Molecular Genetics (Katherine Ford 25-30) Flashcards
(48 cards)
Give 5 reasons why we use model organisms?
1) Easy to grow and maintain in the lab.
2) easy to manipulate.
3) short generation times.
4) small, sequenced genomes.
5) can extrapolate from simple organisms to complex ones.
What are a typical gram-negative bacteria used as a model organism?
E.coli
- 20 min generation time!
- can be frozen/stored
- used to study conjugation 1946
What are a typical gram-positive bacteria used as a model organism?
Bacillus subtillis
- found in soil & digestive systems of ruminants.
- spores are tough
- easily secretes proteins.
Why is Saccharomyces cerevisiae a good model organism?
- doubles every 90 mins
- full genome available
- several plasmids to work with, including integrating plasmids.
Why are modified plasmids good cloning vectors?
- easy to transfer between cells
- easy to miniprep/ isolate DNA
- abundant in cell
- easy to screen for recombinants
(Note: PCRII-TOPO)
What are the three types of artificial chromosomes that can be used as vectors?
- BACs (circular)
- YACs (Linear/ can take the largest fragments)
- PACs
-used for genomic libraries
Name an advantage and a disadvantage of using YIPs as vectors.
- Yeast integrating plasmid = integrates into the chromosome.
- produces stable transformants
- but, transformation rate is slow.
How do Lambda bacteriophages help with producing transformants?
Lambda replacement vectors are from a bacteriophage and can complete their lifecycle even with foreign DNA inserted into them.
- The ds linear DNA has two cohesive ends (Cos sequences), once in the cell, these are cleaved off and the DNA circularises.
- The middle of the vector is replaced with a multiple cloning site.
What are Cosmid vectors and how do the work?
They have highly modified Lambda vectors but everything between the cos sites is replaced instead.
- DNA up to 45Kb can be inserted
What is Gateway cloning?
Exploits Cosmid vectors and can clone up to 4 DNA fragments.
- ccdB region is replaced with the gene of interest.
PCR product is taken up by the vector and clonases swaps the gene of interest into lots of destination vectors.
What method is useful if we want to clone more than 4 fragments?
Using Shuttle vectors as they have more selection markers.
List the steps required for transforming vectors into yeast.
- LiAOc makes yeast competent.
- Add PEG, the plasmids and PCR product.
- culture and heat shock
- plate out on nutritional medium (auxotrophic selection) and recover plasmids via miniprep.
List the steps required for transforming vectors into E.coli.
- permeablise the cell wall by calcium chloride and heatshock & ice or by electroshock.
- Blue-white screening
- miniprep.
How can you transform fungi?
Electroshock, PEG-mediation or via Agrobacterium.
Which fungus is used for citric acid production?
Aspergillus niger produces citric acid with the same chemical composition.
Which microbe is often used to produce amino acids?
Corynebacterium glutamicum
- accumulates glutamate under limiting conditions.
- also, mutant forms which could withstand high lysine conditions (limiting) have been used to produce Lysine, an essential amino acid which is added to animal feed.
Describe how vitamin C is produced by biotechnology.
- initially, Gluconobacter Oxydans was used alongside chemical synthesis.
- Using Ewrwinia & Corynebacterium together is found to be more useful but they require different growth conditions.
- Can GM the Corynebacterial gene into Erwinia so that all is required is acid treatment.
What are three disadvantages of using E.coli in recombinant technology and what can be done to overcome them?
1) Codon bias (AAA codes for lysine 75% of the time)
- Overcome by site-directed mutagenesis/ synthesising the DNA/ adding the missing tRNAs/ integrating relevant genes into E.coli.
2) Poor Secretion
- can be overcome by fusing protein to a bacterial protein that is usually secreted
- adding additional secretory sequences.
- expressing the protein in a gram-positive bacterium instead.
- modification of E.coli to give it better extracellular secretion.
3) Post-translational modifications are different to eukaryotes. (70% of human genes are glycosylated and add glycosyl groups to N-terminal groups; whereas prokaryotes at them to side chains.
- overcome by adding glycosylation genes to E.coli.
What model plant species is often used when investigating crops?
Arabidopsis thaliana
- has a similar genetic response to stress and diseases.
- crops are complex
What are the three types of tissue culture used when producing transgenic plants?
1) Plant leaf discs
2) callus culture (immature embryos or meristems-totipotent)
3) suspension culture (immature protoplasts/ pollen.)
What pathogen is exploited as a tool in plant transgenics?
Agrobacterium tumefaciens
- gram-negative
- attracted to plant wounds
- induces the plant to produce excess auxin and cytokinin.
- galls contain opines synthesised by a tumour inducing (Ti) plasmid.
How does A.tumerfaciens act as a pathogen and how is this exploited by transgenics?
- wounded plant cells produce signal molecules which are recognised by receptors.
- Agrobacterium attaches to plant cell and activates VIR proteins which produce T-DNA.
- T-DNA complex forms and transfers into cells, where it moves into the nucleus.
- T-DNA is then expressed by bacterial proteins.
-we utilise this by inserting a selectable marker and gene of interest into the T-DNA.
How does Agrobacterium tumefaciens regulate VIR proteins to produce T-DNA?
- VirA detects wound and autophosphorylates.
- VirA then phosphorylates VirG (a transcription factor) which initiates transcription of genes Vir B-E.
- VirD1 and VirD2 are DNA nickases which cut and releases ssT-DNA.
- VirD2 attaches to the 5’ end of the T-DNA and VirE2 coast it.
- T-DNA exits the bacterium, into the plant and is moved to the nucleus.
How can plant transgenics be used for herbicide tolerance?
- make a crop resistant to glyphosate (roundup)
- glyphosate inhibits synthesis of some amino acids by binding to EPSPS
- by making mutant ESPS, glyphosate can’t bind to it.
