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Construction of a recombination plasmid used in gene therapy to treat a genetic disorder

Use the simulation provided to construct a recombination plasmid used in gene therapy to treat a genetic disorder. The sequence in the cell DNA will simulate the gene for the normal version of the mutated gene for the disorder you are working on. The gene you are looking to change is one you identified from Newborn Screening Ontario. (the diseases – genes are as follows: Metabolic Diseases, Endocrine Diseases, Sickle Cell Disease (SCD)
Cystic Fibrosis (CF), Severe Combined Immune Deficiency (SCID), Spinal Muscular Atrophy (SMA), and Critical Congenital Heart Disease (CCHD)
As you prepare for, perform and explain the simulation, look for opportunities to practice good Organization skills. Also, look for an appropriate format to show how the shape and structure of DNA changes in a clear and organized fashion. Refer back to other ways that you have shown steps in changes earlier in the course. Practice using vocabulary related to molecular genetics bio technologies to describe the steps in creating this gene therapy vector.
Simulation: Using Recombinants
Materials Needed:

a printout of the three pages with plasmid DNA, cell DNA, and restriction enzymes (using specified colours of paper is optional but helpful.)
https://niagaracatholic.elearningontario.ca/content/enforced/16980908-EL_2021__OTH_SBI4UBa-997587_01_ELO/SBI4UU03/SBI4UU03A02/_teacher/U3A2RecombinantPlasmid.pdf?_&d2lSessionVal=RmR9y3rh59zYS765Cdus2WY79&ou=16980908 – link for them
scissors
tape

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Procedure:

Cut out the PLASMID strips from pink sheet #1. Set aside ANY TWO of the strips (except for the strip which contains the “origin of replication” site (see code at bottom of pink sheet). Shuffle the strips and tape the end of one to the end of another in any random fashion (as long as the letters are going in the same direction). After you have taped the four strips into one long strip, tape the two remaining ends together, to form one long circular paper plasmid. Save the key about antibiotic resistance, from the bottom of the pink sheet for later use. Save extra plasmid pieces until you have successfully completed #step 8
Cut out the CELL DNA strips from goldenrod sheet #2. They must be taped together in the order indicated at the bottom of each strip. That is, strip 2 is taped to the bottom of strip 1, strip 3 is taped to the bottom of strip two, etc. Note where the DNA code for the normal gene (protein gene) is located.
It is time to begin testing the various restriction enzymes that you have in your laboratory. Cut out ENZYMES from green sheet #3. There are 8 restriction enzymes given for cutting the DNAs and one ligase for fusing the DNAs together when done. Note that on each of the restriction enzyme rectangles, there is the name of the enzyme (such as Ava II) and a short DNA sequence that shows exactly what sequence that enzyme cuts.
Use a pen to mark on the PLASMID where each enzyme will cut. Draw the line accurately showing exactly where the bases will be cut apart (and leave the “sticky” ends). Label each. If you have no enzymes that will cut your plasmid only once, then reconstruct your plasmid.
Mark directly on the CELL DNA strip where the enzymes will cut. Draw the line accurately showing exactly where the bases will be cut apart (and leave the”sticky” ends). Write the name of the enzymes next to each line you draw.
Your job as a geneticist is to find a restriction enzyme that will cut open your plasmid at ONE site only (this may or may not be possible depending upon how you constructed your plasmid). The same enzyme should be able to cut your cell DNA at TWO sites, one above and one below the normal gene. It is very important that you find an enzyme that cuts as close to this gene as possible. Some of the enzymes cannot cut open your plasmid, some can. Some of the enzymes cannot cut your cell DNA at two sites, some can. If they don’t meet your needs they are not usable.
After you have completed testing the enzymes, select which ONE enzyme you would use to cut the plasmid and the cell DNA. Use scissors to make the cut in your plasmid and the cell DNA. Be careful to make the cuts in the staggered fashion made by the actual enzyme. This will expose the “sticky” ends where joining will be possible.
Since one enzyme was used, all “sticky” ends will be compatible. Use tape to splice the normal gene into the plasmid chain. You have created a recombination plasmid.
You are interested in confirming that your plasmid contains the normal gene for the disorder you are working on. Describe a test you could use to analyze your DNA.
The recombinant plasmid is now added to a test tube with the monomers to build the protein coat of viral particles.
The recombinant virus is now ready to be tested in medical trials to see if it cures patients and if it has any side effects.

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