Biotech - Recombinant DNA Flashcards
Recombinant DNA
This is the producto f insertion of a DNA fragment into a vector capable of independent replication in a host cell.
Why is recombinant DNA useful?
Large quantities of the inserted DNA can be obtained by replication of the recombinant in a host.
Lambda Phage
This is a tailed bacteriophage, with E.Coli its natural host
What is the step after DNA replication in a host?
Isolation of the recombinant then isolation of the fragment itself by restriction endonuclease digestion, then GE.
Why is Lambda Phage useful?
It can undergo transduction.
Transduction
This is the transfer of genes from one bacteria to another, and is non-pathogenic to humans.
Why are buffers used in RE cleavage?
To optimise the enzyme and ensure efficient proceeding of the reaction.
How can cloning be used to udnerstand reaction enzyme catalyses?
Clone generated then excising the gene from the gene segment, atatch it to a vector and replicate the product in a microorganism
What is Recominbant DNA generated by
RE and DNA ligases
Function of RE and Ligases?
RE cleave DNA at specific sites
Ligases link DNA molecules together
What is the mechanism of RE?
Recognise DNA sequences between 4-8 BP and cut dsDNA leaving one strand overhanging.
PAR genes
(Protein Associated With RNA) are involved in a variety of cellular processes like cell polarity, mitosis and embryonic development.
Where are PAR genese used in biotech?
Control localizationg and expression of recombinant proteins.
Example of a PAR gene?
PAR-4 localises heterologous proteins in mammal cells so when fused with a protein of choice, protein can be targeted to specific locations.
What can PAR genes be designed with?
Biocatalysts, that being biological systems speeding up chemical reactions.
Bacteria Artificial Chromosomes
These are vectors accomodation DNA fragments up to 300kb
What are the elements of BAC?
A selectable marker, a ORI site, a polylinker region and a BAC backbone with genes allowing maintenance in bacterial cells.
What is the polylinker region in BAC used for?
Cloning of target DNA fragment containing many restriction enzyme recognition sites.
Yeast Artifical Chromosomes
These can accomodate 2 MB fragments, containing same elements as BAC but with centromere and telomere.
Transformation
This is DNA uptake by creating pores within the membrane by heat shock, electroporation or chemicals.
What are the steps of transformation?
Insertion through membrane.
Donor DNA protected
DNA transportation to nucleus or nucleoid for integration into host genome.
DNA replication and expression.
How does heat shock facilitate transfomraiton?
Increases membrane fluidity and creating temporary gaps, by disrupting lipi molecule interactions.
How does electroporation facilitate transformation?
Create electrical potential difference disrupting lipid bilayer in dielectric breakdown.
Dielectric Breakdown
This is a sufficiently strong electric field breaking down the insulating properties of an insulator to allow flow of charge.
Transmembrane Potential
This is the difference in electric potential between interior/exterior of a membrane.
What is the mechanism of electroporation in the membrane?
Hydrophilic head grooups allign to the electric field, disrupting the organisastion, by electrical field dipoles and dipole moment allignment.
What causes the dipole in lipids?
Polar head group negative charge on one end and positive charge on the other., due to polar covalent bonds(OH and NH2
How does Calcium Chloride promote membrane porosity?
Ca2+ binding negatively charged phosphate groups of membrane reducing repulsion between negative charged DNA.
What is the importance of antibiotic resistance genes in Recombinant?
Positive selection for bacteria containing the vector.
What does the LacZ gene do?
Reveal which cells contain plasmids through conversion of colourless molecules to blue products.
Blue-White Screening
This is a method of identificaiton of recombinant bacteria through beta-galactosidase nezyme and lactose to glucose to galactose cleavge.
What is the function of the LacZ gene?
When galactose is present, LacZ operon results in beta-galactosidase enzymes metabolising lactose
How is blue-white screening set up?
Plasmids are ligated with LacZ gene, whilst hosts contain mutated version.
Gene contains RE site, which is split if vector is introduced
How is it known that no vector insert has occured?
Functional LacZ gene produces functional enzymes
How is Blue-White screening identification done?
Agar plate with a chromogen called X-gal
What does B-Galactosidase do to Xgal?
Hydrolyses forming 5-bromo-4-chloro-indooxyl which spontaneously hybridises into an insoluble blue pigment
When is the blue pigment produced?
Where failutre of vector insertion has occured
