Lab Final

• Genetically modified organism (GMO): An organism whose genetic material has been altered in a way that does not occur naturally by mating or recombination
• reasons to have genetically modified crops?

  Growing human population needs more food, loss of farmable land due to urbanization, remedies the soil, and gmo have enriched nutrients
• Desirable traits of GMO:

  • Pest resistance
  • Herbicide tolerant
  • viral resistance
  • drought resistance
  • increased nutritional value
  • improved fruit quality and production
  • altered ripening
• Non-desirable factors or GMO:

  • creates super-pests that are resilient to pesticide
  • create super weeds (same thing as above just herbicide instead of pesticide)
  • loss of natural biodiversity/biodiversity in general
  • biotechnology companies are able to control agriculture
  • health concerns
• Method for genetic modification of crops:

  1. Choose a pre-existing desirable trait
  2. Clone that gene responsible for wanted trait
  3. engineer the gene
  4. transform gene into a plant
  5. back cross GM plant into a high yielding crop to promote reproduction and passing on of desirable trait
• Choosing a desirable trait example :

  • the protein found inside of Bt is a delta endotoxin capable of killing corn borers, which makes it a great pesticide
• transforming a gene into plant:

  • isolate the cells of the plant
  • grow undifferentiated callus
  • transform the cells
  • select from the transformed cells
  • redifferentiate callus
  • grow transgenic plant
• US approval for GM crops:

  1. corn
  2. soy
  3. papaya
  4. canola
  5. potato
  6. chicory
  7. rice
  8. squash
  9. sugarbeet
  10. tomato
• Very reliable food source for viable plant DNA:

  • Fresh corn
  • fresh papaya
  • corn bread mix
  • soy flour
• Reliable food source for viable plant DNA:

  • Veggie sausages
  • Tortilla chips
  • flavored tortilla chips
  • puffed corn snacks
  • meatballs and burgers containing soy protein
• Less reliable food source for viable plant DNA:

  • veggie burger
  • fried corn snacks
  • popcorn
  • fries
  • potato chips
• Not reliable food source for viable plant DNA:

  • Oil
  • salad dressing
  • cereal
  • wheat flour
• How to test for GMO using PCR:

  1. grind food
  2. extract DNA from sample
  3. test sample DNA for viable plant DNA
  4. Test sample DNA for genetic modifications
• PCR is used to amplify small amounts of DNA into large enough quantities to be analyzed
• Why amplify a plant gene?

  conformation that viable DNA was extracted and that negative GM result isn’t due to to a non-viable template.
• CaMV 35S - Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus.

  Used because it functions in every plant cell
• NOS- Sequence for nopaline synthase terminator from soil bacterium

  Agrobacterium tumefacians

  Used because it evolved to be recognized in most plants
• The PCR Reaction:

  1. template - the DNA to be amplified
  2. Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence
  3. Nucleotides - dATP, dCTP, dGTP, dTTP
  4. Magnesium chloride - enzyme cofactor
  5. Buffer- maintains pH and contains salt
  6. Taq DNA-polymerase - thermophilic enzyme from hot spring
• How does the PCR reaction work?

  • Heat to denature DNA strands
  • cool back down to anneal primers to template
  • warm back up to activate taq polymerase, which extends primers and replicates DNA
  • repeat cycle 40 times
• What makes CRISPR-Cas9 so powerful is the combination of its precision and simplicity.
• "Clustered regularly interspaced palindromic repeats" (CRISPR) are sequences in the genomes of some prokaryotes that act as a genomic record of previous viral attack.
• CRISPR-associated (Cas) proteins, bacteria use the sequences to recognize and disarm future invading viruses.

  Scientists have adapted this system for genetic engineering purposes.
• • Cas9 enzyme (Cas9) — a bacterial endonuclease that forms a double-strand break (cuts) DNA at a specific site within a larger recognition sequence, or target site.
• • The Cas9 recognition sequence includes a 20-nucleotide sequence called the protospacer that is determined by a guide RNA bound to the enzyme
• • Single guide RNA (sgRNA) — an engineered form of guide RNA that forms a complex with Cas9.The sgRNA is an approximately 100 nucleotide-long fusion of two regions that occur as separate RNAs in nature:
• • Guiding region — part of the CRISPR RNA or cRNA in nature, a typically 20-nucleotide region that is complementary to the target DNA sequence and that defines where Cas9 cuts.
• • Scaffold region - called the transactivating CRISPR RNA or tracrRNA in nature, a region that forms a multi-hairpin loop structure (scaffold) that binds tightly in a crevice of the Cas9 protein.The sequence of this region is typically the same for all sgRNAs
• • Protospacer adjacent motif (PAM) — a sequence motif immediately downstream of the protospacer sequence in the Cas recognition sequence that is required for Cas9 function. Cas9recognizes the PAM sequence 5'-NGG where N can be any nucleotide (A, T, C, or G). When Cas9 binds the PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA. If the sgRNA is complementary to that sequence, Cas9 cuts the DNA
• • Cas9 binds an sqRNA

  Cas9 recoqnizes and binds the scaffold

  tracthiNa reson or an serina. Ine nucleotide sequence of the scaffold region determines its structure, which is tailored to fit within the Cas9 protein like a key fits into a lock.
• The guiding region of the sgRNA binds to the target DNA sequence.

  The guiding region of the gRNA attempts to base-pair with the DNA.

  If a match is found, the process continues.

  Unerwise, ine complex releases and atemors.

  to oind anoter rAM and target UNA secuence
• Cas9 makes a double-stranded

  break in the DNA three base pairs

  upstream of the 5'-NGG PAM sequence.
• Gene editing involves two steps: cutting double-strand DNA at a desired location and then directing

  DNA repair to produce a desired sequence change.
• • Nonhomologous end joining (NHEJ) — specific proteins reconnect the ends of the double-stranded break back together. This process may randomly insert or delete one or more bases and can cause mutations that can disrupt gene function or expression
• • Homology directed repair (HDR) — enzymes patch the break using donor template DNA, which is required for HDR. Researchers design the donor template DNA, which may include a desired sequence flanked on both sides by "homology arms" that match the sequence upstream and downstream of the cut. A complementary DNA strand is created during repair
• enzyme called B-galactosidase (B-gal), which catalyzes the hydrolysis of the sugar lactose into its component sugars. B-gal can also hydrolyze a sugar analog called X-gal, which produces a blue pigment after it is hydrolyzed.
• In nature, lactose induces the expression of the lac operon. But because the lac operon allows bacteria to use lactose itself as a food source, they consume it, which then stops expression.
• In these bacteria, expression of the HDR DNA repair system is controlled by an arabinose-inducible promoter; when the bacteria are exposed to arabinose, they express, or "turn on," the HDR DNA repair machinery. Only then can the bacterial cells can use donor template DNA to repair double-strand breaks.
• The cells that have been exposed to arabinose will retain the enzymes needed for HDR even if they are transferred to a plate with no arabinose. Their daughter cells, however, will not produce HDR enzymes unless they are exposed to arabinose.
• • pLZDonor - (control) includes a donor template DNA sequence that will be used by the HDR machinery to fix double-stranded DNA breaks. The donor template DNA includes an insert sequence, which will be inserted into the lacZ gene and impair its function
• • pLZDonorGuide - includes both the donor template DNA sequence from pLZDonor and a sequence that codes for the sgRNA. Once transcribed, the sgRNA will direct Cas9 where to cut lacZ