Microbiology MOOC Newsletters: March 2012

Friday, March 30, 2012

Daily Newsletter March 30, 2012

Daily Challenge: Eukaryotic Microbes and Humanity

In our continued discussion of the Eukaryotic Microbes, today you are to discuss the relationship of Eukaryotic Microbes to Humanity.

This is an open-ended challenge. You could talk about the environment, diseases, evolution, industry, or just about anything as long as it deals with the interaction between Humanity and Eukaryotic Microbes.

The goal of this is for you to explore this complex and diverse grouping of organisms and find those items that fascinate or inspire you. Your not expected to write about EVERYTHING, just about what you find as fun.

Thursday, March 29, 2012

Daily Newsletter March 29, 2012

Today's Topic: Protozoa
Suggested Reading: Introduction to Protozoology

The Kingdom Protista (Protoktista) is currently undergoing massive restructuring based upon genetic analysis. New classification schemes are being proposed and debated. These schemes break up the algae (protophyta) into different phylla, and even rearrange some families and genera. The same is occurring for the non-photosynthetic unicellular eukaryotes commonly referred to as the Protozoa ('proto' meaning earliest form of, and 'zoa' meaning animal).

The award winning evolutionary biologist Thomas Cavalier-Smith has been at the forefront of the changes in taxonomic structure for over twenty years. His reclassification of the protozoa is now well regarded among parasitologists and evolutionary biologists. Below is a 'Tree of Life' commonly used by parasitologists, and is based on the work of Cavalier-Smith.

Note that animals, plants and fungi are reclassified as Kingdom Opisthokont. This is due to the idea that animals, plants and fungi have genetic and metabolic commonalities that separate them from all other life (they are a monophyletic group).

Do you see how the genetic revolution has altered our appreciation of the diversity of life?

Why is it important for scientists to create a tree of life that is really based on genetic and metabolic relatedness?

While it is critical to recognize that the classification of protists is being rethought, it is important to understand some of the main protozoal groups. The proposed kingdoms most linked to Protozoa are:

Rhizaria
Excavata
Amoebozoa
Aveolates (not listed in above chart)

Daily Challenge:Take one of these protozoal groups and write about them. What are some of the species now held in this new grouping? Why are they now being considered related to each other, but different from other protists? Are there any species that are relevant to medicine, industry, food?

Wednesday, March 28, 2012

Daily Newsletter March 28, 2012

Today's Topic: Fungi
Suggested Readings: Doctor Fungi (a great site for general medical mycology).
The Web of Life: Fungi (Good general introduction).

I like fungi. Their fun, do some amazing things, and perform critical function in our ecosystems. They also tend to be the least discussed in biology classes. Why? Few people work on them. They are not as high profile as viruses, or not as well known as the pathogenic bacteria. Save for the fungi used in food, we usually think of fungi in a negative light (you have mold! Kill it, Kill it, Kill it).

Fungi though have been the work horse of genetics, and yeast has been used for baking and brewing for thousands of years. So what is so interesting? Here is a small list:

Ascospores can be used to demonstrate meiosis, and provides a great visual of Mendelian genetic at work.
Fungi are great models of eukaryotic genetics and molecular processes.
Most of our antibiotics are derived from Fungi.
Fungi can be used to fight insects (biocontrol)

Some fungi can even lasso nematodes in the soil (the purpose is to acquire nitrogen)
http://youtu.be/0n04wCkIpuQ

Fungi are used as food and to prepare food.
Some fungi spoil food (bread mold)
Fungi are even now being used as replacements of Styrofoam: http://green.blogs.nytimes.com/2009/04/13/using-fungi-to-replace-styrofoam/
They are decomposers (natural and use in industrial settings).
Mycorrhizal fungi help plants to grow.
Some fungi are plant pathogens.
Pathogenic Fungi of animals and mycotoxins.

Daily Challenge: Pick one topic about fungi, and write about it.

Tuesday, March 27, 2012

Daily Newsletter March 27, 2012

Today's Topic: Harmful Algal Blooms

Algae inhabit many areas of the world. There are marine and freshwater algae, soil algae, and those that form symbiotic relationships to produce lichens. All algae have photosynthetic capabilities, which means that they are primary producers (first level of a food chain), and are oxygenic (produce oxygen).

There are secondary metabolites that algae can produce, for example the Rhodophyta (Red Algae) produce Agar. This gelatinous substance is used for microbiological media, and in cooking. As a culinary agent, it is used as a substitute for pectin to produce jellies, jello, and other gelatinous confections. It is also used as a gelling agent in ice cream. (What is the chemical structure(s) of Agar?)

Not all phytoplankton secondary metabolites are so useful or desirable. Some algae produce toxins, most often neurotoxins. At low concentration levels, they are of minimal risk. The problem is that they can bioaccumulate in marine organisms, most notably shellfish.

The diatom Nitzschia navis-varingica produces the toxin domoic acid, which in high doses, causes permanent loss of short term memory and brain damage. Death is a possible outcome with high concentrations of the toxin.
The dinoflagellate Alexandrium fundyense produces saxitoxin, the toxin responsible for paralytic shellfish poisoning (PSP). PSP has multiple symptoms, that usually start with abdominal cramps and diarrhea, but progress to mental functions and loss of coordination. As the name implies, paralysis is an advanced symptom.
Karenia brevis, a dionflagellat, produces brevetoxin, which causes neurotoxic shellfish poisoning (NSP). NSP has no reported fatalities, unlike the previous two toxin based diseases, but can cause painful abdominal problems, slurred speech, and other neurological problems.

Harmful algal blooms are often reffered to as Red Tide, a phenomena where the algal population is so large that it becomes visible on the surface of marine environments. Red Tides can be beautiful, but harmful algal blooms are not always red. The blooms also don't follow tides, they generally stay in an area (the cells produce a film that congeals them).

Daily Challenge: Pick one of the harmful algal blooms above and discuss it in more detail. Describe the algae and it's habitat. When and why would the algae experience a bloom? Describe the toxin, and the affect it has on human phisiology. Why doesn't the toxin affect shell fish? Describe the signs and symptoms of the disease condition (signs are observable, while symptoms are personal to the patient; e.g., patient core temperature or blood pressure is a sign, nausea or a feeling of unease is a symptom).

Monday, March 26, 2012

Daily Newsletter March 26, 2012

This week's topic: Eukaryotic Microbiology

When we talk about microbiology, most peoples thoughs immediately turn to bacteria and viruses. There are though a number of Eukaryotic microbes. It is important to remember that microbiology is ultimately the study of organisms too small to see with the naked eye.

Why then do we spend so much time on bacteria? The answer is rather simple. Most of your training has been on eukaryotic cells. Principles of Biology, as well as Cell and Molecular Biology, focus on eukaryotic systems. As you have seen, bacterial and archaeal systems are different. We need time to go through those differences, and also talk about the importance and impact of these other two domains of life.
Are Eukaryotic Microbes as important as bacteria? YES!

Phytoplankton, which is made up of both photosynthetic bacteria and algae (photosynthetic microscopic eukaryotes) produce most of the world's oxygen.
Fungi are important decomposers, and are needed to recycle nutrients in the environment.
The mycorrhizal fungi are needed for the healthy growth of many plants.
Yeasts are needed in bread making and brewing.
Protists (microscopic animal-like eukaryotes) are important predators of other microbes.
Protists are also medically important.

Daily Challenge: Common Characteristics
When doing taxonomy, it is important to have a clear idea about the common characteristics that seperates one group from another. To that end, you are to compile a list of common characteristics for the Algae, Protists and Fungi. Make sure you discuss these characteristics! Questions to start you off:

Is the organism heterotrophis or autotrophic?
Is the organism predatory or a detritivore?
What is the organization of the cell?
Does the cell have a cell wall, and if so, what is it made of?
Any special cellular characteristics, such as unique lipids in the membrane?
Any special metabolic features?
Remember Alternation of Generations? How would you classify the organism?
How does the organism reproduce?
Are there any important reproductive structures or strategies common to the group?

Friday, March 23, 2012

Daily Newsletter March 23, 2012

Daily Challenge: Archaea
Pick one Archaea that you find interesting, and write about it. Write up a "personal profile" of the organism. Beyond the basics of taxonomy and habitat, highlight the special features of the cell.

Summer Course:
BIOL 4930, CRN 53934, is a topics class with a focus on Medical Mycology. The instructor, Dr. Errol Reiss, is retired from the CDC and is a mycologist of note. As we have few classes that focus on Fungi, I would strongly suggest that you take this opportunity to learn some of the medical implications of Fungi.

Thursday, March 22, 2012

Daily Newsletter March 22, 2012

Daily Challenge: As with bacterial diversity, archaeal diversity is best handled by looking over the organisms. Today I would like you to review and write about the Methanogens. Describe their taxonomy, habitat, and metabolic features. Include a discussion methane production.

Wednesday, March 21, 2012

Daily Newsletter March 21, 2012

Today's Topic: Archaea

The Archaea represent the third domain of life, and as such posses characteristics that make them different from either Bacteria or Eukarya. They are prokaryotic, but with some unique features. Your goal today is to learn what makes Archaea different from Bacteria.

You may have heard in the past that Archaea are extremophiles, living only in extreme environments. This is a lovely fiction. While many Archaea live in extreme environments, it is not a requirement. Some even live in moderate environments. So what then makes them different?

First, they are prokaryote, which makes them different from Eukarya (there is more than just this, but let's wait for a moment). Like Bacteria, they have cell walls, but their cell walls are not based on peptidoglycan. Instead, the Archaea form an S-Layer to act as the cell wall.

The Archaea produce transmembranal proteins to create a schaffold for globular proteins. These globular proteins are then arraied to form a protective coating similar to "chain mail" (see the image below). Some Archaea, such as members of the Order Methanobacteriales add pseudomurein to add additional support and strength to their cell walls. Pseudomurein is similar to peptidoglycan, but lacks the ordered arrangement of glycan chains.

Another major difference between the Archaea and both the Eukarya and Bacteria is in the formation of their cell membranes. You will recall discussions of phospholipids and cell membranes. You may remember that we always discuss phospholipids as having ester linkages between fatty acids and the gylcerol backbone. This is not the case with Archaea. Archaea use an ether linkage between fatty acids and the glycerol backbone. In addition, archaeal fatty acids can be connected across the membrane, resulting in monolayers (instead of bilayers). A molecule with two polar heads seperated by a non-polar region is known as a bolaamphiphile.

Consider the Archaeal phospholipid: What advantage would an ether linkage provide? What advantage would a bolaamphiphile provide? Would you make these the same way you would make a bacterial phosophlipid? What type of metabolic pathways would you have to have? Based on this physiological difference, can you compare the evolutionary history of these two organisms?

You will also note in the above diagram that the fatty acids are also different. Archaeal fatty acids are based on isoprenoids and they can have cyclopropane and cyclohexane ring structures. While there are other differences to Archaeal phospholipids, digest on what we have mentioned for now. One thing to note: while the chemical architecture may be different, Archaeal phospholipids serve the same function as other phospholipids, and their membranes have the same function as other cellular membranes.

Unlike Bacteria, but like Eukarya, Archaea use Methionine on the initator tRNA. There are also some structural differences between the Archaeal tRNA, and those found in Bacteria and Eukarya. As with bacteria, Archaea lack introns, and do not undergo splicing.

They have 70S ribosomes, but their ribsomes are different enough from Bacteria that the ribosome effecting antibiotic chlramphenicol does not affect Archeal ribosomes. Conversely, the Eukaryotic ribosomal toxin anisomycin also affects Archaeal ribosomes. So while they are bacterial in size, they so similarities to the Eukaryotic ribosome.

Another anomylous protein is DNA-dependent RNA polymerase (RNA polymerase that makes mRNA). While it is a single enzyme, like Bacteria, it is a complex protein that more closely resembles the subunit patterns of Eukaryotic DNA-Dependent RNA Polymerase. Also, the antibacterial compound Rifampicin, which blocks mRNA transcription in bacteria, does not work on either Archaea or Eukarya. Arhaea also have RNA polymerase II type promoters, just like Eukarya.

Archaea are neither Bacterial or Eukaryal, but they share some characteristics. They also have some unique characteristics.

Archaea have a single circular genophore (molecule of DNA), just like Bacteria. They also have a process analogous to conjugation for horizontal gene transfer. It has been shown though that Archaea possess histones, and while they don't function just like Eukaryal histones, they share a common ancestry.

Daily Challenge: Archaeal Characteristics
In your own words, describe the general characteristics of the Archaea. It has been hypothesized in the past that the Archaea are related to the Proto-Eukaryotic cell. What is your view on that with what you have read? Justify your answer.

Tuesday, March 20, 2012

Daily Newsletter March 20, 2012

Daily Challenge: Bacterial Diversity

Today, there are four bacteria for you to look at:

Genus Bdellovibrio
Genus Agrobacterium
Genus Rickettsia or Wolbachia
Genus Anabaena

The task today is to discuss the special features of these organisms. As you will note, I'm only asking you to look at the special features of the Genus level (not individual species). Review them, and think about how this may influence our understanding of microbiology, biology in general, or biotechnology.

Special Announcement:

It's Spring Today!

Monday, March 19, 2012

Daily Newsletter March 19, 2012

Special Video: In honor of St. Patrick's Day, here is a little biology video. Can you describe all of the biological processes the singer presents? Could you describe all of these if asked?

Today's Topic: Diversity

After our discussion on bacterial evolution, it is time to turn our attention to the wealth of bacterial diversity found on Earth. As you are hopefully starting to see, there are more bacteria on Earth than any other life form. Even a human being is 10 times more bacterial than eukaryotic. Many bacteria have unique biochemical pathways and interesting metabolic processes. The metabolic diversity of bacteria is incredible, with bacteria and archaea being able to perform chemical processes that do not occur in eukaryotes. Some of these chemical transformations scientists believed only humans could manage in a lab.

The only way to learn about bacteria diversity is to start learning about the different organisms. Today and tomorrow will be fairly straight forward. I want you to discuss bacteria. On Wednesday, we'll move to Archaea.

Daily Challenge: Diversity
Below, you will see three groups of organisms. Pick one organism from each group and write up a synopsis of the bacteria.

Important Information:

Taxonomy.
Habitat.
Human Interaction (Medical, Environmental, Research, Industrial)
One important metabolic feature.

Group 1: Purple Phototrophic Bacteria, Nitrifying Bacteria, Sulfur Oxidizing bacteria, Iron Oxidizing bacteria, or Methanotrophs.

Group 2: Neisseria, Vibrio, Shigella or Mycobacterium

Gropu 3: Bacillus, Streptococcus, Streptomyces, or Flavobacterium

Administrative NOTE: Milestone Paper 2
You have until tomorrow night to finish your paper reviews. Authors can then go back and leave comments for their reviewers.

Friday, March 16, 2012

Daily Newsletter March 16, 2012

Today's Topic: Bacterial Evolution
As we have discussed this week, bacterial evolution is slightly different from Eukaryotic evolution. Natural selection is the same; what changes is the inheritance patterns. Hardy-Weinberg equilibrium (equation)does not hold for haploid organisms, and we can not follow inheritance through Mendelian genetics.

So, how does it compare to Eukaryotic evolution? Can we use information from bacterial evolution to inform eukaryotic evolution? The answer is YES.

Bacteria are a simple system to use in labs. The genome is smaller than eukaryote, and they grow faster. This means faster replication. As you may remember, the greatest risk of mutation is in the replication event. Outside of replication, you mutate only due to chemical or extreme environmental conditions. Replication errors are the main source of mutation. So why is mutation important? It is the foundation for all variation (allelic variation within a gene).

First off, as the article demonstrates, we can use bacterial models to show us how a beneficial mutation arises, and how long it takes for such a mutation to occur. It takes a sequence of multiple mutations to make a beneficial variation, and most of the time the mutations just lead to neutral results. There is also the loss of individuals who had negative mutations. In therms of time frame, we are looking at 10's of thousands of generations. Can this be compared to mutation rates in eukaryotic cells? Yes. It helps us to understand how we get radical phenotypic variations among individuals of a population or species.

Second, we can use bacteria as models for genomic comparisons. Looking for similarities and differences in a genome is laborious, but can be done. Using a simple system, such as bacteria, allows researchers to test procedures and hypotheses, as well as building algorithms to run the mathematical comparisons. In other words, we can create research systems in bacteria that we could transfer to studying eukaryotic genomes.

Take some time and look around the internet. Look at bacterial evolutionary studies. What do you find? What interests you? Provide references for what you find interesting, and talk about it. Ultimately, your question is: How do studies in bacterial evolution inform our understanding of bacteria?

Wednesday, March 14, 2012

Daily Newsletter March 14, 2012

Today's Topic: 16S rRNA based Phylogeny

Suggested readings for the day:

In class today, we talked about mechanisms of Bacterial evolution and using rRNA and other gene sequences to determine phylogenetic relationships among bacteria. The above resources should help you learn a little more regarding bacterial phylogenetic trees.

Daily Challenge: Why is it important?
Why are phylogenetic trees important? Why do we need to know evolutionary relatedness among bacteria? Why do we need to appreciate the mechanisms of bacterial evolution?

Admin Note: Remember that you need to upload your papers into SWoRD http://sword.lrdc.pitt.edu/sword/

As stated in class, just in case it closes tonight, I have given a late window to make sure you can get it uploaded. Make sure you upload it by tomorrow night.

Tuesday, March 13, 2012

Daily Newsletter March 13, 2012

Today's Topic: Is Prokaryotic Evolution Different?

Yesterday, you were asked to review the theory of evolution. Fresh off of that review, here is a question: Is evolution the same for prokaryotes as it is for eukaryotes?

Consider the following:
Do prokaryotes have an alternation of generations?
Do they move between haploid and diploid states?
Do they follow Mendelian Genetics?
Do Mendel's first and second laws work with bacteria?
How does prokaryotic evolution fit with the Modern Synthesis of Evolution?
Do bacteria experience natural selection?

Reflect on this statement: Bacterial evolution represents a sequence of random mutations and accumulation of foreign genes, with resulting surviving generations acquiring further genes or gene modifications providing stronger adaptation potential.

How does this statement fit what you know of evolution? Is it correct? Are there problems with it?

Remember: Evolution is not goal oriented, and there is no "Final Product". Evolution is a population level phenomena, not an individual event.

Read the abstract, introduction and conclusion of the following article:

Blount ZD, Borland CZ, Lenski RE. Inaugural Article: Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proceedings of the National Academy of Sciences. 2008;105(23):7899–7906.

(you can gain access through GSU library)

A helpful website is Bacterial/Prokaryotic Phylogeny.

Today's Challenge: Describe Prokaryotic Evolution
From your textbook, your review of evolution yesterday, and information from today, write up a discussion on the mechanism of bacterial evolution. Two things to consider while writing: 1) The source of all new genetic variation is mutation, and 2) bacteria experience horizontal gene transfer.

Administrative Note: The Website for Milestone 2 will be open later today. You will receive a special notice regarding the new website when it is ready to accept papers.

Monday, March 12, 2012

Daily Newsletter March 12, 2012

Administrative Note: Upon the recommendation of a colluege, you are going to try a different peer review service. You will be given an opportunity after this review to comment on which you found more useful and esier to use.

More information about the service will come when the site opens after 5pm tonight. You will have until Wednesday night to upload your papers. You can continue to review until Sunday night (you must review all papers given you to).

Today's Topic: Bacterial Evolution

What do you remember about evolution?
Think for a minute. Could you define evolution? If someone asked you to explain evolution, could you? Today, I want you to do an evolution refresher.

Go through the tutorial sections An Introduction to Evolution and Mechanisms: The Process of Evolution. This tutorial entitled Evolution 101 is part of an effort by Berkeley University to increase awareness and understanding of evolution. It is an excellent resource to refresh your understanding of evolutionary theory and a great place to find out about modern evolutionary research.

I would also recommend reading Problem Concepts in Evolution, for a good discussion of misconceptions and rebuttal against perceived problems with evolutionary theory. The Evolution FAQ from PBS is another excellent resource. A final site of interest is the Index of Creationist Claims, which includes rebuttals to each claim.

Take time to consider evolution. You will need a good foundation for what we will be talking about this week.

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Daily Challenge: During a debate on whether Evolution should be taught in high schools, you have been asked to provide a 10 minute explaination on how biologists view evolution and why the concept of evolution is considered central to biology. NOTE: your doing this from the perspective of a biologist. If you hold religious opinions, you may share them, but only after you explain the scientific theory of evolution. (Remember a theory is a robustly supported hypothesis, and thus it is based on accumulated data, i.e., facts.)

Friday, March 9, 2012

Daily Newsletter March 9, 2012

Daily Challenge: At this stage of the course, you have gone over a number of different features of bacteria, from their genetics to metabolism, and now growth. Today, I want you to consider the following:

A few weeks ago, there was a discussion of an organism that could produce Polyhydroxyalkanoates (PHA). You looked at the metabolism of PHA, and three different strains of Bacillus subtilis that could produce PHA.

From this week, we went over microbial growth. Yesterday's assignment had you looking at concepts of fermentation. With this in mind, consider the following:

Some bacteria have the ability to produce large carbon polymers for energy storage that appears as inclusion bodies within the cell. Polyhydroxyalkanoates (PHA) is a commercially important linear polyester that is produced by certain bacteria. PHAs can be used as either a thermoplastic or an elstomeric material, and is biodegradable. Many biodegradable plastics are now made from PHA.

You have been asked to maximize the production of PHA. One pathway for PHA biosynthesis involves the use of acetyl CoA:

It is known that b-ketothiolase is inhibited by unbound CoA. Coenzyme A is unbound when there is free NAD⁺ in the cell. All three proteins in this pathway are constitutively produced.

You have three strains of Bacillus subtilis (Gram +, neutrophile, mesophile, chemoorganoheterotroph) that are capable of PHA biosynthesis:

GSU14PHA – has a knock-out of the gene for NADH oxidase (the first step in the electron transport chain), is a fermentative anaerobe, and a generation time of 90 minutes.
GSU92PHA – is a facultative anaerobe with a pyruvate decarboxylase knockout. It has a generation time of 15 minutes. This strain requires that the fermentation conditions change when you reach production level population by going to a low oxygen level.
GSU03PHA – is a facultative anaerobe that was the first in house successful PHA producer. It has a generation time of 10 minutes. This strain requires that the fermentation conditions change when you reach production level population by going to a low oxygen level, adding excess carbon, with minimal nitrogen and phosphorus (phosphate).

The organism will begin producing PHA when the population density has reached 10⁹ cells/ml. The metabolic information can be found here.

NOTE: The question sets below are for your CONSIDERATION. Take notes on the questions, but don't sit and try to answer them fully.

QUESTION Set Alpha:

Describe the general characteristics of B. subtilis, and the specific characteristics of GSU14PHA and GSU92PHA.
Explain the consequences of having an NADH oxidase knock-out. How could the cell maintain a proton motive force without NADH oxidase?
What is the consequence of having a pyruvate decarboxylase knockout?
Describe the typical fermentation pathway (to lactic acid). In describing the purpose of fermentation, explain why there is a need to oxidize NADH + H⁺.
Give a brief account of quorum sensing, and how it affects cell physiology.
Given 100ml of a starter culture with 10⁶ cells/ml, a 40L fermentation take, and a desired population of 10⁹ cells/ml, how long will it take you to get to the production level? Show all of your work. Make sure that you tell how long each strain will take to get to production levels.
Based upon growth to production level and metabolic characteristics, which strain is best suited for production? Explain and justify your answer. You must be able to justify using numbers, and explain what the numbers mean.
Describe the concept of a rate limiting step, and using examples from the next page, show how a rate limiting step can alter the production of a desired end product.
In the introduction, it states that you have to change conditions for GSU92PHA and GSU03PHA before production can begin. Why would you start with one condition (such as to reach production population levels) and then change conditions to start producing PHA?

To improve production, you have a number of genetic alterations that you can perform with in-house plasmids and materials.

Plasmid pPHA12 holds a PHA operon, which contains coding regions for all of the genes needed to convert Acetyl CoA into PHA. In addition, the promoter is changed, having a transcriptional rate of 2.8.
Plasmid pPHA13 holds a Phophoreductase that can use the reducing power of NADH.
Plasmid pAN34 has a restriction site that will knockout NADH oxidase, and holds a phosphoreductase that can be used to oxidize NADH.
Plasmid pPHI32 has restriction sites that allow integration into the chromosome, it holds the PHA operon, but knocks out hexokinase.
Plasmid pPH132 acts as a conversion to the PHA operon, holding mutations of the genes with the following changes in Kinetics: 10⁸M sec^-1, 10⁹M sec^-1, 10⁹M sec^-1.

QUESTION set Beta:

Using the strain selected above, which alterations will maximize production? Explain and justify your answer.
Describe an operon, and discuss the advantages a bacterium has by using an operon instead of the way in which eukaryotes transcribe genes. What advantage does the eukaryote have?
What is a transcriptional rate, and how does it influence the proteome and metabolome?
What is a phosphoreductase, and will it provide an advantage to our B. subtilis system as it tries to make PHA?
What is hexokinase, and what complications can arise with this knockout? How would you have to change your Fermentation process? How would it change bacterial growth?
What is a restriction site, and how is it used in genetic engineering?
What changes will you have to make, if any, to your system to compensate for any genetic alteration?
What changes will you have to make when you up-scale your fermentation to 1,000L? What do you have to consider when moving from 400L to 1,000L?
Describe other genetic alteration you could use that would result in greater production. Be sure to explain your answers.

The Challenge: After considering these questions, reflect on what you have learned. With all the information above, how would you OPTIMIZE this system to produce PHA? Giving a brief description of the organism is important, and the reasons why you selected a specific organism. Provide any genetic modifications you think would be important, and any information as to the culture conditions that would enhance production. This is a thought questions! Take your time and reflect.

For this blog, your word limit is set to 150 words.
You can get up to 8 points for a well considered discussion.
You'll get 5 points for an average disucussion.
You'll get 2 points for just the minimum.

Thursday, March 8, 2012

Daily Newsletter March 8, 2012

Daily Topic: Variations in plant photosynthesis
As discussed in class, there are variations in how plants experience photosynthesis. The C₄ plants have a spacial separation of CO₂ acquisition and utilization. The CAM plants have a temporal seperation of CO₂ acquisition and utilization. In bacteria there are even more variations, as some species can fix carbon through a reverse TCA process.

The image below is one example of a reverse TCA (Kreb's Cycle).

Daily Challenge: Alternative Carbon Fixation routes
Your task today is to look at different ways organisms can fix carbon. Focus your discussion on the C₄ and CAM plants. Discuss how they separate CO₂ acquisition and fixation. Discuss the habitats of plants with these modifications. Discuss why this modification is helpful (hint: discuss photorespiration). Finally, take a moment to discuss why bacteria may have reverse TCA reactions.

Learning objectives: What are you suppose to learn from this exercise? Why are these modifications important? Is it just about knowing different routes of carbon fixation? What is the main goal of learning about different routes of carbon fixation?

Daily Newsletter March 8, 2012

Daily Challenge: Bacterial Growth
You are to work with the bacterial growth equations and your understanding of bacterial growth factors to answer the following questions.

You have been hired to maximize the production of indene by Rhodococcus. Indene is a precursor used in the manufacturing of the AIDS medication Crixivan™. Given conditions of growth are 4% lactose, 3% yeast extract, oxygen saturation of 18%, and a pH of 6.8. You have ten 10ml stored cultures of the organism, with an OD that is equivalent to 10⁵ cells/ml for this organism. Your fermentation vessel holds 15L, and is aerated and agitated to prevent the formation of biofilms (these cells need to be planktonic). The cells have a lag period of 1 hour when first introduced to a new culture from storage, and will enter late log phase when there are 10⁸ cells/ml. From looking at a colleague’s research notes, you find that the bacterium has a generation time of 25 minutes. How long will it take to reach late log phase?
Hint: You are adding stock cultures to a 15L tank. Have you diluted these cultures? What then is your starting culture?

You have been told that you must maintain late log phase for 48 hours in order to produce a sufficient quantity of Indene. How would go about doing this?
Hint: You will need to go back to the textbook and other references to look at fermentation and continuous cultures. This is not about a "right" answer as much as it is about your thought process.

You have been asked to upscale the production to a 10,000L fermentation tank. What complications could arise when upscaling from a 100L fermentation vessel?
Hint: What problems would come up if you had more liquid or a larger volume? Give this some thought. Think about pH effects of metabolizing organisms, temperature differences in a large body of liquid, oxygenation issues.

Someone suggests that you double the lactose and yeast extract concentrations. What effects could this have on your culture (explain)? How would you go about seeing if this was a good or bad idea?

Give a brief description of Rhodococcus. What is a biofilm?

Learning objectives: What are you being asked to do in these questions? Is this about just using the growth equations? How do conditions affect bacterial growth? Is it important to be sensitive to culture environment and conditions when growing bacteria?

Wednesday, March 7, 2012

Daily Newsletter March 7, 2012

Today's Topic: Bacterial Growth Formula

Today we are going to do a little math. Don't get scare, it is simple, but powerful. A critical concept that all microbiologists, and even cell biologists, must deal with is the rate at which cells grow. Even if you never use these equations again, going through them will build a mental picture of rate at which bacterial cells can multiply, and how large populations can become. On a practical side, it is critical as it helps you to understand when your population reaches a point that would be equivalent to a late log stage population; a working culture as it were.

The core bacterial growth formula is N_t = N_o x 2ⁿ

N_o is the original culture, or the 0 generation.
t is time.
n is the number of generations that bacteria goes through
N_t is the final population size (the population at a specific time interval).

This formula is based on the binary fission form of bacterial growth, and describes the log phase of growth.

Thus, it describes the doubling of a bacterial population at every time interval. This time interval is dependent upon the organism, and the environmental conditions.

Using this formula, you can solve more meaningful problems than just the doubling of bacteria. Question: What would be a more meaningful question to ask about bacterial growth?

n = the number of generations a population has undergone.

n = (log N_t – log N_o) / log 2 = (log N_t – log N_o) / 0.301

So if you know the original population size, and the final population size, you can discover the number of generations a bacteria has gone through. Through mathematical manipulation, we can solve for other problems. In the next formula set we look at the generations per hour of a given bacterial culture.

k = the mean growth rate of a bacterial population (generations per hour).

k = n/t = (log N_t – log N_o) / (0.301 x t)

If we know the original and final population size, we can extrapolate n, the number of generations that have occured in the population. If we divide n by the time it took, you will get k, the number of generations that occur per hour. Why would you want to know the number of generations per hour?

An inverse of k allows us to learn how many hours it takes to have one generation. This is also known as a generation time. If you ever read that an organism has a generation time of x minutes, you are looking at the solution to the problem below. As you can see, this provides a powerful tool in understanding an organism.

g = the mean doubling time of a bacterial population (hours per generation).

g = 1/k

Will an organism have the same g in all environmental conditions? Why?

Daily Challenge: Problems
Here are some problems to help you go through these equations. Show your work and answer the associated questions.

Problem: You have been asked to growth, in batch culture, a population of Rhodococcus spp. (the designation ssp represents a generic species; either the species is unknown, unnamed, or in this case unimportant). You are using lactose as a carbon source, and providing a complete array of other essential nutrients by adding Yeast Extract to the nutrient broth. Rhodococcus has a doubling time of 20 minutes in this environment. You start with 100 ml of a 104 culture. Your fermentation vessel is 10L (so it contains 10L of broth, including the starting culture). How long will it take to reach a population of 10^9,?
HINT: What are you looking for? number of generations? generation time? Something else? Which formula will give you the answer?

Question: Using the same system, an assistant attempts to replicate your system, but instead of lactose, they use sucrose as the carbon source. After 72 hours, they have a population of 10⁷. What was the growth rate of this particular species of Rhodococcus in this environment? Was this a more effective environment?

Question: Using the original fermentation equipment, you are asked to grow a population of Rhodococcus spp. to 10^{8,/sup> in 24 hours. Is this possible? Explain.

Learning Objectives:

What would you describe as the learning objectives today? Are you being asked to memorize these equations? Are you being asked to understand these equations? Are you being asked to use these equations? How could you use these equations?}

Tuesday, March 6, 2012

Daily Newsletter March 6, 2012

Today's Topic: Binary Fission
Bacteria do not undergo mitosis or meiosis. They lack a nucleus, thus there is no nuclear division.

When we have an organism that undergoes a replication event followed by cellular division, the process is called binary fission (splitting into two). The assumption is that the two daughter cells will be genetically identical (plasmids that don't replicate can weaken this assumption).

There are some questions you should consider:

What does a cell need to do before it can divide? (Hint: Biomass)
In eukaryotic cells, there is a signal that controls the cell cycle (CDK), are their signals that control bacterial division?
Without a nucleus, how do you move the genophore (DNA molecule) to opposite sides of the cell?
How long does this process take? (Hint: Each species is different)
Is the time to division constant, or is it variable? Why? (Hint: Conditions)

Whenever you come to a topic like cellular division, you need to "think like a cell". Cells ultimately want to survive, and leave healthy daughter cells that survive. You want to increase your population (that is the biological imperative at a cellular level). So how do you do it, and make sure that the daughter cells are fit for survival?

Daily Challenge: Binary Fission
Describe how a bacterium divides into two daughter cells. Look at possible signals, environmental conditions, and even nutritional requirements that may affect the rate of division. See if you can find one bacterium that is considered slow growing and one fast growing. What is different about them?

Monday, March 5, 2012

Daily Newsletter March 5, 2012

Administrative Note: There are two notes today.
1) This is a milestone week. On Thursday you will have your milestone exam.
2) We have passed the midpoint of the semester, and this is when a planned change is to occur. You will notice that each news letter has guiding comments or questions to help you build your own Learning Objectives for this week.

Today's Topic: Vocabulary

Generally your not asked to work on vocabulary as a sole topic, but in this case it is important to become familiar with various terms used to describe optimal conditions for bacterial growth. Your challenge today will involve working through this vocabulary.

Today's Challenge: Vocabulary.
Provide a definition and example for each of these terms:

Defined media
Complex (undefined) media
Broth (liquid) culture
Solid (agar) culture
Aseptic Technique
Pure Culture
Chemostat
Batch culture
Psychrotroph
Psychrophile
Mesophile
Thrmophyle
Hyperthermophile
Acidophile
Alkaliphiles
Halophiles
Osmophiles
Xerophiles
Obligate Aerobes
Facultative Anaerobes
Obligate Anaerobes
Microaerophiles
Aerotolerant anaerobes*
Superoxide
Fastidious
Growth Factors
Trance Minerals

*Aerotolerant Anaerobes: This is an oxygen utilization term that is often down played. There are bacteria that do not require oxygen as a terminal electron acceptor, but they are able to survive in atmospheric oxygen. This is different than the obligate anaerobes which are killed (or inhibited) in atmospheric oxygen. Look to the Streptococci as aerotolerant.

Learning Objectives:
This first newsletter has an easy objective, learn the terms listed above. By writing out a definition and an example, you will have a better grasp on these terms. You will see these terms repeatedly, as they help us to identify growth conditions for bacteria. It would benefit you to learn them.

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