Daily Newsletter March 9, 2012

Daily Newsletter March 9, 2012

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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:

1. Describe the general characteristics of B. subtilis, and the specific characteristics of GSU14PHA and GSU92PHA.
2. Explain the consequences of having an NADH oxidase knock-out.  How could the cell maintain a proton motive force without NADH oxidase?
3.  What is the consequence of having a pyruvate decarboxylase knockout?
4. Describe the typical fermentation pathway (to lactic acid).  In describing the purpose of fermentation, explain why there is a need to oxidize NADH + H⁺.
5. Give a brief account of quorum sensing, and how it affects cell physiology.
6. 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.
7.  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.
8. 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.
9.  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:  10⁸ M sec^-1, 10⁹ M sec^-1, 10⁹ M sec^-1.

QUESTION set Beta:

1. Using the strain selected above, which alterations will maximize production?  Explain and justify your answer.
2. 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?
3. What is a transcriptional rate, and how does it influence the proteome and metabolome?
4. What is a phosphoreductase, and will it provide an advantage to our B. subtilis system as it tries to make PHA?
5. 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?
6. What is a restriction site, and how is it used in genetic engineering?
7. What changes will you have to make, if any, to your system to compensate for any genetic alteration?
8. 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?
9. 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.