Topic 21: Cell factories with biotechnology

Question 1

Identify microbes and their roles in biotechnology

Answer 1

• Viruses:
  + vectors to carry genes of interest into the new host
  + useful enzymes (e.g. T4 ligase used in joining DNA pieces)
• Bacteria: excellent host for cloning and expressing genes of interest
• Archaea: produce thermostable polymerase enzymes used in copying DNA
• Fungi: yeasts and molds
  + yeasts are great host for cloning and DNA expression
  + molds: antibiotic synthesis
• Algae: convert CO2 into biofuels like ethanol

Question 2

Define host cells and their features

Answer 2

• Have plasmids that can easily be extracted, added genes of interest and put back
• Able to make copies of the plasmids quickly and produce the desired proteins
• Receives simple input (sugars, phosphate, etc.) to generate more complex compounds

Question 3

Explain what a plasmid is and its features

Answer 3

Plasmids circular DNA strands floating in cytoplasm and containing genes that may not be essential, but still crucial in certain cases.

Vectors to carry foreign genes to cell hosts.

Lab tamed plasmids are much easier to handle and conduct experiment on.

Able to exchange plasmids horizontally

• Features:
  + Replication functions: own origin of replication -> able to replicate and self-sustain in cell host
  + Cloning site: area where restriction enzymes can cut open plasmids and insert foreign DNA
  + Selectable markers: carry genes that can help to select plasmids and force cells to take up that plasmids and acquire specific phenotypes

Question 4

Identity 2 host cells most commonly used in biotech and their features

Answer 4

• Bacteria E.coli:
  + easy to extract plasmids and add foreign DNA
  + fast growing -> desired genes can be replicated quickly
• Fungi: yeasts - Saccharomyces
  + more similar to genes in humans -> better for expressing eukaryotic genes
  + post-translational modifications
  + generally recognized as safe

Question 5

Explain how recombinant DNA and GMOs are made, and especially which enzymes do which jobs in this process

Answer 5

• Amplification: DNA of interest is replicated and extracted from another organism by thermostable enzymes
• Digestion: DNA strands are cut into smaller pieces by restriction enzymes
• Ligation:
  + Restriction enzymes cut open plasmids at cloning site
  + T4 ligase joins the foreign DNA pieces to the plasmids
  –> ligation mixture containing: recombinant plasmids, non-recombinant plasmids and non-ligated pieces of DNA
• Transformation: add mixture into cloning host by either
  + Horizontal exchange or
  + Electrical change
• Selection: select cells that contain the plasmids based on the selectable marker
• Screening: identify the genes of interest by either:
  + Sequence-based screening: look for the DNA directly or
  + Phenotypic screening: look for the phenotypes characterized by the genes
• The host cell containing the plasmids with the foreign DNA is now called the GMO - genetically modified organism

Question 6

How is the gene of interest expressed in the GMO?

Answer 6

• promoter region located right up the foreign DNA sequence in the plasmid signals RNA transcription
• mRNA is then translated to make desired proteins which can either
  + be the end-product itself or
  + substrate to make the end-product
• The end-product is then purified for further processing.

Question 7

Explain how vaccines is created by GMO host method

Answer 7

• Genes of the viruses can be extracted and combined with plasmids to produce recombinant plasmid containing virus’s genes.
• The host cell when received the recombinant plasmids produce proteins from the genes associated with the viruses.
• The proteins are then extracted to produce vaccines. when vaccines containing the proteins is injected in humans, the proteins can activate immune system, just enough to recognize the viruses on later encounter.

Question 8

Why is E.coli recognized as generally less safe than yeasts?

Answer 8

E.coli is adapted to survive in human intestinal tract.