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Assume you are doing a project where you need toDNA obtain a genomic clone of the pBluescript plasmid membranes that to proteins and nucleotides, red color enzymes bind to antibodies enzyme that adds sticky ends. antibodies that will only bind to the red fluorescent protein (RFP) from coral and a cDNA clone of the green fluorescent nucleotides stick to protein (GFP) gene ofradioactive a jellyfish. Fluorescent proteintofrom Recombinant plasmid with an material to label nucleotides, color enzymes bind tocoral antibodies insert that is a clone of the jellyfish an electroporation green fluorescent protein gene machine antibodies that will on Fluorescent protein fro a. What item above is your starting material for your library that will contain the RFPpBluescript plasm Other materials clone? a genegene gun DNA from coral cells zapper!! cd player,membranes that proteins and nucleotides stick to country western cds, b. What item above is your starting material for your library that will contain the rock cds GFP gene clone? an electroporation machine mRNA from jellyfish a gene gun Recombinant p insert that is a c green fluoresce pBluescript plasmid pBluescript plasmid zapper!! c. What restriction enzyme will you use to cut your pBluescript plasmid? cd player, BamHI, EcoRI, or KpnI country western cds,(it must cut in the Lac-Z region) zapper!! rock cds Recombinant plasmidDNA withfrom an the d. What enzymes will you need in order to make double stranded insert that is a clone of the jellyfish starting material for your GFP library? green fluorescent protein gene Reverse transcriptase, DNA polymerase e. Will the lab strain of E. coli you start with be susceptible or resistant to the AMP antibiotic (this will be before transformation)? Susceptible Recombinant plas insert that is a clo green fluorescent f. After you transform your E. coli, how could you tell the difference between bacteria that are transformed and those that are not transformed? Transformed bacteria will be able to grow in the media with antibiotics because the plasmid carries a gene for resistance. g. When will you use ligase in your gene cloning work? To ‘seal in’ the DNA that you inserted into the plasmid. h. How will you transform your bacteria? Electroporation (or heat shock) i. Assume that jelly fish have 10,000 genes in total, but they express ~1,000 genes in most of the specific tissues. Which type of gene library would require the most bacterial colonies to isolate a clone of your gene? Genomic library j. Assume the petri dishes below contain colonies growing on Amp/Xgal obtained from transforming amp sensitive bacteria with the plasmids you made in #1. Circle all of the colonies you would keep if you wanted to obtain a clone for your Genomic gene library. All the white colonies 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? All the colonies would be white. You would know the colonies had plasmids, but not if they contained added pieces of DNA in those plasmids. 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? It would be a lawn of bacteria. All the bacteria would be able to grow (transformed or not) and all the colonies would be white. 2. Assume that after cloning the GFP you form a company that makes the plasmid below. Arabinose sugar positively regulates the expression of GFP. Arabinose sugar After transformation you plate some transformed and some untransformed bacteria on the media types shown below. 1 2 3 4 A. Match the outcome below with the plate number above _2_ will contain green glowing colonies _3_ will contain no bacteria growth _4_ will be a continual lawn or bacteria where colonies grew together _1_ will contain only white colonies of bacteria that do not glow green araC Transcription Factor protein Arabinose sugar B. Arrange these steps in the proper order to describe the function of the araC and the GFP genes on this cloning vector. Hint: GFP is under negative regulation. _3_a. araC transcription factor protein binds to GFP promoter _1_b. araC gene is transcribed _4_c. arabinose sugar binds to the araC transcription factor protein _5_d. araC protein falls off the GFP promoter _2_e. araC mRNA is translated _6_f. GFP is transcribed, GFP mRNA is translated, GFP glows green inside the E. coli cell C. If you were going to use the pGLO plasmid above as a cloning vector and use the restriction enzyme HindIII, where would you want the HindIII restriction enzyme cutting site? ___a. Amp gene _X_b. GFP gene ____c. araC gene D. Which colonies would you select from the plates to make your gene library using the vector? ___ a. green glowing colonies _X_b. colonies that DID NOT glow green _____E. 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 3. If you sequenced a gene cloned from your GFP library would you be able to determine the amino acid sequence of the protein encoded by this gene? Why? Yes, the gene can be read in codons to determine the amino acid sequence because each codon codes for only one type of amino acid. 4. How about your RFP gene library? Remember, both jellyfish and coral are eukaryotes. No, because it is a genomic clone. This means the introns have not been spliced out and we have no way to know what areas are introns and what are exons. The bacteria itself is incapable of splicing out the introns. 5. 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. Yes, the DNA would still be inserted and it would still interrupt the reporter gene on the plasmid. The plasmid would still give the bacteria resistance. You could not, however, screen the library with an antibody because the E. coli would not make the proper protein. A B 6. Make sure you view the animation “Screening a DNA Library” to help you answer the following questions. a. Which molecules above would we be trying to detect if this library was being screened with the antibody that binds to the RFP from coral. __X_ protein ___DNA sequence b. Which molecules above would we be trying to detect if this library was being screened By sequencing followed by database analysis? ___ protein _X__DNA sequence c. Which molecules would we be trying to detect if this library was being screened by PCR followed by gel electrophoresis? ___ protein _X__DNA sequence
