Team:Amsterdam/Project/Phy param/Synechocysytis

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 Q_p in the growth coupled producer Δacs remains constant over the time (figure 2).
• We have estimated Q_p and growth rates for six Synechocystis (Table 1).

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Figure 1. - Growth and Q_p of strain SAA023 over 900 hours of continuous culture showing the drop in production.

Figure 2. - Growth and Q_p of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.

Table 1. - Estimated Growth and Q_p 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.

Figure 3. - How a turbidostat works for dummies.

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.

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).

Figure 4. - The multicultivator.

Luckily 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 that 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.

Methods summary

Cultivation conditions

The strains producing glycerol, lactate and acetate (Δacs) were grown in turbidostats for 900 hours under constant LED lights providing 20 μE/ m²* s * OD.

The Pta1, Pta2, Δacs and Ack strains were cultured on photostats. This systems, also implemented by Joeri Jongbloets in the MC, is basically a batch culture where the light intensity per OD provided to the cells is maintained constant. In this case the light intensity per OD was set to 20 μE/ m²* s.

In both cases the culture medium was BG-11 supplemented with NO_3- and TES buffer {link to recipe}.

Measuring product

Samples were taken from the cultures and centrifuged (5 min, 14.500 rpm) to obtain the supernatant. Product concentration was then estimated by enzymatic assays {links to protocols} in the case of lactate and acetate and by HPLC for glycerol {links to protocols}.

Estimating parameters

Growth rate

Growth rates were estimated by fitting a regression model to the logarithm of the OD over the time during the exponential phase of growth. The slope of this model is considered as the growth rate since it shows how much the OD changes per unit of time. Its unit is 1 / h since.

Production rate

Q_p are obtained by dividing the the concentration of measured product by the biomass of the culture estimated from OD (1 OD unit = 0.2 gDW). This value is in fact the production yield, that is, how much product the cells excrete per amount of biomass. To get the production rate, Q_p, we multiply this value by the estimated growth rate. Its units are amount of product (in mmol) / gDW * h.