Cloning

Question 1

Reasons for cloning genes, transcripts and DNA

Answer 1

-PCR is for making small amounts of DNA or gene transcript

• reasons for cloning DNA in living cells
  1. To make more DNA with high fidelity for further study or manipulation (living cells copy DNA with more accuracy than PCR)
  2. To produce substances of scientific or commercial value from genes (eg. Numerous enzymes, synthetic hormones etc)
  3. To modify the genomes of plants or animals to introduce new, desired traits

Question 2

Cloning requires a vector

Answer 2

• plasmids are commonly used as cloning vectors
• a plasmid is a stable, self-replicating molecule circular DNA molecule
• plasmids contain:
  
  • origin of replication
  • selectable markers (traits that enable cells containing the vector to be recognized)
  • unique restriction enzyme cleavage sites

Question 3

pUC19

Answer 3

• pUC19 is a typical and commonly used bacterial vector
• contains a portion of the LacZ+ gene, with a restriction site linker that contains numerous unique restriction enzyme cut site (unique site not found anywhere else on plasmid)
• antibiotic resistance gene (ampR) for ampicillin

Question 4

Inserting foreign DNA sequence into a plasmid

Answer 4

• cut foreign DNA with a restriction enzyme
• cut plasmid with same restriction enzyme, or one that produces compatible fragment
• mix cut foreign DNA and cut plasmid DNA
• use DNA ligand to deal sugar-phosphate backbone

Question 5

Transforming bacteria and selecting for recombinant

Answer 5

• “competent” bacteria are E.coli made receptive to transformation by chemical or electrical treatment
• they are also lacZ-, which means they lack the portion of the lacZ gene that is present in the plasmid
• ligated plasmids containing DNA inserts are used to transform competent cells
• transformed bacteria are plated on agar containing ampicillin and X-gal
• bacteria with no plasmid: do not grow
• bacteria with non-recombinant plasmid: produce B-galactosidase, resulting in blue colonies
• bacteria with recombinant plasmids (lacZ gene disrupted by insert) do not produce B-galactosidase, resulting in white colonies

Question 6

Bacterial expression vectors

Answer 6

• include an operon and regulatory sequences to allow expression of gene in bacteria
• good for production of many enzymes, especially those that originate from bacteria (eg. Taq DNA polymerase, commercially available REs etc)
• not good for gene products that requires post-translational modification (as do most eukaryotic proteins)

Question 7

Agrobacterium Tumefaciens

Answer 7

• naturally transforms DNA of higher plants
• can be co-opted to introduce new genes to plants

1. Natural gene transfer, the Agrobacterium invades the plant at a wound
2. Part of the Ti plasmid is transferred to the plant cell
3. Where it integrates into on the of plant chromosomes

-integrated Ti plasmid DNA in plant chromosome is transcribed and translated, producing enzymes that aid the bacterium

Question 8

General approach to using A tumefaciens to introduce foreign DNA into plants

Answer 8

1. Foreign DNA inserted into a plasmid vector
2. Plasmid is then transferred to A tumefaciens with the Ti plasmid
3. Helper Ti plasmid is required for infection
4. The plasmid vector along with any foreign DNA that it carries is transferred to a plant cell
5. The plasmid integrated into a plant chromosome

Question 9

Introducing Bt gene

Answer 9

• Bacillus thuringiensis produces a protein, Bt toxins, lethal to many insects
• desirable feature: toxicity specific to some insects, not toxic to humans/animals, and is biodegradable
• steps:
  1. First, cloned Bt gene into E. coli plasmid to produce larger amounts of gene sequence for use
  2. use REs to produce DNA sequences containing various portions of Bt genes, which are then ligated to a neo gene (confers resistance to kanamycin)
  3. Constructs inserted into an expression vector
  
  • vectors contained promoter and polyA consensus sequences needed for proper gene expression in plant cells
    
    4. Neo+Bt-contains plasmids used to transform A tumefaciens bacteria.
  • recombination between expression vector and Ti plasmid occurred inside the bacterial cells
    
    5. Tobacco plant cells infected with transformed A tumefaciens bacteria
    6. Whole plants regenerated from plant cells
    7. Recombinant plants screened for Kanamycin resistance
    8. Plants fed to tobacco hornworms to assess toxicity.
    9. Most-insect toxic plants had 2/3 of Bt gene inserted, full length gene was not effective (maybe not well expressed by plant)
    10. Further breeding demonstrated that Bt gene was stably integrated in plant genome
    11. Similar approach used for plant species

Question 10

Creation of trans-genic Atlantic salmon

Answer 10

• created at MUN in 1989
• combined growth hormone gene (cDNA) from chinook salmon with promoter and terminator sequence for antifreeze gene from ocean pout
• chinook salmon GH similar to Atlantic salmon GH
• ocean pout antifreeze promoter shown to be constitutively active in Atlantic salmon, in contrast to normal GH promoter in salmon which is only active in response to environmental cues (day length)
• gene construct injected directly into salmon eggs
• some salmon resulting from this had accelerated growth
• used pUC19 plasmid
• further breeding shows that ocean pout promoter, chinook GH construct was stably integrated into Atlantic salmon genome, allowing creation of a strain that constitutively expresses chinook salmon GH
• the gene construct rearranged itself inside the Atlantic salmon genome (part of promoter moved downstream making it slightly less effective)

Question 11

AquaBounty

Answer 11

• company that trademarked AquAdvantage salmon
• several measures taken to reduce risk of transgenic salmon escaping and interbreeding with wild salmon
• farmed salmon will be triploids (made by pressure treating eggs)
• triploid females are sterile
• males can produce sperm, but risk is controlled by using neo males: sex reversed females created by methyl testosterone treatment
  
  • all offspring will be females
• salmon will be grown in closed systems on land (feasible because they grow much faster than regular salmon