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Lesson #12 Gene cloning Genetics Agronomy 315 Questions due Monday, April 9th, 2012 11:59 pm Lab materials DNA from jellyfish cells DNA from Zebrafish cells DNA from coral cells mRNA from mRNA ground up coral cells Ground up jellyfish mRNA from Fin cells of a zebrafish materials to make bacterial growth plates with Amp + Xgal no antibiotic amphicillin (Amp) The following DNA modifying enzymes and biochemicals: restriction enzymes: EcoRI, KpnI, JelliI, CoralVI, BanHI E. coli Bacteria susceptible to all antibiotics other enzymes: Ligase, reverse transcriptase, DNA polymerase, RNA polymerase enzyme that adds sticky ends. radioactive material to label nucleotides, color enzymes to bind to antibodies antibodies that will only bind to the red Fluorescent protein from coral Other materials membranes that proteins and nucleotides stick to an electroporation machine pBluescript plasmid a gene gun zapper!! cd player, country western cds, rock cds Recombinant plasmid with an insert that is a clone of the jellyfish green fluorescent protein gene 1. Assume you are doing this to obtain a genomic clone of the red fluorescent protein (RFP) from coral. You are also trying to obtain a cDNA clone of the green fluorescent protein (GFP) gene from jellyfish. a. What item above is your starting material for your library that will contain the RFP gene clone? b. What item above is your starting material for your library that will contain the GFP gene clone? c. What restriction enzyme will you use to cut your plasmid? d. What enzymes will you need to make double stranded DNA from the starting material for your GFP library? e. Will the lab strain of E. coli you start with be susceptible to the AMP antibiotic or resistant to the antibiotic (this will be before transformation)? f. After you transform your E. coli, how could you tell the difference between bacteria that are transformed and those that are not transformed? g. When will you use ligase in your gene cloning work? h. How will you transform your bacteria? i. Assume that jelly fish or coral have 10,000 genes and they express 1000 genes in most of the specific tissues they have in their bodies. Which gene library will you need to make the largest in terms of number of bacteria colonies in order to be likely to contain a clone of your gene? Assume the petri dishes below contain colonies growing on Amp/Xgal obtained from transforming amp sensitive bacteria with the plasmids you made in #1 . j. Circle all of the colonies you would keep if you wanted to obtain a clone for your Genomic gene library. k. How many petri dishes like those shown above should you plan to work with if you want your library to obtain the desired cloned gene? ____ 2 ___ 12 ____ 100 ____ 1000 ___ 1,000,000 l. What would your Petri dish look like if you had forgotten to put X-gal into the media but plated the same number of bacteria onto the plate? m. What would your Petri dish look like if you had forgotten to put amp and X-gal into the media but plated the same number of bacteria onto the plate? Assume that after cloning the GFP, you form a company that makes the following plasmid to use in educational kits to teach students the gene cloning process. The plasmid used in this experiment is… 2. Arabinose sugar positively regulates the expression of GFP. Procedure: After transformation 1 2 3 4 3a. . Match the outcome below with the plate number above ___ will contain green glowing colonies ___ will contain no bacteria growth ___ will be a continual lawn or bacteria where colonies grew together ___ will contain only white colonies of bacteria that do not glow green 3b. Assume you want to make genetically engineered zebra fish that glow either green or red. You will use the gene you are cloning in your oral question (#1) 1. replace the promoter that is a part of your gene clone with a mouse cancer cell promoter 2. replace the promoter that is a part of your gene clone with a fish fin promoter. 3. add a fish fin promoter to your cloned gene 4. add a mouse cancer cell promoter to your cloned gene. 5. breed your transgenic zebra fish with non transgenic zebra fish to produce your new product for the pet owner market. 6. follow the fate of these cancer cells in your lab mice by observing the red or green fluorescing cells. 7. electroporate the clone and modified gene into a fertilized zebra fish egg 8. inject the cloned and modified gene into a fertilized zebra fish egg 9. combine the cloned and modified gene with a virus that invades fish cells and introduce this virus to zebra fish eggs. 10. combine the cloned and modified gene with a virus that invades cancer cells and introduce these cells into laboratory mice. 11. put the eggs in the right environment for development to fry. a. From the list of steps below, select the four that would be done to perform Genetic Engineering List their order below: First _____ then _____ then ______ followed by ______ b. Assume that cancer researchers use your cloned gene to follow the growth and spread of cancer cells in lab mice. Select the steps that would be done to perform this gene therapy procedure. List their order below: First _____ then _____ followed by ______ 3.c. Explain why even though we are transferring the jellyfish or coral gene to mice or fish and expecting them to make the fluorescent protein, we do not need to transfer ribosomes or tRNAs from jellyfish or coral to the genetically engineered fish or mice. _____3.d. Assume that you do an in vitro translation experiment with the following materials. Ribosomes from coral, tRNAs from jellyfish, mRNAs from zebra fish, amino acids from mice. The proteins made in vitro should be the same proteins found in the cells of.. a. coral b. jellyfish c. mice d. Zebra fish 4. If you sequenced a gene clone from your GFP library would you be able to determine the amino acid sequence of the protein encoded by this gene? 5. How about your RFP gene library? Remember, both jellyfish and coral are eukaryotes. 6. If the genetic code was not universal but all organisms used DNA as their genetic material, would the gene cloning process we are using in the problem set still work to help us identify bacteria that have a jellyfish or a coral gene? Assume the AMP resistance gene on our cloning plasmid uses the E.coli gene code. Explain. A B 7. Make sure you view the animation “Screening a DNA Library” to help you answer the following questions. a. Which molecules above that we would be trying to detect if this library was being screened with the antibody that binds to the RFP from coral. ___A ___B b. Label the molecules above that we would be trying to detect if this library was being screened with a cloned gene that was similar to the clone we were trying to find our DNA library. ___A ___B c. Screening a library with an antibody is similar what other molecular test we have learned about? ___ lateral flow strip test ___ PCR d. Screening a library with a similar cloned gene is based on the concept of “complementary sequences” . What other molecular test we have learned about that is based on this concept as well? ___ lateral flow strip test ___ PCR