4e Flashcards
whatis recombinant DNA technology for genetic engiennering
where Gene of interest is cut using restriction enzymes and pasted into a vector such as the plasmid of a bacteria
Bacterial genesfunction
bacterial genes can be used to insert genes into bacteria to repurpose ribosomrs
Plasmids
Plasmids are circular DNA that are able to replicate independently to the chromosome.
bacterial transformation process
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
advantages of using plasmids as vectors
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
Process of making a recombinat chromatid-how are recombinant chromatids made?
/transformaiton of bactiera
=recombinant plasmid
- The recombinant plasmid is placed into the bacteria. Not all will take up the plasmid
6.
Selection of bacteria that have been transformed
What does it mean when bacteria is tranformed
Some bacteria will take up the plasmid??
In order for bacteria to take up the plasmid, some techniques are used:
Heat shock
electroporation
what is heat shock
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
Electroporation
an electrical current is passed through the solution allowing the plasma membrane to become more permeable allowing the plasmid to enter
Selection of transformed bacteria/ how to determine if transformation has been succesful
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
identification of cellsIdentification of cells that have successfully incorporated the recombinant plasmid
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 –
how is resistance to ampillicin created
Antibiotic-resistance gene such as ampR – encodes resistance to ampicillin
gene can easily be indentigied when determing the identication of the cells/if transomfraiton was succesful
a gene that produces coloured or fluorescent proteins using gfp gene
How are target proteins produced
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
The transformed bacteria are cultured and induced to produce the target protein, what can be produced
insulin can be produced to treat diabetes
structure of insulin
Insulin has a quaternary structure – made up of secondarytwo polypeptide chains. One chain is known as “alpha”, the other “beta”
Uses of bacterial transformation for protein production
insulin for diabetes
Erythropoietin to treat anaemia
Growth hormone
Interferon for treatment of some cancers
Hepatitis B vaccine
function of insulin
Plays a role in regulating blood glucose levels. and
Those with diabetes are unable to produce their own insulin
Plays a role in regulating blood glucose levels.
Those with diabetes are unable to produce their own insulin????
Steps involved in producing human insulin:
Creating the recombinant plasmid
Creating transformed bacteria
Producing Insulin protein
Processof producing insulin via bacteria transofmriaton
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
- 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
- Recombinant plasmid with lac Z is added to E.coli
- 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
- Membranes of selected bacteria are broken down. Insulin is extraced and cyanogen bromide is added to seperate include from β-galactosidase
- The 2 insulin chainsare mixed to make a quatnery structure. Disfulide bonds connects the 2 to make a functioning protein
how are transformed bacteria selected
selected based on their ability to survive in the presence of antibiotics that target cells lacking the recombinant plasmid.
