Prescott's Microbiology
10th Edition
ISBN: 9781259281594
Author: Joanne Willey, Linda Sherwood Adjunt Professor Lecturer, Christopher J. Woolverton Professor
Publisher: McGraw-Hill Education
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Chapter 13, Problem 3CHI
DNA polymerase I (Pol I) of E. coli consists of three functional parts (domains): an N-terminal domain with 5' to 3' exonuclease activity required for removal of the RNA primer, a central domain responsible for 3' to 5' exonuclease proofreading, and a C-terminal domain with polymerase activity. Pol I is thought to simultaneously remove RNA primers and fill in the gaps that result (figure 13.14). A group of proteins known as RNaseH also have 5' to 3' exonuclease activity and can thus remove RNA primers. However, they lack the other two functions observed for Pol 1. Predict the ability of the following mutants to replicate DNA: (1) a strain with a mutant gene encoding Pol I such that it no longer has polymerase activity (but retains both types of nuclease activities); (2) a strain without RNaseH proteins; (3) a strain with a mutant gene encoding Pol I such that it no longer has 5' to 3' exonuclease activity (but retains 3' to 5' nuclease and polymerase activities); (4) a strain with the mutant Pol I described in (3) and a strain lacking all RNaseH proteins. Explain your reasoning for each.
Read the original paper: Fukushima, S., et al. 2008. Reassessment of the in vivo functions of DNA polymerase I and RNaseH in bacterial cell growth. J. Bacteriol. 189:8575.
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1)Which plate did you see purple/pink/blue bacterial cells? Why did you see this growth? Explain your answer in terms of transformation and plasmids?
2) Calculating Transformation Efficiency
For the +DNA/+Amp/+IPTG plate, record the following:
Number of transformants (colonies): _________________
Nanograms of plasmid DNA added: 50 ng
Final recovery volume: 0.50 mL
Volume plated: 0.25 mL
Transformation efficiency equation:
Transformation efficiency = Number of transformants / µg of DNA x Final volume at recovery (mL)/ volume plated (mL)
3) Using the equation above, calculate the transformation efficiency.
4) Describe the success of the transformation efficiency of this demo based on the calculation you did above?
1) Look at the ideal results. Were your predictions accurate, and how did they compare with your results?
2) You used aseptic technique during this lab. Why is it important to work in a sterile manner when working with bacteria in the lab?
3) Why are the cells incubated at 42°C?
Overview of Transformation Protocol
-Prepare competent bacteria for transformation:
Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure.
Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed.
-Transformation procedure:
Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA.
Add CaCl2 to both tubes.
Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube.
Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes.
Add recovery broth to both tubes.
Incubate both tubes in a 37 C water bath for 5 minutes.
Questions:
1) What differences would you expect to see between the…
Chapter 13 Solutions
Prescott's Microbiology
Ch. 13.1 - MICRO INQUIRY Based on what we now know about...Ch. 13.1 - Retrieve, Infer, Apply 1. Briefly summarize the...Ch. 13.1 - Retrieve, Infer, Apply 2. Explain how protein was...Ch. 13.2 - MICRO INQUIRY To which carbon of ribose...Ch. 13.2 - MICRO INQUIRY How many H bonds are there between...Ch. 13.2 - Prob. 3MICh. 13.2 - Prob. 1RIACh. 13.2 - Retrieve, Infer, Apply What does it mean to say...Ch. 13.2 - Retrieve, Infer, Apply Amino acids are described...Ch. 13.3 - MICRO INQUIRY What provides the energy to fuel...
Ch. 13.3 - MICRO INQUIRY What is the difference between...Ch. 13.3 - MICRO INQUIRY Why cant DNA polymerase I perform...Ch. 13.3 - Retrieve, Infer, Apply How many replicons do...Ch. 13.3 - Retrieve, Infer, Apply Describe the nature and...Ch. 13.3 - Retrieve, Infer, Apply Outline the steps Involved...Ch. 13.3 - Retrieve, Infer, Apply What is the end replication...Ch. 13.4 - Why is the nontemplate strand called the sense...Ch. 13.4 - Retrieve, Infer, Apply The coding region of a gene...Ch. 13.4 - Which strand of a gene has sequences that...Ch. 13.4 - Briefly discuss the general organization of tRNA...Ch. 13.5 - MICRO INQUIRY Are the -35 and -10 regions...Ch. 13.5 - Retrieve, Infer, Apply Outline the transcription...Ch. 13.5 - Retrieve, Infer, Apply What is a polycistronic...Ch. 13.5 - Retrieve, Infer, Apply What is a consensus...Ch. 13.5 - Tabulate the similarities and differences between...Ch. 13.6 - Prob. 1MICh. 13.6 - Retrieve, Infer, Apply List the punctuation codons...Ch. 13.6 - What is the difference between a codon and an...Ch. 13.6 - Retrieve, Infer, Apply What is meant by code...Ch. 13.6 - Retrieve, Infer, Apply Is the genetic code truly...Ch. 13.7 - MICRO INQUIRY Why is simultaneous transcription...Ch. 13.7 - MICRO INQUIRY What would be the outcome if an...Ch. 13.7 - MICRO INQUIRY Why would it be impossible for...Ch. 13.7 - MICRO INQUIRY What provides the energy to fuel...Ch. 13.7 - Retrieve, Infer, Apply In which direction are...Ch. 13.7 - Retrieve, Infer, Apply Briefly describe the...Ch. 13.7 - Retrieve, Infer, Apply What are the translational...Ch. 13.7 - Retrieve, Infer, Apply Tabulate the nature and...Ch. 13.7 - Retrieve, Infer, Apply How many ATP and GTP...Ch. 13.8 - MICRO INQUIRY What are two distinguishing features...Ch. 13.8 - Retrieve, Infer, Apply What are molecular...Ch. 13.8 - Retrieve, Infer, Apply Would an intein-containing...Ch. 13.8 - Retrieve, Infer, Apply Give the major...Ch. 13.8 - Retrieve, Infer, Apply Which translocation or...Ch. 13.8 - Prob. 5RIACh. 13 - Streptomyces coelicolor has a linear chromosome....Ch. 13 - You have isolated several E. coli mutants: Mutant...Ch. 13 - DNA polymerase I (Pol I) of E. coli consists of...Ch. 13 - Prob. 4CHI
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- Overview of Transformation Protocol -Prepare competent bacteria for transformation: Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure. Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed. -Transformation procedure: Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA. Add CaCl2 to both tubes. Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube. Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes. Add recovery broth to both tubes. Incubate both tubes in a 37 C water bath for 5 minutes. Questions: 1)What is the selectable marker in this experiment? How…arrow_forwardBased on your results, which suspect's DNA best matches the DNA found at the crime scene?arrow_forwardIn oxidase test with Pseudomonas aeruginosa, the cell cultures on the slide turn colorless to be purple after tetra-methyl-p-phenylenediamine dihydrochloride (TMPD) is added. In the reaction, OTMPD is electron acceptor O cytochrome c is the electron source oxygen is terminal electron acceptor OH2 produced is electron donorarrow_forward
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