IGEM:IMPERIAL/2006/project/Oscillator/project browser/Full System/TestingValidation/chemostat

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Outline

Chemostat2.JPG

This is a very breif summary of how a chemostat functions.

The Chemostat is a piece of biological equipment. It maintains a constant flow of media into the growth chamber and allows the waste to overflow. The waste contains cells and any proteins made by these cells. This keeps the population of cells constant as new cells are washed away.

Unlike a batch culture the bacteria’s growth is limited by the amount of nutrients entering the chemostat. This means that at low flow rates through the chemostat the bacteria will grow slowly, due to low nutrients, and at high flow rates the bacteria will grow fast. Similarly at low flow rates the bacteria will be washed out at a low rate and at high flow rates the bacteria will be washed out quickly. This means that the total number of bacteria inside the chemostat will remain constant. By changing the flow rate you only change the growth rate of the bacteria inside the chemostat. This has been extensively verified elsewhere and can be read about in more detail in (1).

The effect of dilution rate on proteins Produced by the bacteria

The effect of changing the dilution rate on the concentration of a protein will be the same weather it is secreted or not. This is because it is the media + cells which is being removed. Any arguments that there will be a diference in behavior due to secretion as there is a constant OD and the media is washed out are flawd. This is because both cells and media are washed out of the chemostat.


Growth Linkage

There are three ways that the concentrations of a protein produced by bacterial cells can behave. Which category the protein falls into depends on the way in which the production of a protein is related to the growth of bacterial cells. There are three categories.


  • Growth linked This is where the production of a protein is directly linked to the growth of a cell. Examples of this could be cell wall proteins where the production of the protein is a requirement for growth so is obviously growth linked. Most proteins are growth linked
  • Non Growth Linked and Pseudo Non Growth Linked. This is where the bacterial cells produce a set amount of protein per cell regardless of growth rate. Examples of non growth linked proteins this are synthesis of a few specific enzymes and some antibiotics. Many growth linked proteins can behave in a non growth linked fashion in some situations inside a chemostat. For example if the growth linked protein requires a specific metabolite for it’s production and that is at a sub optimal concentration then it will be produced at a maximum rate limited by the media and not the activation at it’s promoter or the energy charge of the cell and therefore not be linked to growth.
  • Partially Growth linked. Many proteins are growth linked for some growth rates and non-growth linked for others. This category is essentially for all proteins whose expression profile does not fit into the above two categories. Many examples of this are quorum sensing systems, virulence factors, conjugation genes or genes related to competency. It is incredibly difficult to make a predictive model of these systems as there is no way other than by experiment to test what will happen when you vary the growth rate.

Behaviour of Growth linked proteins

These will behave in exactly the same way as OD. Ie they will reach a steady state where the concentration inside the chemostat is not dependent on the flow rate through the chemostat. The mathematical proof is available (on page 158 of reference 1).

Behaviour of non-growth linked proteins

The concentration of these in the media will fall as dilution rate in the chemostat increases. This is due to washout of the protein and a constant production rate so you usually get an exponential decrease in the concentration of the protein.

Behavior of Partially growth linked proteins

These will show a combination of the above behaviours with short growth linked phases and short non growth linked phases.

Controlling OD

In a chemostat the OD is limited by the nutrient content of the medium being pumped in. LB is not a defined medium therefore the limiting nutrient may vary in concentration so the same portions of ingredients may lead to different ODs so to get round this we make an LB+Glycerol medium. So the glycerol is the main carbon source and the limiting nutrient. The LB simply provides other salts and ions. A second crucial difference between the chemostat and the batch culture situations is that in a batch culture the cells are showing unlimited growth and growing at their maximum rate. In a chemostat there is one limiting factor which is slowing the growth rate of the culture. If this is a carbon source (such as glycerol) then the cells will be limited by the amount of energy they have available and behave in a similar way to the unlimited cells (but Slower). If it is any other nutrient then the cells will display unusual behaviors to burn off the excess. So it is very important that the limiting nutrient is a carbon source.

This lead to a series of media experiments

Designing a media is said by many to be a dark art. It is almost imposible to accuratly predict how a bacteria will respond to any given media so we devised a series of ecperiments. First there was an LB dilution to determine the concentration of LB needed to produce a very low OD. Glycerol was then added on top of this dilute LB

Final iCOLI media

To make 1 litere

  • Glycerol 0.065%
  • Tryptone - 0.01%
  • Yeast Extract - 0.05%
  • (0.1M) MgSO4 - 8ml
  • (0.5M) CaCl2 - 80ul
  • NaCl - 2.5g
  • NH4Cl - 5g

To autoclave separately

  • Na2HPO4 - 33.906g
  • KH2PO4 - 15g

References

1. S. John Pritt Principles of microbial and cell cultivation (1975) Blackwell scientific publications