Difference between revisions of "Team:Amsterdam/Project/Phy param/Synechocysytis"

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One faces two main problems when trying to study the stability of the aforementioned parameters in <i>Synechocystis</i>. One is how to maintain a culture originated from the same population growing for many generations giving the opportunity to the cells to accumulate mutations and evolve. One option is to grow the cells on a flask until they reach the lag phase then reinoculate a fraction of this culture to a new fresh medium. Although this method allows the population to evolve it can take a long time to observe mutations because during the  lag and the stationary phase of bacterial growth cells do not divide. This can seems trivial but due to the slow growth rate of  <i>Synechocystis</i> it can make a difference.
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Turbidostat are culture devices that allow to maintain the cells in constant exponential growth. This is achieved by automatically diluting the culture, i.e. pumping in fresh medium and pumping out culture medium, when a certain threshold is reached. Its name refers to the fact that the turbidity of the medium, so  the amount of cells, it maintained (-stat from static) on a certain range. This requires a three compartment system where the first contains the cultures, the second holds a reservoir of fresh medium and the third collect the medium extracted from the cultures. Pumps move the medium between containers. The cell density is recorded at regular intervals by a spectrophotometer and based on this value a software decides when the culture is diluted.
 
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  <img src="https://static.igem.org/mediawiki/2015/d/db/Amsterdam_turbidostat.gif" alt=" Turbidostat">
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  <figcaption>Figure 3. - How a turbidostat works for dummies. </figcaption>
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  <figcaption>Figure 4. - The multicultivator. </figcaption>
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This system allow us to reduce the time of observing a mutation but now it comes the second problem. How do we know if a mutation leading to a change in the parameters has happened? The first option that comes to our synthetic biologist mind is by sequencing periodically the genome of the population and screen it for mutations. The major drawback of sequencing is that it does not inform whether the mutation affects the parameters or not. The remaining solution is then to estimate the physiological parameters with high time definition to spot when the mutation has occurred. Using the turbidostat it would be possible to store all the values recorded by the spectrophotometer and calculate the growth rate from these data. This is done by fitting a linear model to the logarithm of the OD over the time. The production rate can obtained by periodically measuring the amount of product in the medium (link to protocols for enzymatic assays and HPLC).
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Luckly for us  when we came to the lab there was a master student, Joeri Jongbloets{link}, who had transformed a multicultivator(MC) like this{link} in a 8 channel turbidostat. For his internship he wrote the program connect with the MC hardware and control the pumps to dilute the culture when necessary. In addition the software stores the measurements from the MC spectrophotometers in a database and analyzes it to obtain the growth rate. Thanks to this impressive piece of software engineering we were able to run long term experiments and observe evolution.
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Revision as of 01:25, 18 September 2015

iGEM Amsterdam 2015

Synechocystis physiological parameters

Counting cells, counting molecules

Overview

Background

Estimating physiological parameters is necessary for building meaningful models that can help us to rationally design our consortium. In addition production stability can be assessed by estimating this parameters over a long period of time give.

Aim

We aim to use different cultivation strategies to Characterize growth and production rates of carbon producing Synechocystis strains their stability.

Approach

We have used turbidostat cultivation system to determine the stability of the physiological parameters

Results

  • We have demonstrated the instability of classical engineering strategies. As shown in figure 1 the strain SAA023 lose the ability to produce lactate after 300 hours of continuous culture.
  • We show that the Qp in the growth coupled producer Δacs remains constant over the time (figure 2).
  • We have estimated Qp and growth rates for six Synechocystis (Table 1).
.

Lactate Qp and growth
Figure 1. - Growth and Qp of strain SAA023 over 900 hours of continuous culture showing the drop in production.
Acetate Qp and growth
Figure 2. - Growth and Qp of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.
Qp and growth
Table 1. - Estimated Growth and Qp for different Synechocystis strains.

How to measure stability?

One faces two main problems when trying to study the stability of the aforementioned parameters in Synechocystis. One is how to maintain a culture originated from the same population growing for many generations giving the opportunity to the cells to accumulate mutations and evolve. One option is to grow the cells on a flask until they reach the lag phase then reinoculate a fraction of this culture to a new fresh medium. Although this method allows the population to evolve it can take a long time to observe mutations because during the lag and the stationary phase of bacterial growth cells do not divide. This can seems trivial but due to the slow growth rate of Synechocystis it can make a difference.

Turbidostat are culture devices that allow to maintain the cells in constant exponential growth. This is achieved by automatically diluting the culture, i.e. pumping in fresh medium and pumping out culture medium, when a certain threshold is reached. Its name refers to the fact that the turbidity of the medium, so the amount of cells, it maintained (-stat from static) on a certain range. This requires a three compartment system where the first contains the cultures, the second holds a reservoir of fresh medium and the third collect the medium extracted from the cultures. Pumps move the medium between containers. The cell density is recorded at regular intervals by a spectrophotometer and based on this value a software decides when the culture is diluted.

Turbidostat
Figure 3. - How a turbidostat works for dummies.
Turbidostat
Figure 4. - The multicultivator.

This system allow us to reduce the time of observing a mutation but now it comes the second problem. How do we know if a mutation leading to a change in the parameters has happened? The first option that comes to our synthetic biologist mind is by sequencing periodically the genome of the population and screen it for mutations. The major drawback of sequencing is that it does not inform whether the mutation affects the parameters or not. The remaining solution is then to estimate the physiological parameters with high time definition to spot when the mutation has occurred. Using the turbidostat it would be possible to store all the values recorded by the spectrophotometer and calculate the growth rate from these data. This is done by fitting a linear model to the logarithm of the OD over the time. The production rate can obtained by periodically measuring the amount of product in the medium (link to protocols for enzymatic assays and HPLC).

Luckly for us when we came to the lab there was a master student, Joeri Jongbloets{link}, who had transformed a multicultivator(MC) like this{link} in a 8 channel turbidostat. For his internship he wrote the program connect with the MC hardware and control the pumps to dilute the culture when necessary. In addition the software stores the measurements from the MC spectrophotometers in a database and analyzes it to obtain the growth rate. Thanks to this impressive piece of software engineering we were able to run long term experiments and observe evolution.