Overview

In order to meet the requirements of iGEM, models of our projects are dedicated to solving the problems in experiment and providing guidance for the products. Our project is mainly based on bacteriostasis and incense production, while PLA is the major bacteriostatic material, so we have built two models about mechanism and yield of PLA with innovation and practicability of development as the following.The first model is about Mechanism of PLA .The second model is about PLA yield curve. The result of our models has an application.

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Mechanism of PLA

Effect of different concentration of PLA on the number of Staphylococcus epidermidis.

Introduce
we had planned to established a dynamic equation to describe the effect of PLA on staphylococcus epidermidis which is the main bacteria that cause the armpit order. We gave several candidate forms of function for describing this process and finally found the best one which fit the experimental data quite well. By using the data from the experiment，we continually refine our models and guide our project through this models. Besides, this model, of which type has never been built before, also makes us learning the mechanism of PLA and solved problems as following:
(1) How many PLA should we put in our products that can kill the desired amount of staphylococcus epidermidis?
(2) What is the effect of concentration of PLA on the number of staphylococcus epidermidisover time.
（3）What is the growth of bacteria at any concentration of PLA?

Method
To solve the problems above, we used different concentration of PLA to react with Staphylococcus epidermidis, then we measured the rest of bacteria every hour for a total of 8 hours. Through fitting the initial data, we got the following chary and found the appropriate function to describe this figure.

dx/dt=(-1.0836*(t-1.4028) ^2+1.7601) *x(t) -(a*dCp ^n)*x(t)
dCp=Cp-Cp* Through this model, we have known that PLA would not have any effect on the bacteria until reaching a certain concentration, so we designed two parts of equation for this model

(1) Cp dx/dt=[-a(t-b)2+c] * x(t)
When the concentration of PLA in the system are less than the critical value, the bacteria are able to grow naturally, so we use a dynamic equation of growth rate of Staphylococcus epidermidis.

(2) Cp>Cp*
△Cp=Cp-Cp*
dx/dt= f(△Cp) *x(t)
f(△Cp) = axn
In this situation, Cp more than the value of Cp*, PLA has an inhibitory effect on Staphylococcus epidermidis. The mortality is set as f(△Cp) and the death rate are expressed by dx/dt= f(△Cp) *x(t). According to the data analysis, mortality should meet the following two conditions:
①The function value should increase with the value of (Cp- Cp*), and the function need to be super linear.
②When Cp -Cp*=0，the value of f(△Cp) should equal to zero.
From the above conditions, we set the f(△Cp) as the exponential function: axn
By combining these two formulas, we can describe the survival of Staphylococcus epidermidis at different PLA concentrations over time. And we have concluded that the PLA concentration is 6.7106mmol/l, which has an inhibitory effect on the epidermidis.

Expand
In order to verity if there is a multiple relationship between inhibitions of PLA on different amounts of bacteria. We changed the parameter value of starting optical density equal to 2kbs and 0.5kbs, then we got the figures as following and concluded the same critical value of PLA. Later, we have used experiment to verity those charts correct.

Through these two figure, we have learned that the same concentration of PLA has the same effect on the different number of bacteria, which means that PLA kill the bacteria in proportion to the whole population. This conclusion proves that the formula about applies to any number of bacteria.

Optimize
Based on the above formulas, we had obtained the range of the critical value and lethality of PLA preliminarily, which has instructed the following experiment. We subdivided the PLA concentration in the above range to make it work on Staphylococcus epidermidis to validated the model and obtained more accurate parameters.

Achievements
①Using 6.7106mmol/l PLA will have an inhibitory effect on Staphylococcus epidermidis.
②PLA mechanism: kill bacteria in the proportion.
③Any kinds of bacteria can grow naturally in a certain range of concentration of PLA under the critical value, which can be used in screening and isolation of strains.

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Methods

  We followed this protocol to do the Interlab Study.
  When we completed three of the calibration measurements, performing the cell measurements. Used the same plates, volumes and settings that we used in calibration protocol. We transformed E.coli DH5α competent cells with the 8 plasmids and picked 2 colonies from each of plates into 5 mL LB medium + Chloramphenicol. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0, 6 hours.

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Result

Calibration
Calibration 1: OD600 reference point

Calibration 2: Particle Standard Curve

Cell measurement

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Analyse

  A standard curve in a linear relationship can be obtained by calibration experiments.
By comparing and analyzing the fluorescence values and Absorbance value of the different test devices at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences in the 6h fluorescence measurement results. Among the six different test equipment, Device 4 has the strongest fluorescence, while Device 3 has the lowest fluorescence. Compared with the relation between absorbance and fluorescence value, we can't get the relationship, and we need to improve on the control variables during the experiment.

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Summary

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