white screening what substrate is adding the show the effects of beta galactosidase and why ?

Answer 38

X-gal It is a colourless compound but when it is hydrolysed by beta galactosidase it produced a blue pigment called 5,5'-dibromo-4,4'-dichloro-indigo Therefore E. coli containing the plasmids, x-gal and IPTG will form blue colonies whereas if x-gal and IPTG are not present then white colonies are formed

Question 39

What happens if the gene of interest is inserted into the multiple cloning site of pBluescript?

Answer 39

This means the lacZalpha gene will be disrupted so beta galactosidase will not be produced so even if x-gal and IPTG are present blue colonies can be produced so white colonies form instead This is used to see if ligation of the inserted gene has been successful

Question 40

What is pBluescripts replication capabilities ?

Answer 40

Replicates about 500 cells per cell

Question 41

Why did we use a 1% agarose gel instead of the usual 0.08% ?

Answer 41

Because although the resolution is better in 0.08% the gel can be floppy and break easily

Question 42

What growth occurred and explain why when the plate contained no antibiotic and was a control ?

Answer 42

Clear bacterial growth which was impossible to distinguish between the colonies Because they were able to grow even though they didn't contain the plasmid because there was no antibiotic present in the growth medium

Question 43

What growth occurred and explain why when the plate contained ampicillin, IPTG, X-gal and a plasmid with no gene of interest ?

Answer 43

Blue colonies Because the multiple cloning site in the pBlurscript was not disrupted so it produced beta galactosidase which hydrolysed x gal and produced the blue pigment The bacteria were able to grow in the ampicillin because they had a plasmid which conferred antibiotic resistance

Question 44

What growth occurred and explain why when ampicillin, IPTG, x-gal and plasmid b containing gene of interest was present ?

Answer 44

White colonies Because the gene of interest present in pBluescript was present in the multiple cloning site so it disrupted the production of beta galactosidase so even though x gal and IPTG were present the x gal couldn't be hydrolysed because beta galactosidase wasn't produced therefore x gal remained colourless

Question 45

What growth occurred and explain why when the plate contained ampicillin, IPTG, x-gal and no plasmid ?

Answer 45

No bacterial growth Because the bacteria hadn't taken up a plasmid and therefore they didn't contain a gene which conferred antibiotic resistance so due to the presence of ampicillin it prevents the bacteria growing