Recombination and transformation Flashcards
what are plasmids
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
how can plasmids move in and out of bacterial cells
by rupturing the cell walls of the bacteria, they can even be transfered between different species of bacteria
what are recombinant plasmids
plasmids that have been genetically changed to include dna from another organism/species
what are recombinant plasmids called
vectors because they are transferring a gee from one organism to another
how are recombinant plasmids made
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
what is tranforming bacteria
the act of making bacteria contain forgein dna, a vector is used (e.g. a virus or plasmids)
what is heat shocking
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
what is the first step of transforming bacteria
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
what is the second step of transforming bacteria
the recombinant plasmids are added to a bacterial culture, they are heat shocked and the plasmids are taken up by some bacteria
what is the third step of transforming bacteria
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
why is transformed bacteria used for insulin production
transformed bacteria can be cultured and induced ro produce target proteins which can then be extracted and purified
what is insuliin
a protein hormone that is responsible for the regulation of blood glucose levels
why does insulin need to be synthesised artifically
people who suffer from diabetes do not produce insulin or do not respond to insulin so require insulin to be administered
what is the structure of insulin
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
what is the first step for creating the recombinant plasmids for insulin
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
what is the second step for creating the recombinant plasmids for insulin, after gene has been identified
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
how were insulin genes inserted into the plasmids different to the regular insulin gene
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
what is the first step of creating transformed bacteria for insulin
the created recombinant plasmids added to s solution of E. Coli bacteria and some of the recombinant plasmids were taken up by the bacteria
how was it determined if the bacteria had taken up the first round of recombinant plasmids for insulin (step 2)
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
why are the recombinant plasmids not resistant to tetracycline
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
what does the tetracycline (tetR) act as
a reporter gene
what is the third step of creating the transformed bacteria for insulin
the identified plasmids were cut open using ECORI to insert another gene called lacZ (minus its top codon)
why was the lacz gene added as a protein
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
recombination
joining dna together from different sources
transformation
changing a bacterium by introducing a plasmid
how can the specific gene to be inserted be made without introns
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
antibiotic
any of a number of compounds that inhibit bacterial cell growth in some way
why was lacZ added as a gene
the two genes (lacZ and insulin) will be transcribed and translated together as there is no stop codon
why was methionine (ATG) added to the start of insulin subunit a and b
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
what is the final step of creating the insulin protein
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
how are the a and b chains held together
by two di-suulfide bonds (strong covalent bonds that form between the sulfur in one base and cysteine in another)
in vitro
“in glass” a process carried out in a laboratory, in a petri dish, test tube, etc
what is the fourth step for creating iinsulin transformed bacteria (after lac z is added)
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.
what is the fifth step of creating transformed bacteria for insulin (how to check which bacteria contain recombinat plamids containing lacz and subunit)
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.
how come the transformed bacteria turned blue in the presence of xgal
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.
what is the sixth step for creating transformed bacteria for insulin (after bacteria containing lacz have been identified)
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.
what is the seventh step fotrr creating transformed bacteria for insulin (after protein hhas been exttracted)
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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