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13 Cards in this Set

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What type of medium is used for plating the conjugation mixture in the Transposon Experiment, Part a?
- LB Amp
- LB Cm
- LB
- LB DAP Kan
- LB DAP
LB DAP
What type of medium is used for plating the diluted cell suspension in the Transposon Experiment, part b?
- LB DAP Kan
- LB Amp
- LB Kan
- LB DAP
- LB
LB
What will be the results of the Idole test for true Serratia marcescens mutants?
Negative
Which of the following elements are necessary for replication of the pRL27 plasmid?
- tnp gene
- oriR6K
- Pi protein
- tra genes
- oriT
oriR6K
Pi protein
Which of the following elements are characteristics of the recipient strain used for conjugative transfer of the pRL27 plasmid?
- It is Kan r (Kan resistant).
- It can replicate the pRL27
plasmid.
- It cannot replicate the
pRL27 plasmid.
- It is E. coli.
It cannot replicate the pRL27 plasmid.
Which of the following were the negative controls used in the transposon mutagenesis experiment?
- The conjugation mixture was plated on LB_DAP medium.
- The donor and recipient cells were plated separately on LB medium.
- The conjugation mixture was plated on LB-Kan medium.
- The donor and recipient cells were plated separately on LB-Kan medium.
- The donor and recipient cells were plated separately on LB-DAP medium.
The donor and recipient cells were plated separately on LB-Kan medium.
What is the function of the oriT site?
- This is the site where the plasmid DNA is nicked prior to transfer.
- This is the site to which the Pi protein binds.
- This is the origin of replication of the pRL27 plasmid.
- This is the target site for transposon insertion.
- This is the excision site of the transposon.
This is the site where the plasmid DNA is nicked prior to transfer.
In the Transposon Mutagenesis Characterization experiment, cells from a white colony were analyzed using various tests. All of the following test results might suggest a contaminant rather than Serratia marcescens EXCPET which one?
- unable to hydrolyze
esculin into esculetin.
- coccal shaped
- stained purple in the gram
stain.
- no fermentation in the Of-glucose test.
Unable to ferment lactose.
Usually a naturally occurring conjugal plasmid has most the genes and DNA sites needed for conjugation (Note: pRL27 was constructed in the lab and is not a naturally occurring conjugal plasmid). However, what enzyme is needed for the conjugation process to take place that is encoded by genes found on the chromosomes of the donor and recipient cells?
DNA polymerase (and other enzymes involved in DNA synthesis)

Both the donor and recipient cells need to contain genes that encode DNA polymerase. During conjugation, a ssDNA plasmid is transferred to the donor, while the other ssDNA strand remains in the recipient. DNA polymerase is needed to synthesize the second strand producing a complete copy of the plasmid in both donor and recipient.
What important step preceded the step of combining the donor and recipient cells for mating and why was that step important?
Centrifuging the tube containing the donor cells and removing the supernatant. The pelleted cells were then resuspended in LB broth.

This step was important as the donor culture medium contained kanamycin and the recipient cells are sensitive to kanamycin. The kanamycin must be removed or else the recipient cells would be killed by the antibiotic.
How does the aph gene confer antibiotic resistance to the bacterial cell that possesses it?
The aph gene encodes an enzyme that modifies kanamycin by transferring a phosphate group to the antibiotic. This modification causes kanamycin to no longer bind to the small ribosomal subunit so it can no longer inhibit ribosome function and protein synthesis.
In the transposon mutagenesis experiment, explain how the selection was performed and how it prevented specific bacterial cells from growing.
The selection involved plating the cells from the conjugation mixture onto a medium containing kanamycin.

The recipient cells do not carry pRL27 (or the aph gene) that confers kanamycin resistance, so they are sensitive to this antibiotic and will not grow (protein synthesis is inhibited).

My Answer: The selection medium was an LB-Kan plate. Since the selection plate contains kanamycin, the only cells that can survive are the cells that contain the pRL27 plasmid, coding kanamycin resistance. The recipient cells that successfully received and transferred the plasmid into their chromosome through conjugation will survive along with the donor cells. The counter selection medium lacked DAP. Since the counter selection media doesn’t contain diaminopimelic acid (DAP), the only cells that can survive are the cells that are capable of synthesizing DAP. DAP is a component of the peptidoglycan layer, strengthening the cell walls.The donor strain of E. coli are not able of synthesizing DAP and cannot survive on a media that does contain DAP to survive. The only cells that can survive the selection and counter selection medium are recipient cells that successfully encode kanamycin resistance.
Where is DAP mainly found and what is a function of DAP?
DAP (diaminopimelic acid) is found in the cell wall or peptidoglycan or peptide crosslinkages.

Its function is to connect the peptidoglycan strands together to strengthen the cell wall to prevent osmotic lysis and death of the cell.