Team:Groningen/Model

iGEM Groningen 2019 :: Results

Modelling

At the start of the project we set out several goals so our eventual protocol would be easy to use, robust, reproducible and safe.
Goals:
  1. Use a mathematical model to predict optimal growth conditions for V. natriegens.
  2. Determine optimal media for V. natriegens based on the mathematical model we developed

Findings:
  1. We found that high NaCl concentrations delay the stationary phase and increase the carrying capacity of the organism of interest.
  2. We confirm that v2 salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) increase the growth rate of V. natriegens

1. Introduction

The main focus of our project is to grow Escherichia coli and Vibrio natriegens in the shape of the QR code. The growth of E. coli in various types of media has been very well characterized. For our project it is important to know what media compositions are ideal for growth and sustainability of V. natriegens. However, only a few articles report the growth of V. natriegens under different media compositions. This prompted us to model the growth dynamics of this microorganism to investigate which media is optimal for its growth. V. natriegens is a halophile marine bacterium that is found in salty environments. We modelled the growth of V. natriegens based on Lysogeny broth (LB), brain heart-infusion (BHI) media basis with varying NaCl concentrations and v2 and v3 ocean salts.To model the growth of the bacterial population over time, we considered the logistic differential equation:
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Growth function
where:
  1. r - growth rate of the population (1/min)
  2. K - carrying capacity (OD units)
  3. y - population of bacteria (OD units)
  4. t - time (min)
This mathematical model deals with the growth characteristics of a bacterial population (Figure 1). We can use this equation to model growth for V. natriegens.
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Figure 1 - Logistic growth model, Hande Acar Kirit DOI: 10.1093/molbev/mst187
The fastest exponential growth combined with a delayed deceleration phase would be ideal conditions for the growth of V. natriegens. Our goal was to find optimal growth media to optimize bio-ink production by increasing the responsiveness of the vibrio cultures. We do so by comparing growth curves for V. natriegens in different media with different salts and salt concentrations.

2. Experiments with LB media

2.1 Media Components

The experiments were conducted with the following variations of LB:
  • LB with NaCl concentrations of 0.5 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 22.5 g/L, 25 g/L, 27.5 g/L, 30 g/L, 32.5 g/L, 35 g/L, 37.5 g/L, 40 g/L, 45 g/L
  • LBv2
  • LBv3
The recipes involved were:
  • For LB with varied salt concentration: LB broth: 10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 0.5 g/L NaCl.
  • For LBv2: LB broth supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)
  • For LBv3: LB broth + v3 salts: LB broth supplemented with additional salts (475 mM NaCl, 9.7 mM KCl, and 54 mM MgCl2)
The experiment was carried out in triplicates for a period of 6h30min. The graph below (Figure 2) is a representation of the results of the OD measurements.
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Figure 2 - LB data points

2.2 Results & discussion:

We observe from Figure 2 that every media composition leads to a different exponential growth. As we can see in the graph, the steepness (exponential phase) and the deceleration phase of the growth curve with increase in salt (NaCl) concentration. After a certain concentration (20 g/L) of NaCl in LB, we observe that increasing the concentration has no positive effect on the growth of V. natriegens. As concentration of NaCl increases stagnation of growth occurs. It is important to note that the growth curve of V. natriegens is most favorable for LBv2. This satisfied our need for the steepest curve and tallest exponential phase to increase the responsiveness of the bacteria. In conclusion, for LB media, it is optimal to use 20 g/L of NaCl concentration or v2 salts (204 mM NaCl, 4.2 mM KCl and 23.14 mM MgCl2) concentration.

To further verify our conclusions we plot carrying capacity against salt concentrations (Fig 3). It is not a good idea to plot growth rate against salt concentrations unless the growth rate for every concentration curve is measured at the exact time interval.

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Figure 3 - Carrying capacity (K) against NaCl concentration
The plot above shows that the maximum population size is achieved at 15 g/L and 20 g/L of NaCl concentration (Kmax=1.4 OD600) . But as seen previously (Fig2), the 20 g/L curve is more optimal than 15 g/L curve. This indeed reinforces our claim that for LB based media the use of 20 g/L of NaCl concentration is indeed optimal for efficiently growing and transforming V. natriegens. Although the use of LBv2 is more effective practically, the use of NaCl is preferred considering cost effectiveness.

3. Experiments with BHI media

3.1 BHI media components

We performed similar experiments with BHI based media by varying the salts and their concentrations as follows:
  • BHI with NaCl concentrations of 0.5 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 22.5 g/L, 25 g/L, 27.5 g/L, 30 g/L, 32.5 g/L, 35 g/L, 37.5 g/L, 40 g/L, 45 g/L
  • BHIv2
  • BHIv3
The recipes for this media involved:
  • For BHI with varied salt concentration: Brain Heart Infusion Broth. 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500) + 20 g/L NaCl
  • For BHIv2: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500) supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)
  • For BHIv3: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500) supplemented with additional salts (475 mM NaCl, 9.7 mM KCl, and 54 mM MgCl2)
The data was collected in triplicates over a period of 6h30min. The graph below (Fig. 4) is a representation of the results of the OD measurements.
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Figure 4 - BHI data points

3.2 Results & discussion

In this case, LBv2 has the steepest exponential phase but a shorter deceleration phase as compared to NaCl concentrations. This suggests that even though the carrying capacity of V. natriegens is less in LBv2, the responsiveness of the culture is high. In case of NaCl concentrations, even though 35 g/L has a delayed exponential phase, we observe a prolonged deceleration phase. This translates to delayed yet better responsiveness than other NaCl concentrations. Also, the carrying capacity for this curve is the highest. In conclusion we can say that, for BHI based media it is optimal to use 35 g/L of NaCl concentration or v2 salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) concentration.
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Figure 5 - Carrying capacity (K) against NaCl concentration.
We observe that the 35 g/L NaCl concentration curve yields the highest value of carrying capacity (Kmax=1.4 OD600). This confirms our claim that for BHI media, the use of 35 g/L NaCl solution is optimal for growth of V. natriegens. In this case the 35 g/L NaCl concentration works better than the v2 salts.

4. Conclusion

Using modelling we evaluated several BHI and LB formulations as estimations for an optimal media for growth of V. natriegens. For our bio-ink it is important that bacteria stay responsive. It is also interesting to know what medium supports the fastest growth with effective responsiveness. The experimental data collected on LBv2 is in agreement with our estimations given by the model when compared with BHIv2 (KBHIv2= 1.29OD600). For media based on NaCl concentrations, LB with 20 g/L of NaCl (KLBNacl= 1.402OD600) is a better option than BHI with 35 g/L NaCl (KLBNacl=1.404OD600) since BHI media would require more amount of NaCl for the same results.The cost of BHI is about 11 euro per liter (Sigma Aldrich) and LB is about 2 euro per liter (Sigma Aldrich), hence LB is the optimal choice. According to our data we conclude that LB with 20 g/L of NaCl concentration is the optimal choice as the media for growing V. natriegens.