Question

CASE STUDY Part 1: You are a researcher working for the USDA. After 2 years of effort, you have isolated in pure culture a new, highly virulent bacterium from duck feces that is responsible for several major outbreaks of deaths in mammalian wildlife from contaminated pond water in the Southern US states. Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the gram-negative bacterium V. cholerae, and you have named the new strain Vibrio birdsii. Using high-throughput/Next-Gen DNA sequencing methods, your research group has completely sequenced and annotated the genome of V. birdsii. You have subsequently determined that V. birdsii is sensitive to ampicillin (i.e., it cannot grow in its presence). You have also obtained the following molecular "tools" that can be used in V. birdsii: (1) a Vibrio transposon that you can use to generate a transposon mutant library of V. birdsii; (2) a STM transposon system that you can use to generate a signature-tagged mutagenesis (STM) library of V. birdsii; (3) a "shuttle vector" that encodes ampicillin resistance and can replicate in both E. coli and V. birdsii; (4) a plasmid encoding ampicillin resistance that will integrate into the V. birdsii genome by homologous recombination (it integrates at a site known not to be involved in virulence in vivo). Furthermore, mice appear to be an excellent animal model for the disease caused by V. birdsii, which results primarily from uptake through drinking contaminated water, followed by colonization of the gut and bloody diarrhea, and then by invasion of intestinal cells with spreading to lymph nodes and spleen, and finally death by dehydration and/or organ failure. You are interested in identifying virulence factors associated with disease in mice. Part 2: Using a STM strategy to identify putative virulence genes in V. birdsii, a total of 2 putative virulence gene mutants were identified. Furthermore, a third virulence gene was also identified by screening individual mutants of a "regular" transposon mutant library for attenuated virulence in vivo. Sequencing of the chromosomal DNA immediately flanking the inserted transposon combined with bioinformatics tools yielded the following putative identification and information: Gene name (Based on annotated V. birdsii genome) --> Predicted function (based on sequence homology to known virulence factors of other bacteria) toxA --> Secreted enterotoxin sigY --> Alternative sigma factor that regulates genes involved in iron acquisition orf2559 --> Predicted outer membrane protein, no known function QUESTION 1. What technique could you use to determine the amount and different types of immune cells (T-cells, B-cells, etc) present in mice infected with the toxA mutant strain? A) Flow cytometry B) Biophotonic imaging C) RNAseq D) IVIAT QUESTION 2 Which of the following are not advantages to using signature-tagged mutagenesis (STM) to identify bacterial virulence genes important for pathogenesis in vivo? A) Since STM is essentially a competition experiment between many mutants growing in a single host, trans complementation effects could be observed. B) STM bypasses the need for initial in vitro screening for virulence phenotypes. C) A relatively small number of animals are utilized in screening STM mutants D) STM is a method that can be applied to a wide variety of bacteria.

Name: case study part 1 you are a researcher working for the usda after 2 years of effort you have isolated in pure culture a new highly virulent bacterium from duck feces that is responsible for 59297
Uploaded: 2023-06-20T07:52:07-08:00
Duration: 3 min 20 s
Channel: Adi S
Description: case study part 1 you are a researcher working for the usda after 2 years of effort you have isolated in pure culture a new highly virulent bacterium from duck feces that is responsible for 59297

CASE STUDY

Part 1:
You are a researcher working for the USDA. After 2 years of effort, you have isolated in pure culture a new, highly virulent bacterium from duck feces that is responsible for several major outbreaks of deaths in mammalian wildlife from contaminated pond water in the Southern US states. Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the gram-negative bacterium V. cholerae, and you have named the new strain Vibrio birdsii.

Using high-throughput/Next-Gen DNA sequencing methods, your research group has completely sequenced and annotated the genome of V. birdsii. You have subsequently determined that V. birdsii is sensitive to ampicillin (i.e., it cannot grow in its presence). You have also obtained the following molecular "tools" that can be used in V. birdsii: (1) a Vibrio transposon that you can use to generate a transposon mutant library of V. birdsii; (2) a STM transposon system that you can use to generate a signature-tagged mutagenesis (STM) library of V. birdsii; (3) a "shuttle vector" that encodes ampicillin resistance and can replicate in both E. coli and V. birdsii; (4) a plasmid encoding ampicillin resistance that will integrate into the V. birdsii genome by homologous recombination (it integrates at a site known not to be involved in virulence in vivo).

Furthermore, mice appear to be an excellent animal model for the disease caused by V. birdsii, which results primarily from uptake through drinking contaminated water, followed by colonization of the gut and bloody diarrhea, and then by invasion of intestinal cells with spreading to lymph nodes and spleen, and finally death by dehydration and/or organ failure. You are interested in identifying virulence factors associated with disease in mice.

Part 2:
Using a STM strategy to identify putative virulence genes in V. birdsii, a total of 2 putative virulence gene mutants were identified. Furthermore, a third virulence gene was also identified by screening individual mutants of a "regular" transposon mutant library for attenuated virulence in vivo. Sequencing of the chromosomal DNA immediately flanking the inserted transposon combined with bioinformatics tools yielded the following putative identification and information:

Gene name (Based on annotated V. birdsii genome) --> Predicted function (based on sequence homology to known virulence factors of other bacteria)
toxA --> Secreted enterotoxin
sigY --> Alternative sigma factor that regulates genes involved in iron acquisition
orf2559 --> Predicted outer membrane protein, no known function

QUESTION 1.
What technique could you use to determine the amount and different types of immune cells (T-cells, B-cells, etc) present in mice infected with the toxA mutant strain?
A) Flow cytometry
B) Biophotonic imaging
C) RNAseq
D) IVIAT

QUESTION 2
Which of the following are not advantages to using signature-tagged mutagenesis (STM) to identify bacterial virulence genes important for pathogenesis in vivo?
A) Since STM is essentially a competition experiment between many mutants growing in a single host, trans complementation effects could be observed.
B) STM bypasses the need for initial in vitro screening for virulence phenotypes.
C) A relatively small number of animals are utilized in screening STM mutants
D) STM is a method that can be applied to a wide variety of bacteria.

Added by Encarnacion M.

Biology for AP Courses

Julianne Zedalis, John Eggebrecht

Instant Answer

Solved by Expert Adi S

06/20/2023

Step 1

The best technique for this purpose is: A) Flow cytometry For Question 2, we are looking for the option that is NOT an advantage of using signature-tagged mutagenesis (STM) to identify bacterial virulence genes important for pathogenesis in vivo. The correct Show more…

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CASE STUDY Part 1: You are a researcher working for the USDA. After 2 years of effort, you have isolated in pure culture a new, highly virulent bacterium from duck feces that is responsible for several major outbreaks of deaths in mammalian wildlife from contaminated pond water in the Southern US states. Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the gram-negative bacterium V. cholerae, and you have named the new strain Vibrio birdsii. Using high-throughput/Next-Gen DNA sequencing methods, your research group has completely sequenced and annotated the genome of V. birdsii. You have subsequently determined that V. birdsii is sensitive to ampicillin (i.e., it cannot grow in its presence). You have also obtained the following molecular "tools" that can be used in V. birdsii: (1) a Vibrio transposon that you can use to generate a transposon mutant library of V. birdsii; (2) a STM transposon system that you can use to generate a signature-tagged mutagenesis (STM) library of V. birdsii; (3) a "shuttle vector" that encodes ampicillin resistance and can replicate in both E. coli and V. birdsii; (4) a plasmid encoding ampicillin resistance that will integrate into the V. birdsii genome by homologous recombination (it integrates at a site known not to be involved in virulence in vivo). Furthermore, mice appear to be an excellent animal model for the disease caused by V. birdsii, which results primarily from uptake through drinking contaminated water, followed by colonization of the gut and bloody diarrhea, and then by invasion of intestinal cells with spreading to lymph nodes and spleen, and finally death by dehydration and/or organ failure. You are interested in identifying virulence factors associated with disease in mice. Part 2: Using a STM strategy to identify putative virulence genes in V. birdsii, a total of 2 putative virulence gene mutants were identified. Furthermore, a third virulence gene was also identified by screening individual mutants of a "regular" transposon mutant library for attenuated virulence in vivo. Sequencing of the chromosomal DNA immediately flanking the inserted transposon combined with bioinformatics tools yielded the following putative identification and information: Gene name (Based on annotated V. birdsii genome) --> Predicted function (based on sequence homology to known virulence factors of other bacteria) toxA --> Secreted enterotoxin sigY --> Alternative sigma factor that regulates genes involved in iron acquisition orf2559 --> Predicted outer membrane protein, no known function QUESTION 1. What technique could you use to determine the amount and different types of immune cells (T-cells, B-cells, etc) present in mice infected with the toxA mutant strain? A) Flow cytometry B) Biophotonic imaging C) RNAseq D) IVIAT QUESTION 2 Which of the following are not advantages to using signature-tagged mutagenesis (STM) to identify bacterial virulence genes important for pathogenesis in vivo? A) Since STM is essentially a competition experiment between many mutants growing in a single host, trans complementation effects could be observed. B) STM bypasses the need for initial in vitro screening for virulence phenotypes. C) A relatively small number of animals are utilized in screening STM mutants D) STM is a method that can be applied to a wide variety of bacteria.