MFD5 Flashcards
What is recombinant DNA technology?
Genetic engineering and cloning
E.Coli K12 Genome
1) What is the shape of its chromosome?
2) a) How many base pairs?
b) How many proteins does that code for?
3) What percentage of genes are of unknown function?
4) The average protein has ___a___ amino acids.
1) circular
2) a) 4.6 million base pairs.
b) 4,400
3) 40%
4) 300
What are operons?
2) Name a genome where this occurs:
1) the clustering of genes that encode enzymes of a single biochemical pathway.
2) E.Coli K12 Genome
1) What are they?
2) What sort of information do they carry?
e. g.
3) What releases plasmids?
4) What absorbs plasmids?
1) Small circular DNA
2) not essential to survive but are useful
e. g. e.g. genes which make bacteria resistant to antibiotics
3) are released by dead bacteria
4) absorbed by those still living thus genetic information is exchanged
Horizontal Gene Transfer in bacteria
Occurs naturally by :
- Transformation
- Transduction
- Conjugation
Define
horizontal gene transfer via Transformation:
the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane
Define
horizontal gene transfer via Transduction:
injection of foreign DNA by a bacteriophage virus into the host bacterium
Define
horizontal gene transfer via • Conjugation:
transfer of genetic material between two bacterial cells in direct contact
Is the following transformation, transduction, pilus-mediated transfer, conjugation:
DNA uptake
transformation
Is the following transformation, transduction, pilus-mediated transfer, conjugation:
Phage-mediated DNA transfer
transduction
Is the following transformation, transduction, pilus-mediated transfer, conjugation:
Mating
pilus-mediated transfer; conjugation
DNA is incorporated into the chromosome of the recipient cell via ____a____ after horizontal gene transfer has occured.
homologous recombination
What 3 thing ford biotechnology produce:
Production of recombinant proteins e.g. insulin
Develop vaccines e.g. hepatitis B
Generate improved probiotic bacteria
What is recombinant DNA technology?
3
- Creation of recombinant DNA
- Construction of new combinations of unrelated genes in the test tube - Cloning of recombinant DNA:
–Amplifying the new DNA many times by introducing it into living cells- produces many identical copies (clones). - Using recombinant DNA, example:
-Expressing a cloned gene to produce a protein
Why do molecular biologists have this in there toolbox:
enzymes
To manipulate the DNA/RNA
Why do molecular biologists have this in there toolbox:
Vectors
Act as a vehicle to carry recombinant DNA into host cell
Why do molecular biologists have this in there toolbox:
Taq polymerase
the raw material- usually purified from tissue/cell culture
Why do molecular biologists have this in there toolbox:
Reverse transciptase
To amplify –and sometimes express- the recombinant DNA
What are restriction enzymes?
+e.g.
1) molecular scissors
e. g. Restriction endonucleases:
1) How many base pair palindromic sequences do restriction enzymes recognise?
2) What is a palindromic sequence?
1) 4-8
2) sequence made up of nucleic acids within double helix of DNA and/or RNA that is the same when read from 5’ to 3’ on one strand and 5’ to 3’ on the other, complementary, strand
Restriction endonuclease
1) What do they do?
2) What is it naturally produced by and why?
1) Cleave DNA, usually at very specific sequences
2) Naturally produced by bacteria: self-defence mechanism –cleaves foreign (bacteriaphage) DNA
What are the 2 types of cleavage, and what ends of they produce?
- Symmetrical cleavage = blunt ends
2. Asymmetrical cleavage = sticky ends
What type of ends are easier to anneal together?
2) What enzymes is used to anneal them?
1) Sticky ends are easier to anneal together with
2) ligase.
Give the 5 features of a vector:
- Contain unique restriction sites for insertion of the new DNA.
- Can be introduced into host cell relatively easily
- An efficient origin of replication
- Contain a gene to allow selection of cells which contain the plasmid- eg an antibiotic resistance gene
- May also contain regulatory sequences to allow expression of the inserted gene. (polylinker)
What are commonly used as vectors?
plasmids
Plasmids
1) What size do they come in?
2) Do they replicate at the same time as the bacterial chromosome?
3) What is their function?
1) 2-200kbp
2) no, replication is independent of bacterial chromosome
3) many functions e.g. self-defence- may carry genes for antibiotic resistance
What is a polylinker?
multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids
A) Other than plasmids what else can be used as vectors: (2)
B) Why may they be more useful?
A)1)Bacteriophage
2) Cosmids/ phagemids:
b) for cloning larger fragments of DNA
What are bacteriophaes?
(viruses which infect bacteria)
•
What are Cosmids/ phagemids
genetically engineered hybrids which replicate as a plasmid but can be packaged as a bacteriophage.
How would we set about cloning a human gene in E.coli?
- Create Vector: Cut DNA molecule using restriction enzymes or restriction endonucleases, and then pasting the DNA of interest into the plasmid at the sticky ends using an enzyme called ligase
- Recombinant plasmid inserted into E.coli host .
- E.coli cells containing plasmid are selectively cultured
- Recombinant plasmid is purified or expressed
How is bacteria containing pUC18 plasmids selectively cultured?
By growing on agar + ampicillin.
How is recombinant plasmid inserted into E.coli host ?
host cell in transformed by CaCl2 on ice +heat shock (42degrees, 2mins) OR electroporation
What are the issues with using animal hormones to replace our lack of hormones?
1) Animal protein not identical to human= side effects
2) Difficult to purify
3) Possible contamination with pathogens eg BSE
Where is insulin made?
2) What is its function?
3) What disease does it cause?
4) How were symptoms alleviated?
1) Insulin made in the beta-cells of Islets of Langerhans in pancreas
2) Vital for controlling glucose levels in blood
3) Insulin defiency results in diabetes mellitus – death if untreated afflicts millions of people worldwide
4) Alleviate symptoms by regular injections of animal insulin e.g. pigs/ cows
1) What is the issue when producing a vector containing the gene for human proinsulin?
2) What is the solution?
1) the human proinsulin gene has 2 introns (non-coding segments)
2) using mRNA for human insulin, it is reverse transcribed proinsulin cDNA (no introns)
How is recombinant DNA technology used to improve oral health?
- Recombinant DNA can be used to manipulate genes to produces GM organisms.
- Creation of a genetically altered strain fo Streptococcus mutans, called SMaRT, which does not produce lactic acid.
- The SMaRT replacement therapy technology: protection against tooth decay!
