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8) What sort of selection marker system will ensure that only E. coli transformed with pGLO will survive?
Can I have an answer for 7 and 8 based on the diagram.
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- Are the bands indicated in Step 6 (SDS-PAGE) likely visualized by western blot or by a general protein stain (such as Coomassie or silver)?For the beta-galactosidase assay, as controls, you plate bacteria known to be able to digest lactose (Lac+) and bacteria known to be unable to digest lactose (Lac-) on LB + X-Gal plates.The cellulase gene of Bacillus licheniformis was successfully cloned into the pET21a vector and expressed in Escherichia coli BL21. The pET21a vector consists of ampicillin resistant gene. To screen for the successful transformants, E. coli BL21 was cultivated on LB agar containing ampicillin (100 pg/mL) and 0.5% (w/v) carboxymethylcellulose, and incubated at 37 0C for 6 hours. After that the agar plate was stained with Congo red solution for 15 minutes and washed twice with sodium chloride solution and the observation is as shown in Figure 3. Answer the following: (i.) Briefly explain why ampicillin was added to the LB agar. (ii) Indicate the function of carboxymethylcellulose in the LB agar. (iii) Conclude how the researchers were able to identify the E. coli BL21 that carried the cellulase gene.
- According to Research and Development department analysisresults, it was concluded that a two-stage control strategy wasoptimal for antibody production. In the first stage, conditions wereoptimized for cell growth. In the second stage, conditions werechanged to halt cell growth. Please answer the following questions in 1-2 sentences. (a) Why did they choose this two-stage approach? (b) How did they cause the cells to shift from a growth phase to a quiescent (non-growth) phase? (c)Please discuss which factors influence the choice of bioreactor design for optimal antibody production (Hint: The production of antibody which is anchorage-dependentsystems from continuous cell lines can be accomplished usingseveral bioreactor configurations, each of which was designed tosatisfy differing operational, product quality, economic, andproduction scale requirements.)Please DESCRIBE, in outline form, the method you will use to select for bacterial cells that have taken up the pL311 plasmid, and to screen those cells for the presence of plasmids that are likely to contain a cloned gene. Be sure to mention the specific media you will use. In addition, please explain the rationale behind this specific selection and screening procedure. (Remember that you have available the following types of media: (i) media containing neither kanamycin nor X-gal, (ii) media containing BOTH kanamycin and X-gal, (iii) media containing tetracycline, and (iv) media containing ampicillin.A plaque assay is performed beginning with 1 mL of a solution containing bacteriophages. This solution is serially diluted 4 times by combining 0.1 mL of each sequential dilution with 9.9 mL of liquid medium. Then 0.1 mL of the final dilution is plated in the plaque assay and yields 21 plaques. What is the initial density of bacteriophages in the original 1 mL? Recall that initial phage density = (plaque number/mL) ×× (dilution factor).
- What internal references can be used in western blot of plasma membrane protein,expect Na-K ATPase??Salmonella strains developed for the Ames test contain many special phenotypes that make them more useful for these techniques. Many of these phenotypes, including UvrA and rfa, make these strains mutate more easily, and therefore make the assays more sensitive. In 1-2 sentences each, state how the UvrA and rfa phenotypes make Ames test strains more sensitive to mutations.A student said, “The approach shown by the Chicago researchers is just conventional gene therapy with biomaterial carrier”. Is he or she correct? Why? based on “Targeted polyelectrolyte complex micelles treat vascular complications in vivo”, PNAS 2021, Vol. 118 No. 50 e2114842118;107:110210; https://doi.org/10.1073/pnas.2114842118
- Differentiate the advantages and disadvantages of messenger ribonucleic acid (mRNA) by RT-PCR and protein via ELISA in cytokine analysis•There are two figures- one for vector control plate & another PCRinsert+vector experimental plate. Control plate = 10 blue colonies , Ligation plate ( experimental) = 150 white , 8 blue. Interpret what each figure means, what is its importance (control and experimental)? Why do colonies grow on vector control plates? Why are both blue and white colonies in your experimental plate? What were the components of the agar plate & their use (AMP+Xgal+IPTG)? What do these suggest about amp sensitivity and resistance? Why did we use X-gal and IPTG? Can you think of any issue with your result? Is the recombination efficiency good? Is your transformation efficiency good? •Write a concluding sentence about this lab and the data you analyzed.What is the difference between true, intragenic and a second-site reversion? Does the Ames test together with the s9 extract fall into any of these categories?