lab final overhaul

• what organism was genetically edited in the crisp-cas lab?

  E. coli HB101-9pBRK, a bacterial strain with a functional lac-z gene, Cas9, and plasmid encoding genes needed for HDR
• LacZ gene encodes for Beta-galactosidase. This enzyme (beta-galactosidase) catalyzes the breakdown of X-gal into two products, one of which produces a blue pigment
• if beta-galactosidase is expressed in the presence of x-gal, the bacterium will appear blue
• Cas9 (CRISPR associated protein 9) is the enzyme that cuts DNA.
• A guide RNA (gRNA) is needed to locate the target piece of DNA that needs to be cut
• CRISPRs ( Clustered Regularly Interspaced Short Palindromic Repeats) are short, palindromic segments of DNA.
• CRISPR is the fastest and easiest tool in gene editing science
• CRISPR is a natural process found in bacteria and archaea that behaves as an immune system to protect against viral infection
• Naturally occurring Crisper uses two main components:

  1. Short repetitive snippets of DNA sequences called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
  2. Cas (CRISPR associated proteins) that chop up DNA like scissors
• When a virus attacks a bacterial cell, the cat will go in and excise a segment of the invading viral DNA to stitch into the bacterium's CRISPR region. This allows for the viral code to be captured in RNA. This RNA will then bind to Cas9 and search for matching viral codes in the cell. if a match is found, Cas9 recognizes it and swiftly destroys the DNA
• guide RNAs are designed to match a specific gene that is wanted to be edited in lab. it is then attached to Cas9. This RNA will guide the Cas9 to the matching DNA so it can cut it.
• Non-homologous end joining is what happens when proteins called nucleases trim the broken ends of DNA and attempt to put them back together after Cas9 (with its gRNA) has cleaved it. This is prone to mistakes and can result in extra or missing bases.
• Homology directed repair is when a template DNA is used as a blueprint for the building process of DNA after being excised. repairs defective genes and can even replace them with entirely new ones.
• What are some new treatments for diseases that scientists think using CRISPR could help?

  cystic fibrosis and sickle cell anemia.
• Cas9 is an enzyme that can cuts DNA, It will only cut the DNA if it has a guide RNA telling it what DNA to cut.
• The nucleotide sequence of the guide RNA is complementary to the target's DNA nucleotide sequence.
• Homology directed repair (HDR) is a DNA repair mechanism that involves a donor DNA template being placed at the site of chromosomal break.
• in order for HDR mechanism to occur, two things must be present in a cell:

  1. a donor DNA template strand
  2. repair enzymes
• E.coli in the crispy lab is able to express the genes needed for HDR when it is exposed to arabinose
• The starter bacteria for the CRISPR lab that were grown on IX/ARA media (containing arabinose) have functioning HDR/DNA repair system. The ones grown on IX (no arabinose) will have neither.
• What is a bacterial plasmid?

  a small, circular piece of DNA that is not part of a bacteria's chromosome. They contain only a small number of genes. They can replicate on their own and be transferred between bacteria by horizontal gene transfer.
• the two plasmids used in the CRISPR lab are pLZ Donor and pLZ Donor guide
• pLZDonor plasmid contains a donor DNA sequence. If inserted into the LacZ gene, the donor DNA sequence will disrupt Beta-galactosidase function.
• for the CRISPR lab, the pLZDonor plasmid is used as a control because even though it encodes for a donor DNA sequence, the machinery necessary to cut the DNA and insert the donor sequence is not present
• pLZ donor guide contains a donor DNA sequence and a single guide RNA (sgRNA)
• if pLZ Donor Guide is inserted into the LacZ gene, the donor DNA sequence will disrupt beta-galactosidase function.
• The sgRNA encoded on the pLZ Donor guide binds to Cas9 (already available in bacteria) and guides Cas9 to a DNA sequence in the bacterial genome that is complementary to the sgRNA
• The recognition between the DNA sequence in the bacterial genome and the sgRNA initiates cleavage of the DNA sequence via Cas9.
• if bacterium with functioning HDR is transformed with the pLZDonor guide the LacZ gene in these bacteria will be disrupted via CRISPR gene editing.
• sgRNA encoded in the plasmid is transcribed and bound to a Cas9 enzyme.
• Cas9 + sgRNA binds and cleaves a portion of the LacZ gene that is complementary to the sgRNA sequence.
• LacZ gene is cut out and must be repaired or the cell will die.
• HDR DNA repair system places donor DNA sequence into the LacZ gene to repair the breakage.
• The insertion of the donor DNA sequence repairs the break but disrupts the LacZ gene where b-gal will no longer be expressed. All E. coli offspring will be white if this is done.
• How to process food sample before DNA extraction:

  1. Weigh out 0.5 g of certified non-GMO food and put it into the mortar.
  2. Add 3-5 ml of distilled water.
  3. Grind with the pestle for at least 2 min to form a slurry.
  4. Place slurry material into a clean microcentrifuge tube labeled NC (Negative Control).
  5. Repeat steps 1-4 to prepare the test (unknown) food sample. *label the microcentrifuge used in step 4 for the test material U (unknown).
• You will have two sets of tubes. Label one set with NC (negative control, oats) and the other with a U (unknown, mango slice).
• Pipet 250 ml of the negative control ground slurry to the screwcap tube labeled NC.  
• Pipet 250 ml of the test (unknown) ground food slurry to the screwcap tube labeled U.
• Add 200 ml BioFluid & Cell Buffer (tube labeled BFCB) to both screw cap tubes (NC and U) (GMO lab)
• Add 20 ml Proteinase K (tube labeled PK) to both screw captubes (NC and U). (GMO lab)