Bacterial Transformation (13.2) Flashcards
Describe plasmids
Small, circular DNA molecules found in bacterial cells
State what plasmids are often used as
Vectors
Provide an alternative term to describe vectors
Carriers
State the role of plasmids in DNA exchange
Employed as vectors to move target DNA from one organism to another.
State what genes can be inserted into
Plasmids
State by what process genes from unique organisms can be incorporated into bacterial cells
Bacterial transformation
State what the inserted gene can be replicated by
Self-replicating properties of plasmid & bacterial cells
State what an inserted gene can express once replicated
Proteins for which it codes
Describe restriction enzymes
Naturally-occurring bacterial enzyme which can be employed in genetic engineering
Provide an alternative term for restriction enzymes
Endonucleases
State what endonucleases have enabled
- cutting of DNA into smaller, more usable fragments
- isolating regions of interest
State what endonucleases compose
Bacterial cell’s defence system
State what endonucleases target as part of the bacterial cell’s defence system
Foreign DNA that may enter the cell
Describe bacteriophages
A virus that infects a bacteria
State what each restriction enzyme generally targets
Specific sequence of nucleotides (usually 4-6 base pairs in length)
State what a specific sequence of nucleotides targeted by restriction enzymes is referred to as
Recognition site
State what occurs every time a restriction enzyme passes its recognition site
Breaks the phosphodiester backbone once on each DNA strand
State whether or not bacterial cells cut up their own DNA
No.
State what the name of the enzyme in bacteria which adds a methyl group to a specific nucleotide is
Methylase
State where methylase adds a methyl group to a specific nucleotide in bacteria
Within the recognition site of the restriction enzymes made by the bacteria
State what adding specific nucleotides within the recognition site of the restriction enzymes made by the bacterium blocks
Restriction enzymes from binding to and cutting the baterium’s own DNA
State the 2 types of retstriction enzymes
- sticky-end restriction
2. blunt-end restriction
State how sticky-end restriction enzymes leave DNA fragments
Overhanging ends
State where sticky-end restriction enzymes cut the DNA backbone
At different locations on each strand within the recognition site
State what cutting the DNA backbone at different location on each strand within the recognition site results in
A staggered cut, leaving ‘sticky ends’
State what the ‘sticky ends’ left by the sticky-end restriction enzyme action are composed of
Two fragments have bases exposed
State what the exposed bases left by the ‘sticky end’ restriction enzyme can form
Complementary base pairs
State how exposed bases left by the ‘sticky end’ of a restriction enzyme interact
Hydrogen bonding occurs between nucleotides of other DNA molecules that have complementary sticky ends
Provide 1 example of a sticky-end restriction enzyme
EcoRI
Describe blunt-end restriction enzymes
A restriction enzyme that leaves clean-cut ends because it cuts both strands of the DNA molecule at the same location within the recognition site
Describe sticky-end restriction enzymes
A type of restriction enzyme that makes a staggered cut in DNA to leave fragments with overhanging ends
State how blunt-end restriction enzymes leave DNA fragments
Clean-cut ends
State where blunt-end restriction enzymes cut the DNA backbone
At same location on each strand within the recognition site
Provide 1 example of a blunt-end restriction enzyme
HaeIII
State what restriction enzymes cleave
The phosphodiester bonds in double-stranded DNA
State what the base-pairing ability of sticky ends allows DNA from different species to do
Ligate
State what ligation between different species DNA forms
Recombinant DNA molecules
Describe ligation
The process of joining two fragments of DNA using DNA ligase enzymes
State what small variations in DNA sequences occur within a population are termed
Polymorphisms
Describe ligases
A group of enzymes that join fragments of DNA or RNA in a process called ligation
State what DNA ligase joins
Fragments of DNA
State what RNA ligase joins
Fragments of RNA
State the function of DNA ligase
Joins fragments of DNA
State the function of RNA ligase
Joins fragments of RNA
State whether or not DNA ligase is also required to join segments of newly replicated DNA and to repair breaks in DNA molecules
Yes
State what bond ligases forms between two DNA fragments
Phosphodiester bond
State whether or not ligation of sticky-end fragments is specific
Yes.
State why the ligation of sticky-end fragments is specific
Exposed bases of sticky-end fragments first bind to complementary bases
State whether or not ligation of blunt-end fragments is specific
No.
State why the ligation of blunt-end fragments is random
Any two fragments can join if they come in contact and DNA ligase is present to join them
State why blunt-end fragments are difficult to use in DNA manipulation processes
Ligation is random
State whether or not plasmids replicate independently of the chromosome
Yes. Plasmids replicate independently of the choromosome.
State what is formed when DNA from two different sources is joined
Recombinant DNA
State why scientists create recombinant DNA
To clone particular genes
State what scientists commonly use as a vector when creating recombinant DNA
Plasmids
State what scientists insert into the plasmid to produce a recombinant plasmid
Target DNA
State where the recombinant plasmid is placed after formation
Bacterial cell
State what occurs when the recombinant plasmid is introduced back into the bacterial cell
Self-replicating system of the plasmid & cell replicates plasmid genes
State 3 reasons supporting the use of plasmids as vectors when creating recombinant DNA
- small size (ease of manipulation)
- carry range of restriction enzymes
- self-replicate independently once placed in host bacterial cell
State whether or not recombinant plasmid self-replicate at a faster rate than the bacterial’s host chromosomal DNA
Yes.
State 2 factors required of plasmids for the identification of cells that have incorporated the recombinant plasmid
Plasmids need:
- antibiotic resistant gene
- gene that can be easily identified
Describe reporter gene
A gene that allows detection of gene expression in genetic engineering
State the 4 steps involved in the process of creating recombinant plasmids
- Cut target DNA using sticky-end restriction enzyme and isolate
- Cut bacterial plasmid by same restriction enzyme
- Place target DNA and plasmids together
- DNA ligase added to rejoin backbone
State what DNA is employed as target DNA to produce a eukaryotic protein in a bacterial cell
Complementary DNA
Describe cDNA
DNA that has been copied from mature mRNA and contains only exons
State how cDNA is synthesised
Using reverse transcriptase enzyme
State what reverse transcriptase has the ability to make cDNA from
mRNA
State why the ability of reverse transcriptase to make cDNA from mRNA is useful
Mature mRNA has already had the introns spliced out
State whether or not prokaryotic cells are unable to splice out introns
Yes.
Reverse transcriptase allows the synthesis of DNA from what?
RNA in a test tube
State what occurs when cDNA is inserted into a plasmid and thus, incorporated into a bacterial cell
Protein encoded by cDNA will be expressed
State for what purpose regulatory genes may be included in plasmids
Purpose of controlling the expression of the target gene that is inserted into the plasmid
State what the regulatory gene is turned on by
An inducer molecule
State what occurs once the regulator is transcribed
The target gene can be transcribed and translated
State what inducers are important in
Regulating gene expression
Describe inducers
A molecule that regulates gene expression
State what cells that have foreign DNA introduced to them are called
Transformed cells
State the 2 forms of artifical bacterial transformation
- Heat shock
2. Electroporation
Describe heat shock
Involves placing bacterial cells and a mixture of recombinant and non-recombinant plasmids in an ice-cold solution
Describe electroporation
Bacterial cells and a mixture of recombinant and non-recombinant plasmids are subjected to an electrical current that alters the plasma membrane
State, after heat shock and electroporation, whether or not many bacterial cells will be transformed with recombinant plasmids.
No. Very few.
State what genes all plasmid vectors contain
- gene for antibiotic resistance
- gene that displays a particular phenotype
State how it can be determined whether or not bacterial cells have been transformed
Cells are incubated at 37C upon agar plates that contain antibiotic
Those that survive will be those that have taken up the plasmid
Describe genetic transformation
Process that involves the introduction and expression of foreign genes in a host organism.
