In Vivo Gene Cloning - The Use Of Vectors Flashcards
What are the two ways in which a gene can be cloned for medial or commercial use
In vivo - transferring the fragments to a host cell using a vector
In vitro - using the polymerase chain reaction
How do we use sticky ends to
Combine DNA from different sources
Restriction endonuclease cuts out a dna fragment
DNA from other source is added ( DNA from other source cut with same restriction endonuclease)
DNA Ligase joins the two sections
Making recombinant DNA
Why are sticky ends important
Because provided the same restriction is used, we can combine the DNA of one organism with that of any other organism
What are the sequences of DNA that are cut by restriction endonucleases are called
Recognition sites
How is the DNA fragment prepared for insertion
It involves the addition of extra lengths of dna.
What happens for the the transcription of any gene to take place
the enzyme that synthesise mRNA (rna polymerase) must attach to the DNA near a gene
What’s the binding site for rna polymerase
Region of DNA, known as promoter
What is essential if we want our dna fragment to transcribe mRNA in order to make a protein
That we attach it to the necessary promoter region to start the process
What’s a terminator
releases rna polymerase and ends transcription.
Why do we need a terminator
To stop transcription at the appropriate point
What is the vector used for. Give an example
Transport the DNA into the host cell
A plasmid
Where are the endonucleases used to do in the plasmid
They are used at one of the antibiotic resistance genes to break the plasmid loop
The restriction endonuclease used for the plasmid is the same as the one that cut of the DNA fragment. What does this ensure
That the sticky ends of the opened up plasmid are complimentary to the sticky ends of the DNA fragment.
What happens when the DNA fragments are mixed with with opened up plasmids
They become incorporated into them
Where they are incorporated, the join is made permanent using the enzyme DNA ligase. These plasmids now have recombinant dna
What happens during transformation
Introduction of DNA fragment into suitable host cell
Describe transformation
It involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions.
The calcium ions, and changes in temperature, make the bacterial membrane permeable, allowing plasmids to pass through the cell surface membrane into the cytoplasm
What are the reasons for not all bacterial cells possessing the DNA fragments with the desired gene for the protein.
Only a few bacterial cells take up the plasmids when the two are mixed together
Some plasmids will have closed up again without incorporating the DNA fragment
Sometimes the DNA fragment ends join together to form its own plasmid
The first task is to identify which bacterial cells have taken up the plasmid. How do we do this
Using the fact bacteria have evolved mechanisms for resisting the effects of antibiotics, typically by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium. The genes for the production of these enzymes are found in plasmids
Describe the process of finding out which bacterial cells have taken up the plasmids entail using the gene for antibiotic resistance, which is unaffected by the introduction of the new gene
All the bacterial cells are grown on a medium that contains the antibiotic ampicillin
Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance
These bacterial cells are able to break down the ampicillin and therefore survive
The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die
What do marker genes do
They identify whether a gene has been take up by bacterial cells. They all involve using a second, separate gene on the plasmid.
How is the second gene easily identifiable for one reason or another. For example what…?
It may be resistant to an antibiotic
It may make a fluorescent protein that is easily seen
It may produce an enzyme whose action can be identified
What does replica plating do
It identifies those cells with plasmids that have taken up the new gene
It uses the other antibiotic resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene.
What’s a positive of replica plating
It is possible to identify living colonies of bacteria containing the required gene
How are fluorescent markers used from the transfer of a gene from a jellyfish into the plasmid
The gene in question produces a green fluorescent protein. (GFP
The gene to be cloned is transplanted untinthr centre of the GFP gene.
Any bacterial cell that has been take up the plasmid with the gene that is to be cloned will not be able to produce GFP
Bacterial cells that have not taken up the gene will continue to produce GFP and to fluoresce. Unlike the cells that have not taken up the gene, these cells have taken it up will not fluoresce
As the bacterial cells with the desired gene are not killed, there is no need for replica plating.
Results can be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce. This makes the process more rapid
