Genetic Engineering Flashcards
What is recombinant DNA?
Artificial stretch of DNA assembled in a precise way
How can bacteria restrict infection?
If they contain endonuclease’s that bind and cut short DNA sequences in the bacteriophage genome
How does EcoRI work?
Binds to the DNA double helix at GAATTC as a homodimer and uses magnesium ions to catalyse the cleavage of DNA
On palidromic sequences, where do restriction enzymes cleave?
Between the same two nucleotides
What does T4 DNA ligase do?
Catalyses the formation pf phosphodiester bonds in double stranded DNA molecules
What does T4 DNA ligase need to work?
Nearby 5’ phosphate and 3’ hydroxyl groups
ATP as a co factor
What are the basic features of a plasmid?
1- unique cloning site
2- drug resistant gene
3- origin of replication
Wat are the steps for building recombinant DNA with plasmids?
1- select plasmid with cloning sites and selectable marker
2- cut plasmid and DNA using restriction enzyme
3- use DNA ligase to put together
4- put plasmid in bacterial cells using heat shock or electroporation
5- select bacteria off plate that has recombinant DNA
If a gene does not have convenient restriction sites, what should be used?
PCR
What are the steps for using PCR to make recombinant DNA?
1- create primers that match ends of DNA sequence
2- mix the gene, primers, DNA polymerase, dNTPs and buffer soltuion
3- use machine to heat and cool mixture - copy gene over and over
4 - cut plasmid and gene (product) using R.E
5- join gene into plasmid using DNA ligase
6- insert plasmid into bacteria and grow on antibiotic plate
Why is genome engineering usually performed in the zygote?
As changes to the genome at this stage will be inherited by all cells of the organism
Where would you find the ubiquitous transcriptional promotor? What does it do?
Found at pCAGS
-drives expression of downstream gene into all tissues at all times of development
What does the bent arrow on the pCAGS-GFP trans gene indicate?
Indicates site of transcriptional initiation
Where would you find the polyadenylation signal pA? What does it do?
Found in SV40pA
- designates the site of transcriptional termination
-initiates formation of poly(A) tail
What are transgenic mice?
Modified mice that have had foreign DNA (a trans gene) introduced into their genome
What are the disadvantages of DNA micro injection into zygote?
- random transgene integration into genome
- there may be multiple copies of the transgene
-could disrupt an endogenous gene (natural gene)
What are the advantages of genetically engineered embryonic stem cells
Precise integration into genome
Single copy integration
Ideal method to ‘knock out’ genes
What are embryonic cells? What are they derived from?
Immortal cell cultures that are derived from Blastocysts
What is a chimera?
An organisms whose cells are derived from two or more zygotes
If a chimera is bred, what does this give you?
Germ-line transmission
Advantages of CRISPR/Cas9?
Increased speed and efficiency of precise genome engineering
No longer restricted to animals for which es cells are available
What does a plasma membrane do?
Maintains differences between intracellular and extracellular environments
What is an intracellular?
Found in eukaryote and some bacteria
Maintains the environment of intracellular organelles
Functions of membranes
Maintain intracellular environment
Contain receptors for external stimuli
Some generate signals
Energy conservation process (eg photosynthesis)
What does lipids do in a membrane?
Acts as a barrier to the movement of water and water soluble molecules
What do proteins do in a membrane?
Functions include transporting molecules across membranes
What is an amphipathic molecule?
Molecules which have one hydrophobic end and one hydrophilic end
What will a tear in a bilayer create?
A free edge with water - only way to prevent is to form a closed compartment
What does Tm depend on? (Bilayer)
Fatty acid chain length and degree of saturation
What do double bonds create in fatty acids? What does this mean/
Kinks
Harder to pack together, more likely to stay fluid at low temperature
