Frederica Bimmel (the infant from Case 3: Wanda\'s Woes) needs your help! Of cou

ID: 178314 • Letter: F

Question

Frederica Bimmel (the infant from Case 3: Wanda's Woes) needs your help! Of course, she's all grown up now, having survived a rough first year back in 1975. She has no memories of her numerous hospital stays, but over the years her parent’s stories about her early struggles made a deep impression on Frederica, and she developed a keen interest in medical microbiology. This interest motivated her as she completed her M.D. degree, then her Ph.D. degree, followed by a Master's degree in epidemiology. She has become one of the world's leading experts on Streptococcus pneumoniae, and she has arranged an important meeting with Melinda and Bill Gates to try to convince him to fund her research to develop a new vaccine that will protect people from all serotypes of

S. pneumoniae. Like any good scientist, Dr. Bimmel prepared extensively for this meeting, including putting together a PowerPoint presentation. On the cab ride from the airport to the Microsoft campus, she received a phone call from one of the Gate's assistants with these words of advice: "You know, of course, that the Gates absolutely despises PowerPoint presentations, so I hope that you plan on avoiding that...they really likes to see ideas scratched out in pen or pencil on a piece of paper. Is that going to be a problem for you?" You happen to be sharing the cab, and so Dr. Bimmel turns to you for help: "What do you know about S. pneumoniae? Can you help me draw a few simple diagrams and write out some basic definitions and ideas? I have some innovative ideas for how we could reduce S. pneumoniae infections by taking advantage of: (i) its metabolic properties; (ii) microbial ecology; (iii) vaccine development. I’ll just tell you the ideas, and you can write them out on this paper!"

The genome of the S. pneumoniae strain Frederica is studying includes the following features that she needs to highlight in a simple diagram;

I. Several important chromosomal genes including:
a. pspA, a gene encoding pneumococcal surface protein A.
b. plyA, a gene encoding the S. pneumoniae pneumolysin.
c. an operon including ptsA ptsB ptsC ptsD encoding the components of a

phosphotransferase transport system that can transport glucose into the cytoplasm d. a gene encoding lactate dehydrogenase (ldh) and genes for each of the

glycolytic enzymes
e. blaA, a secreted protein that can enzymatically inactivate -lactam antibiotics
f. A lac operon (lacZ lacY lacA) encoding proteins very similar to their counterparts

in Escherichia coli.

II. A plasmid that carries:
a. pilA, a gene encoding the major protein of the pilus that mediates attachment to

epithelial cells in the lungs
b. pilZ, A gene encoding the adhesin for the PilA pilus

Several genes NOT PRESENT in the S. pneumoniae genome include:

those encoding TCA cycle enzymes

those encoding electron transport chain proteins

1. Be able to draw a single labeled diagram of S. pneumoniae that depicts this organism and the location of each of the features specified below:
a. Functional anatomy of S. pneumoniae

the shape of the organism;

the layers of the cell envelope including the capsule (Do label the layers; do NOT

draw individual lipid or carbohydrate molecules that are considered part of

the layers.)

do clearly indicate whether this is a Gram-positive or Gram-negative bacterium)

the location of the attachment pilus

b. Genetic anatomy of S. pneumoniae: Depict the location of pspA and blaA in your cell diagram.

c. Biochemical anatomy of S. pneumoniae – show (on your diagram) where you would expect to find: Teichoic acids, Pneumolysin, -galactosidase (LacZ), PspA, -lactamase (BlaA), PtsABCD (a multiprotein complex), PilZ

2. Frederica realizes that the Gates may not understand the genetic nomenclature contained within her drawing of the cell, so she decides that it would be helpful to prepare an additional figure that would explain the concept of a gene. She decides that her drawing should highlight the ptsABCD gene operon. Prepare a simple figure that shows how information is converted from a DNA molecule to a protein. Include in your figure the correct location of:
a. 5’ and 3’ ends of each molecule
b. the location of transcriptional start and stop sites
c. any promoters
d. ribosome binding sites
e. start and stop codons
f. amino and carboxyl termini of the protein
g. sites of operon regulation.

3. Functional components of S. pneumonia.
a. For each of the S. pneumoniae cell components listed below, briefly indicate how it

could contribute to the ability of this bacterium to cause disease in a human host (~1 sentence for each)

i. Capsule:
ii. Attachment (PilA) Pilus

iii. Pneumolysin iv. PspA

v. Teichoic Acid:
b. What term would a Pathogenic Microbiologist (or an Epidemiologist) use to refer to

the collection of cell components listed above?

c. Below are 3 media recipes. Which of these is (are) “complex”? Justify your answer.

Medium 1

Medium 2

Medium 3

Glucose 20 g

Sodium Citrate 50 g

Lactose 20

Peptone 5.0g

Ammonium phosphate 1.0 g

Beef extract 3.0 g

Sodium chloride 5.0 g

Sheep blood 10 g

Magnesium sulfate 0.2 g

Sodium Chloride 8.0 g

Potassium phosphate 1.0g

Agar 15.0 g

All 20 amino acids (200 g each)

Water 1 1iter

Guanine, adenine, thymine, cytosine, uracil (100 g each)

All known vitamins (25 g each)

Water 1 liter

S. pneumoniae can grow in Medium 1 and Medium 3, but not in Medium 2. Why?

Would E. coli be able to grow in media 2? Justify your answer.

A mutation in what gene would prevent S. pneumoniae from growing in medium 3.

Justify your answer. Why would this mutation have no effect on the ability of

S. pneumoniae to grow in medium 1?

4. S. pneumoniae is highly dependent

upon external sugars to meet its energy requirements (recall that it has neither an electron transport chain nor a TCA cycle). The transport system used by

S. pneumoniae for glucose is depicted in Figure 1.

What term best describes this

transport mechanism?

What is the immediate source of

energy that drives this transport system and allows S. pneumoniae to scavenge low

levels of glucose from its environment?

When S. pneumoniae is growing in the presence of oxygen using glucose as its

energy source, which metabolic pathway (Aerobic respiration, fermentation or

anaerobic respiration) would it use? Justify your answer.

What is the NET GAIN in ATP generated by S. pneumoniae catabolizing one

molecule of glucose during this type of metabolism? Remember to take into

account the cost of glucose transport. Show how you generated this answer.

5. When faced with a wide variety of carbon and energy sources, a bacterium has to make metabolic decisions, opting for preferential use of one source of carbon over another in

order to maintain optimal growth. Simultaneous utilization of all available sugars would be metabolically inefficient and would lead to slower growth. Like E. coli, the preferred sugar for S. pneumoniae is glucose. (The doubling time for S. pneumoniae growing

Figure 1. This is the phosphotransferase system that S. pneumoniae uses for the transport of glucose. (PEP is Phosphoenolpyruvate)

minimal media + glucose is 40 minutes, and the doubling time for S. pneumoniae growing minimal media + lactose is 60 minutes)

Draw three growth curves; one that would reflect the growth of S. pneumoniae

growing in the presence of glucose, one that would reflect the growth of S. pneumoniae growing in the presence of lactose; and one that would reflect the growth of S. pneumoniae growing in the presence of both sugars, where glucose is exhausted 80 minutes into log growth. Dr. Bimmel understands this is a sketch of the results, but is insisting you label your x- and y-axis properly and plot it properly using the data she supplied you.

Explain what is happening in each phase of the growth curve for all three curves.

Dr. Bimmel is concerned about eliminating the carrier state of S. pneumoniae. She is

particularly concerned about finding ways to prevent S. pneumoniae-caused otitis media without the use of antibiotics. Antibiotic use against S. pneumoniae has become problematic. Amoxicillin worked for her as a child, but today there are many antibiotic resistant strains.

Describe how amoxicillin affects bacterial cells. (What is the target site? What does amoxicillin do?)

What is the gene product of the blaA gene?

How does this gene product allow bacteria to survive exposure to amoxicillin?

Does blaA confer resistance to antibiotics other than amoxicillin (like tetracycline

and kanamycin)? Briefly explain

Show (describe) how the blaA gene could be naturally transferred from a

S. pneumoniae cell that is amoxicillin resistant to a S. pneumoniae strain that is susceptible to amoxicillin. Include in your response a description of the selection conditions required to select for amoxicillin-resistant S. pneumoniae.

Is this a genotypic change? Design an experiment to support your answer.

The Gates Foundation is interested in developing vaccines, and supports the Global Alliance for Vaccine Development (GAVI). He would be very interested in funding projects that provide novel delivery mechanisms for vaccines. Frederica has always

wanted to find a way to make a vaccine for S. pneumoniae that targets all of the serotypes! This may be her opportunity! Her goal is one vaccine that would be completely protective against all strains of S. pneumoniae. She has an idea to make an edible vaccine that would contain the antigen derived from S. pneumoniae. Frederica decides to try using yogurt as the delivery mechanism! She wants to clone a gene from S. pneumoniae that encodes an antigen and to express this gene in a microorganism that would grow in yogurt. Eating the yogurt would serve as the vaccine delivery. Consider the various molecules expressed by S. pneumoniae.

Choose one molecule that would be an appropriate choice to serve as the antigen for the vaccine preparation. What molecule have you chosen?

Explain how your choice would allow an immunized person protection from all strains of S. pneumoniae.

For the vaccine she must incorporate the gene for the S. pneumoniae antigen into a bacterium that will survive in the yogurt and will express the gene producing the antigen molecule. One organism found in yogurt is Streptococcus thermophilus. Frederica begins her plan to transform S. thermophilus with a plasmid vector encoding the antigen gene. First Federica must choose a plasmid cloning vector. Frederica understands that a gene

encoding resistance to antibiotics is commonly used to select for recombinant organisms (she does not want to include such a gene in her final construct for fear that it would be transferred to normal flora in the gut of the immunized children). As such, her final recombinant S. thermophilus strain will need some further genetic manipulations. However, for a phase one study, where the vaccine will be tested in the laboratory in animals, it is ok.

c. Prepare a drawing of the cloning vector that you would use. Be sure that your drawing contains all of the essential information needed to demonstrate:

i. That the plasmid will replicate in S. thermophilus
ii. Any other genes that you will need to identify your transformants.

d. Prepare a diagram that shows how to construct a recombinant plasmid that would contain the gene that encodes your antigen.

What is the role of restriction endonucleases in the process of constructing your plasmid?

What is the role of DNA ligase in this process?

Consider the plasmid that you have designed. How will you identify

S. thermophilus cells that have been transformed with this plasmid?

For the vaccine to be functional, the antigen needs to be expressed. Do you need

to add anything to the media to ensure expression? How will you determine if the protein is expressed?

Medium 1

Medium 2

Medium 3

Glucose 20 g

Sodium Citrate 50 g

Lactose 20

Peptone 5.0g

Ammonium phosphate 1.0 g

Beef extract 3.0 g

Sodium chloride 5.0 g

Sheep blood 10 g

Magnesium sulfate 0.2 g

Sodium Chloride 8.0 g

Potassium phosphate 1.0g

Agar 15.0 g

All 20 amino acids (200 g each)

Water 1 1iter

Guanine, adenine, thymine, cytosine, uracil (100 g each)

All known vitamins (25 g each)

Water 1 liter

Explanation / Answer

Answer:

In the medum 3 , where you have sodium citrate in the medium, S. Pneumonie will not grow at all because it lack the enzymes.

Similarly in medium 3, lactose is the growth medium and this bacteria lacks catalase enzyme to use lactose.

This bacteria can grow in the first medium as it has glucose.

Please make the question in a form .

This such a huge assays question ,

Navigate

Frederic Taylor applied the scientific method to management. In many cases, this

Frederick & Co. expects its EBIT to be $100,000 every year forever. The firm can

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.

Frederica Bimmel (the infant from Case 3: Wanda\'s Woes) needs your help! Of cou

Question

Explanation / Answer

Related Questions

Navigate