L14

Question 1

Explain selective strains and the process

Answer 1

• Is the selection of mutant strains that produce something desirable, e.g. High glutamic acid production
  
  • Process
    
    1. Sterile velvet is pressed on the grown colonies on the master plate
    2. Cells from each colony is transferred from the velvet to 2 new plates (one containing histidine)
    3. Plates are incubated
    4. Growth on plates is compared
       § E.g. A colony that grows on medium WITH histidine could NOT grow on the medium without histidine, therefore is auxotrophic (histidine requiring mutant)

Question 2

explain vectors, and what are the 2 types

Answer 2

• Tools for inserting DNA into cells for sequencing or expression
  
  • 2 types
    Bacteriophage vectors + plasmid vectors

Question 3

what are the vector variations

Answer 3

• Shuttle vectors
  ○ Can replicate + stably maintain 2+ unrelated hosts
  ○ E.g. E.coli + yeast
  
  • Expression vectors
    ○ Include regulatory sequences to switch on/off expression of inserted genes
  • Phage vectors
    ○ Phage genome allows for holding large amounts of DNA than a plasmid
  • Cosmid vectors
    ○ Specialised plasmids, based on phage genes added, that uses phage virions
  • Artificial chromosomes
    ○ Bacterial artificial chromosomes (BAC) engineered from conjugative plasmids
    ○ Yeast artificial chromosomes (YAC) replicate in yeast like normal chromosomes + allow large DNA fragment insertion

Question 4

explain site directed mutagenesis

Answer 4

• Mutate a gene at a specific point

1) addition of a short sequence of DNA (oligo) with 1 altered base from the original

2) undergoes replication to allow copies of the plasmid mutation to be made with the new sequence

Question 5

explain LacZ selectable plasmids

Answer 5

• Selectable markers in plasmid
  ○ Ampicillin resistance to select for transformants (e.g. Only ampicillin resistant E.coli will grow on Amp plates)
  ○ Beta galactosidase gene (lacZ) can be detected using chromogenic substrate X-gal, gives a blue product when hydrolysed with beta galactosidase (so all colonies will be blue if Xgal present)
  ○ If multiple cloning site within the beta galactosidase gene, inserted DNA fragment will prevent the production of Beta galactosidase (so colonies with DNA insertion in plasmids will be white)

Question 6

explain biotech applications of plasmid vectors

Answer 6

• Sequencing
  ○ Plasmid vectors allow specific DNA to be rapidly grown in large numbers
  ○ Plasmids can then be re-isolated from the bacteria
  
  • Vectors commonly used as a high quality template for DNA sequencing
    § Because is sensitive to purity, copy number, and uniformity

Question 7

explain transfection

Answer 7

used to insert foreign DNA into eukaryotic cells
e.g. antigens on surface of Hep. B virus cloned and expressed in yeast, results in a vaccine

Question 8

explain cloning process

Answer 8

• DNA, made of exons and introns
  
  • RNA processing in eukaryotic cells
  • process
    
    1. That DNA piece transcribed to RNA by RNA polymerase
    2. Processing involves ribozymes + proteins in the nucleus to remove the intros, and splice together the exons, to make mRNA
    3. After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis

Question 9

explain the pros + cons of common cloning host

Answer 9

euk (e.coli + hay bacillus) vs pro (yeast)

e.coli- best known, periplasm traps protein
hay bacillus- endospore formation, not genetically stable
pro- easy to grow, plasmid not stable + wont grow pro plasmid

genetics + development = y,n,y
proteins = n, y, y
pathogenic = y, n, n

Question 10

issues with cloning mammalian genes in bacteria

Answer 10

○ Post-translational modification
○ Antibacterial products (toxic)

Question 11

process for using microbiology to genetically engineer plants + animals **

Answer 11

• E.g. Agrobacterium tumefaciens- pathogenic to plants because of Ti plasmid
  
  • Process
    1. Remove pathogenesis gene in Ti plasmid
    2. Make a shuttle vector with sections of DNA around, insert that Ti plasmid recognises
    3. Put vector in E.coli + transfer by conjugation to A. Tumefaciens
    4. Put with plant cells, Ti plasmid mvoes DNA from shuttle vector into plant cell -> homologous recombination

Question 12

explain gene therapy

Answer 12

• Insert working copy of a gene into cells to treat disease
  ○ Repair faulty gene
  ○ Express something which will be beneficial
  
  • Delivery challenges- should deliver how/with what
    ○ Virus
    ○ Stem cells
    ○ Coated nanoparticles
  • Note: if insert foreign DNA into wrong spot, can trigger oncogenes, which result in cancer

Question 13

explain RNAi

Answer 13

form of molecular gene therapy

results in gene silencing + downregulation by triggering the destruction of its mRNA

Question 14

what are the challenges of synthetic iRNA

Answer 14

○ Delivery + survival
○ Selecting unexpected targets
- Displacement of natural role in cell