Recombination and transformation

Question 1

what are plasmids

Answer 1

small, circular and very stable pieces of DNA found in bacterial cells annd can be easily engineered to contain restriction sites, promoters and other features that are useful in biotechnology

Question 2

how can plasmids move in and out of bacterial cells

Answer 2

by rupturing the cell walls of the bacteria, they can even be transfered between different species of bacteria

Question 3

what are recombinant plasmids

Answer 3

plasmids that have been genetically changed to include dna from another organism/species

Question 4

what are recombinant plasmids called

Answer 4

vectors because they are transferring a gee from one organism to another

Question 5

how are recombinant plasmids made

Answer 5

an endonuclease is choosen to cut upstream and downstream of the gene, the plasmid and gene of dna are cut with the same restriction enzymes to create complementary sticky ends, dna ligase is then used to assist the 2 different peices to join together

Question 6

what is tranforming bacteria

Answer 6

the act of making bacteria contain forgein dna, a vector is used (e.g. a virus or plasmids)

Question 7

what is heat shocking

Answer 7

when the bacteria is treated with cycles of cooling and heating, which beats up the external coating of the bacterria, making it easier for the bacteria to take up the plasmids

Question 8

what is the first step of transforming bacteria

Answer 8

the plasmid dna and the target dna are cut with the same restriction enzyme, dna ligase binds the target dna into the plasmid sna as they have complementary sticky ends, this generates a recombinant plasmid

Question 9

what is the second step of transforming bacteria

Answer 9

the recombinant plasmids are added to a bacterial culture, they are heat shocked and the plasmids are taken up by some bacteria

Question 10

what is the third step of transforming bacteria

Answer 10

the bacteria containing the recombinant plasmid, which will also contain a second gene for antibiotic resistance will survive on a culture containing the antibiotic and can be selected, these bacteria can then replicate to create many copies with the plasmid

Question 11

why is transformed bacteria used for insulin production

Answer 11

transformed bacteria can be cultured and induced ro produce target proteins which can then be extracted and purified

Question 12

what is insuliin

Answer 12

a protein hormone that is responsible for the regulation of blood glucose levels

Question 13

why does insulin need to be synthesised artifically

Answer 13

people who suffer from diabetes do not produce insulin or do not respond to insulin so require insulin to be administered

Question 14

what is the structure of insulin

Answer 14

a protein with quaternary structure consisting of two polypeptide chains known as alpha and beta subunits. to produce insulin we require two different recombinant plasmids and thus two different bacteria samples

Question 15

what is the first step for creating the recombinant plasmids for insulin

Answer 15

plasmid vectors were prepared which contain the ampR gene to encode for antibiotic resistance to ampicillin and tetR to encode for antibiotic resistence to tetracycline

Question 16

what is the second step for creating the recombinant plasmids for insulin, after gene has been identified

Answer 16

two plasmid vectors used, one used for the insulin subunit a and one for the insulin subunit b WITHOUT INTRONS (produced in a laboratory), using the ecoRI and bamHI, both plasmid samples, the insulin a subunit gene and the insulin b subunit gene are cut to form complementary sticky ends, DNA ligase re establishes the sugar phosphate backbone and creates the two different recombinant plasmids

Question 17

how were insulin genes inserted into the plasmids different to the regular insulin gene

Answer 17

the insulin genes were inserted into a different plamid (one for a subunit and one for b subunit) the insulin gene was created without introns and with an extra codon coding for methionine at the beginning of the insulin gene

Question 18

what is the first step of creating transformed bacteria for insulin

Answer 18

the created recombinant plasmids added to s solution of E. Coli bacteria and some of the recombinant plasmids were taken up by the bacteria

Question 19

how was it determined if the bacteria had taken up the first round of recombinant plasmids for insulin (step 2)

Answer 19

the bacteria cultures were spread and incubated onto agar plates containg the antibiotic ampicillin, colonies that formed were identified to have taken up a plasmid. these identified colonies were then spread onto agar platees containing tetracycline, those that were susceptable to tetracycline were collected

Question 20

why are the recombinant plasmids not resistant to tetracycline

Answer 20

those plasmids that also took up the insulin gene (subunit a/b) interupted the tetR gene (tetracycline resistence) as a regonition site was in this gene, cutting it, thus making the bacteria with the recombinant plasmids susceptable to tetracycline

Question 21

what does the tetracycline (tetR) act as

Answer 21

a reporter gene

Question 22

what is the third step of creating the transformed bacteria for insulin

Answer 22

the identified plasmids were cut open using ECORI to insert another gene called lacZ (minus its top codon)

Question 23

why was the lacz gene added as a protein

Answer 23

lac z produces a large enzyme (b beta galactosidas) when the recombiinant plasmids produce insulin, it will attached to this large enzyme (fusion protein) an increase in its size protects it from digestive enzymes within E.Coli that may destroy it

Question 24

recombination

Answer 24

joining dna together from different sources

Question 25

transformation

Answer 25

changing a bacterium by introducing a plasmid

Question 26

how can the specific gene to be inserted be made without introns

Answer 26

it can be done synthetically in a lab or usingg cDNA (copy DNA) which is made using the enzyme reverse transcriptase to transcribe mrna backwards into dna

Question 27

antibiotic

Answer 27

any of a number of compounds that inhibit bacterial cell growth in some way

Question 28

why was lacZ added as a gene

Answer 28

the two genes (lacZ and insulin) will be transcribed and translated together as there is no stop codon

Question 29

why was methionine (ATG) added to the start of insulin subunit a and b

Answer 29

it makes it possible to seperate the fusuin protein once it is out of the cell as there is no methionine in the insulin a or b chain

Question 30

what is the final step of creating the insulin protein

Answer 30

once the two subunits hhave bee extracted from the cell and the methionine is broken down, the two chains are purified and solubilized and are mixed together in vitro to form insulin through the disulfide bonds

Question 31

how are the a and b chains held together

Answer 31

by two di-suulfide bonds (strong covalent bonds that form between the sulfur in one base and cysteine in another)

Question 32

in vitro

Answer 32

“in glass” a process carried out in a laboratory, in a petri dish, test tube, etc

Question 33

what is the fourth step for creating iinsulin transformed bacteria (after lac z is added)

Answer 33

The recombinant plasmids containing lacZ were added to a new solution of E. coli bacteria and some of these new
recombinant plasmids were taken up by the bacteria.

Question 34

what is the fifth step of creating transformed bacteria for insulin (how to check which bacteria contain recombinat plamids containing lacz and subunit)

Answer 34

To determine which bathe E. coli were plated on agar plates containing ampicillin and X-gal. Colonies that grew and
were blue in colour were identified as containing recombinant plasmids due to the presence of ß-galactosidase. These
bacteria were capable of producing the insulin subunit proteins that were attached to ß-galactosidase.

Question 35

how come the transformed bacteria turned blue in the presence of xgal

Answer 35

The ß-galactosidase enzyme
was known to convert a compound called X-gal, which results in a compound changing from colourless to being blue
coloured.

Question 36

what is the sixth step for creating transformed bacteria for insulin (after bacteria containing lacz have been identified)

Answer 36

Transformed bacteria that contained the
recombinant plasmid were then placed into
conditions to exponentially reproduce before their
membranes were broken down, and the insulin
subunit and ß-galactosidase fusion proteins were
extracted.

Question 37

what is the seventh step fotrr creating transformed bacteria for insulin (after protein hhas been exttracted)

Answer 37

The compound cyanogen bromide was
added to break down the methionine that was
added at the start of the insulin gene. In doing so, it
separated the insulin subunit from the ßgalactosidase, allowing for the isolation and
purification of the insulin subunit.
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