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- Refer to Figure , which describes the base modifications of bacteriophage T4 DNA, and briefly describe some issues that must be dealt with in preparing a restriction map of T4 DNA.Cloning vectors are not just limited to bacterial plasmids. Bacteriophages and M13 phage vectors are also commonly utilized in the cloning process. State any five (5) key criteria to be an effective cloning vector.You first need to create a plasmid. What are the minimal components necessary to develop this plasmid? In addition to these components, please draw the plasmid. In this illustration, I am looking for the components, the direction of transcription (i.e., the direction the genes will be transcribed), and what should be transcribed last? Also, where would you specifically insert the P. falciparum gene in this plasmid, and why are you checking reading frames in your gene? Finally, please name your plasmid using the correct nomenclature too. You are excited you have this new plasmid; you want to transfect it into P. marinus and provide it as an oral vaccine to laboratory mice. However, even though your supervisor enjoys your enthusiastic attitude, they do not allow you to do this quite yet because all you have is this plasmid. Why doesn’t your supervisor allow you to use the laboratory mouse for this research regarding animal welfare guidelines? Your answer should include the 3 R’s of…
- What is the role of X-gal? What color will cells with the plasmid be in the absence of X-gal? Why?A sample of E.coli cells is transformed with a plasmid containing a transcriptional fusion including the promoter of an E.coli housekeeping gene and the LacZ gene. The transformed cells are plated out on selective medium containing X-gal. Which of the following best describes the likely results. a. Colonies will only be blue if lactose is also included in the growth medium. b. Colonies will only be blue if lactose and glucose are included in the growth medium c. Most of the colonies should turn blue. d. Presence of any white colonies may indicate deletion or mutation of the promoter. e. Both c. and d. are correctIn the following "gene library" cloning experiment Digested genomic DNA AmpR gene TCR gene TCR is tetracycline resistant marker, AmpR is ampicillin resistant marker and BamHI is the unique restriction enzyme on plasmid. A PhD student digests/cuts the plasmids with BamHI restriction enzyme and the genomic DNA with EcoRI restriction enzyme. After performing the cloning experiment and obtaining colonies on a selection plate, the obtained cells will be ..... (Hint: this question is even more challenging; the PhD student was later demoted to an MSc student). a) resistant to ampicillin and tetracycline b) sensitive to tetracycline and ampicillin c) resistant to tetracycline and sensitive to ampicillin d) resistant to ampicillin and sensitive to tetracycline e) sensitive to ampicillin and tetracycline BamHI
- Assume you have successfully cloned a small (200 bp) fragment of DNA into the polylinker region of a pUC18 cloning vector. Describe the appearance of transformed colonies you would expect to see on each of the following plates: plain media, media containing ampicillin, media containing tetracycline, media containing ampicillin and X-Gal.Using a flow diagram, elaborate on how you would generate a recombinant plasmid that can be used for the expression of a therapeutic insulin.To determine if the antibiotic resistance in MH1 was carried on a plasmid, you first isolate the plasmid in MH1 using the plasmid DNA purification technique. Then, you transform bacteria that are not resistant to penicillin/ampicillin with the plasmid isolated from MH1. For the bacterial transformation experiment, you set up the three controls listed below. Match each control with its appropriate purpose (i.e. what it is controlling for) Please note: Transformed bacteria are bacteria that received the plasmid from MH1 and untransformed bacteria are bacteria that did not receive a plasmid. Testing to ensure that the bacteria used in the transformation experiment are viable (i.e. can grow on LB media) (Choose) [ Choose ) after transformation. Untransformed bacteria plated on LB only plate Testing to ensure that the bacteria used in the transformation experiment are viable (ie. can grow on LB media) before transformation. Transformed bacteria plated on LB only plate Untransformed bacteria…
- Part 1. Gene Cloning 1) How is a gene inserted into a plasmid cloning vector? Be specific, but be brief. No more than 2 (non-run-on) sentences please, and do not exceed this space. 2) A geneticist has transformed a vector with an ampicillin resistance gene into E. coli cells and plated those cells on media containing ampicillin. She comes back 24 hours later and sees only blue colonies on the media. a) How does she know that the cells growing on the media were successfully transformed with the vector? b) What does it mean that the colonies are blue?A plasmid has been modified such that it carries the gene for beta-galactosidase (lacZ) under the control of the lac promoter. Between the lac promoter and the lacZ gene is an empty multiple cloning site (MSC). This plasmid also carries ampicillin resistance. E. coli are transformed with this plasmid, and grown on nutrient agar containing ampicillin, IPTG, and the lactose analogue Xgal for 18 hours. Following incubation, the plates are examined. What will be the likely colour of the bacteria colonies?Now that you’ve isolated the gene and made lots of copies, you need to insert the gene into something you can manipulate and move into your lab strain of E. coli. You have access to the following vectors, either pBR322 or pUC19 (look them up here to see a map https://www.neb.com/products/dna-plasmids-and-substrates Please note the restriction sites in BOLD appear only once in the plasmid). You may use either vector. What is a vector? Which vector did you choose? Explain why you made that choice. What enzyme(s) will you use to place your fragment into the vector? Explain why you chose these enzymes. How will you move this construct into coli? What phenotype will tell you if the coli have taken up the plasmid? What phenotype will tell you if the coli have taken up the plasmid with the gasP gene?