4e

Question 1

whatis recombinant DNA technology for genetic engiennering

Answer 1

where Gene of interest is cut using restriction enzymes and pasted into a vector such as the plasmid of a bacteria

Question 2

Bacterial genesfunction

Answer 2

bacterial genes can be used to insert genes into bacteria to repurpose ribosomrs

Question 3

Plasmids

Answer 3

Plasmids are circular DNA that are able to replicate independently to the chromosome.

Question 4

bacterial transformation process

Answer 4

Gene of interest is cut using restriction enzymes and pasted into a vector such as the plasmid of a bacteria

Plasmids are circular DNA that are able to replicate independently to the chromosome.

When the desired DNA is joined with the plasmid, the recombinant plasmid is inserted into the bacteria to allow proteins to be produced

Question 5

advantages of using plasmids as vectors

Answer 5

Easy to manipulate – due to small size

Carry a wide range of restriction enzyme sites

Recombinant plasmids replicate independently once they are inserted into a host bacterial cell

Question 6

Process of making a recombinat chromatid-how are recombinant chromatids made?

/transformaiton of bactiera

Answer 6

=recombinant plasmid

5. The recombinant plasmid is placed into the bacteria. Not all will take up the plasmid

6.
Selection of bacteria that have been transformed

Question 7

What does it mean when bacteria is tranformed

Answer 7

Some bacteria will take up the plasmid??

Question 8

In order for bacteria to take up the plasmid, some techniques are used:

Answer 8

Heat shock

electroporation

Question 9

what is heat shock

Answer 9

a technique to transform bacteria by
bacterial cells and recombinant plasmids are placed in an ice-cold solution containing Calcium ions.

The calcium ions help make the plasma membrane of the bacteria more permeable.

The solution is then heated to 37-42°C then cooled in ice to allow the plasmid to enter the bacteria’s cytoplasm

Question 10

Electroporation

Answer 10

an electrical current is passed through the solution allowing the plasma membrane to become more permeable allowing the plasmid to enter

Question 11

Selection of transformed bacteria/ how to determine if transformation has been succesful

Answer 11

To determine if transformation has been successful, cells are incubated at 370C so they can reproduce and form colonies.
Bacterial cells are grown on agar plates containing the antibiotic ampicillin. Cells that survive have successfully taken up the plasmid

Question 12

identification of cellsIdentification of cells that have successfully incorporated the recombinant plasmid

Answer 12

Identification of cells that have successfully incorporated the recombinant plasmid, the plasmid must have the following characteristics:

Antibiotic-resistance gene such as ampR

Gene can be easily identified –

Question 13

how is resistance to ampillicin created

Answer 13

Antibiotic-resistance gene such as ampR – encodes resistance to ampicillin

Question 14

gene can easily be indentigied when determing the identication of the cells/if transomfraiton was succesful

Answer 14

a gene that produces coloured or fluorescent proteins using gfp gene

Question 15

How are target proteins produced

Answer 15

The transformed bacteria are cultured and induced to produce the target protein. As the bacteria make lots of different proteins, the protein of interest is extracted and purified

Question 16

The transformed bacteria are cultured and induced to produce the target protein, what can be produced

Answer 16

insulin can be produced to treat diabetes

Question 17

structure of insulin

Answer 17

Insulin has a quaternary structure – made up of secondarytwo polypeptide chains. One chain is known as “alpha”, the other “beta”

Question 18

Uses of bacterial transformation for protein production

Answer 18

insulin for diabetes

Erythropoietin to treat anaemia

Growth hormone

Interferon for treatment of some cancers

Hepatitis B vaccine

Question 19

function of insulin

Answer 19

Plays a role in regulating blood glucose levels. and
Those with diabetes are unable to produce their own insulin

Question 20

Plays a role in regulating blood glucose levels.
Those with diabetes are unable to produce their own insulin????

Question 21

Steps involved in producing human insulin:

Answer 21

Creating the recombinant plasmid
Creating transformed bacteria
Producing Insulin protein

Question 22

Processof producing insulin via bacteria transofmriaton

Answer 22

Step 1: Creating the recombinant plasmid:

Plasmids contains ampR and tetR are used.

Insulin A and B from plasmid is cut using restriction enzymes and ligase is used to make recombinant chromatids.

Two plasmids used – one for Insulin subunit A, one for Insulin subunit B.

Step 2: Creating transformed bacteria

heat shock is used to transfmr bacteria and are plated in agar for selection

4. Bacteria is transferred onto agar plates containing ampicillin. Those that formed have taken up a plasmid (not necessarily recombinant plasmid).

Another test with tetracycline used. Colonies from ampicillin plate are grown on tetracycline plate.

The bacteria that survived on ampicillin but not tetracycline are selected.

EcoRI is used to cut the recombinant plasmd to insert lac .

This produces β-galactosidase enzyme that attaches to Insulin A subunit

6. Recombinant plasmid with lac Z is added to E.coli
7. Selection of bacteria containing lacZ gene. Those that have been transformed will appear blue due to β-galactosidase converting a compound (X-gal) from colourless to blue

Step 3: Producing Insulin protein and extraction

8. Membranes of selected bacteria are broken down. Insulin is extraced and cyanogen bromide is added to seperate include from β-galactosidase
9. The 2 insulin chainsare mixed to make a quatnery structure. Disfulide bonds connects the 2 to make a functioning protein

Question 23

how are transformed bacteria selected

Answer 23

selected based on their ability to survive in the presence of antibiotics that target cells lacking the recombinant plasmid.