Team:SZPT-CHINA/Model

S Z P T - C H I N A

• Home
• Project
  • Description
  • Design
  • Modeling
  • Results
  • Demonstrate
  • Safety
  • Protocols
  • Notes
• Team
  • Teammates
  • Collaborations
  • Attributions
• Human Practices
  • Overview
  • Human Practices
  • Euducation & Engagement
  • Meet Up
• Parts
  • Overview
  • Characterization
  • Improved Parts
• Achievements

MODELING

1. Introduction

We have established a model of pH-induced promoter regulation on the target protein of Lactococcus lactis MG1363 since we don't know if the pH regulation we designed can work. Then, we performed numerical simulations on the model and sensitivity analysis on some key parameters.

Overall, we regulate the expression of the protein of interest based on the acid-induced promoter rcfB, the LacR repressor and the operator O1 and O2:

(1) When the extracellular pH reduces to 5.5, the pH-induced promoter will trigger the express of LacR. The accumulation of LacR will combine with the operator gene O1 and O2, inhibiting the expression of downstream gene of interest.

(2) When the extracellular pH rises to 5.5, the pH-induced promoter will not trigger the express of LacR. The degradation of LacR will release the operator gene O1 and O2, stimulating the expression of downstream gene of interest.

Fig.1 Gene Regulatory Circuit

2. The Transcription of LacR Gene

The basal rate of the transcription of LacR gene is given by V₀ and increased at a constant rate V_i which is regulated by the pH value. GR stands for the repressor gene concentration. The degradation of the mRNA occurs at the rate of dM. It is also diluted due to cell growth rate at μ. We assumed the decrease of mRNA is first-order proportion to the concentration of M.

(1)

As the mechanisms of pH-induced promoters have not been fully elucidated, we included a generic pH-dependent switch F(pH) which turns on when pH value is below the threshold. The switch takes the form of the smoothed step function, where 5.5 represents the threshold pH level around which the switch occurs. n indicates the steepness of the smooth switch function.

(2)

Assume that the pH is only affected by the environment ph. Experiments under three different pH were taken and we fitted a function as the form of function (3). To the pH data, P₀, P₁ and P₂ are constants. This function was then fed into the model via the switch function F(pH).

(3)

3. Transcription of Target Protein Gene

For N, it is described as function (5). The basal rate is given by V₂ and the transcription is inhibited by LacR. For the part to simulate the regulation rate of LacR, Tmax stands for the maximal rate of target protein gene transcription, S represents the sensitivity of target protein gene transcription to enzyme. G is the concentration of transcription gene. LR represents the Concentration of LacR protein in the cells. C₁, C₂ represent the translation rate of LacR protein and target protein, respectively.

(4)

(5)

The rate at which mRNA is translated into protein is C₂, and the rate of protein degradation is γ.

(6)

4. Parameters

Table 1 Variables Used in the Model

Table 2 List of Parameters Used in the Model

5. Results

5.1 Target Protein Expression Model

5.1.1 Simulated Expression of Target Protein in Acidic Environment

Fig.2 Simulated Expression of Target Protein in Acidic Environment

We simulated a model for the expression of the protein of interest by L. lactis MG1363 under acidic conditions. According to the model, when the pH was less than 5.5, the expression protein of the L. lactis MG1363 was inhibited after 4 hours. It means that during the fermentation of lactic acid bacteria, when the pH of the external environment drops below 5.5, the expression of the target protein will be inhibited after 4 hours. It is beneficial to reduce the energy consumption of the bacteria, and is beneficial to increase the expression of our target protein in the intestinal tract.

5.1.2 Target Protein Mimic Expression in the Intestine.

Fig.3 Target Protein Mimic Expression in the Intestine

When the pH of the external environment rises above 5.5, the expression of the target protein can be observed after about 2 hours, and the expression rate is relatively stable. It indicates that the corresponding protein of interest will be expressed by lactic acid bacteria two hours after it is colonized into the intestine.

5.2 Sensitivity Analysis of Parameters

We used a sensitivity analysis to determine the effect of GR and Tmax on the expression of our target protein. The results are shown below. It can be seen that the low expression of the target protein at both concentrations is also very similar to our parameters, further validating the feasibility of the model.

Fig.4 Sensitivity Analysis of Parameters

Discussion

Excitingly, the model results are consistent with our expected regulatory mechanisms. It indicates that the regulation mechanism designed for the characteristics of lactic acid bacteria fermentation acid production is feasible. We can use it to increase the yield of the target protein as much as possible.

References

1. Ke Liu, et al. The sensitivity and significance analysis of parameters in the model of pH regulation on lactic acid production by Lactobacillus bulgaricus. [J] BMC Bioinformatics 2014, 15(Suppl 13):S5.

2.Ying wang , et al. Comparison between Lineweaver-Burk Plot and Genetic Algorithms Using for Parameter Estimation of Monod Equation.[J] Journal of Qingdao Agricultural University (Natural Science ) 2014 ,31 (3 ): 225~228.