Difference between revisions of "Team:NEU China B/Model"

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 +
<h1>
 +
    Modeling Part Structure
 +
</h1>
 +
<h2>
 +
    Overview
 +
</h2>
 +
<p>
 +
    In the following article, we describe the process of building our model. During the construction, we aim to build a model by combining the data fitting principle in mathematical modeling with the help of MATLAB through multiple experiments, and obtain a relatively successful fitting formula after multiple optimization, so as to reflect the data trend reasonably.
 +
</p>
 +
<p>
 +
    During the experiment, we first controlled the consistency of the concentration of the bacterial liquid so that it would not affect the experimental results. There are 3 groups of experimental statistic, they record 3 kinds of mutant statistic——The independent variable—concentration of lactic acid(lactate)，time of reaction(t), the dependent variable—intensity of fluorescence(Fluorescence). On the basis of reasonable hypothesis and correlation coefficient test, according to the fitting of known data, we get the function and function curve that can reflect data trend reasonably. This curve illustrates the functional relationship between the fluorescence intensity of the dependent variable and the independent variable.
 +
</p>
 +
<h2>
 +
    How do we derive this model?
 +
</h2>
 +
1: According to some reasonable assumptions given in professional knowledge, we believe that the linear function may be a non-linear function between the dependent variable and the independent variable, since the addition of lactic acid and IPTG will tend to influence the growth rate of the organism, and the lactic acid into the cell and the ai-2 generated by the Luxs catalysis also need to be of a certain and unequal time, so that the whole reaction process does not constitute a linear function requirement. So it's a logarithmic function or a polynomial function.
 +
<br>2: Under each reasonable hypothesis, MATLAB is used to perform corresponding fitting. According to the correlation coefficient R to test the fitting effect, the fitting function formula that can reasonably reflect the data trend can be obtained after continuous optimization.
  
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<div class="column full_size judges-will-not-evaluate">
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<br>T7- lldPRD operon promoter-GFP：
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
+
−
</div>
+
  
 +
<br>[IPTG]=1 mM：
 +
<br>t=0 min Relative Fluorescence= 1.496 t 3 -8.148 t 2 + 10.31 t + 8.919 R2= 0.1473
 +
<br>t=5 min Relative Fluorescence= 9.304 t 3 -49.68 t 2 + 63.56 t + 22.25 R2= 0.9464
 +
<br>t=10 min Relative Fluorescence= 1.286 t 3 -12.36 t 2 + 19.78 t + 29.03 R2= 0.9986
 +
<br>t=20 min Relative Fluorescence= 17.26 t 3 -83.37 t 2 + 96.5 t + 19.91 R2= 0.9957
 +
<br>t=30 min Relative Fluorescence= 9.669 t 3 - 48.15 t 2 + 58.23 t + 39.34 R2= 0.8645
 +
<br>t=60 min Relative Fluorescence= 20.96 t 3 - 106.2 t 2 + 134.9 t + 42.16 R2= 0.9482
  
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<div class="clear"></div>
+
<h3>
 +
        Lldr- T7-lldPRD operon promoter-GFP：
 +
</h3>
  
 +
<br>[IPTG]=1 mM：
 +
<br>t=0 min Relative Fluorescence= 6.123 t 3-32.29 t 2+41.51 t+ 34.97 R2=0.4449
 +
<br>t=5 min Relative Fluorescence= 6.669 t 3 -38.13 t 2 + 55.27 t + 13.89 R2= 0.9808
 +
<br>t=10 min Relative Fluorescence= -3.422 t 3 +7.808 t 2 + 8.072 t + 11.18 R2=0.9946
 +
<br>t=20 min Relative Fluorescence= 10.74 t 3-48.53 t 2+57.9 t+ 9.947 R2= 0.9621
 +
<br>t=30 min Relative Fluorescence= 7.02 t 3 - 37.92 t 2 + 55.03 t + 9.921 R2= 0.9998
 +
<br>t=60 min Relative Fluorescence= 9.403 t 3 - 55.39 t 2 + 80.41 t + 8.451 R2= 0.9787
  
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<div class="column full_size">
+
<h3>
−
<h1> Modeling</h1>
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    lldPRD operon promoter-GFP：
 +
</h3>
  
−
<p>Mathematical models and computer simulations provide a great way to describe the function and operation of BioBrick Parts and Devices. Synthetic Biology is an engineering discipline, and part of engineering is simulation and modeling to determine the behavior of your design before you build it. Designing and simulating can be iterated many times in a computer before moving to the lab. This award is for teams who build a model of their system and use it to inform system design or simulate expected behavior in conjunction with experiments in the wetlab.</p>
+
<br>[IPTG]=0 mM：
 +
<br>t=0 min Relative Fluorescence= 2.27 t 3 - 12.95 t 2 +23.4 t+ 8.107 R2= 0.9883
 +
<br>t=5 min Relative Fluorescence= -2.332 t 3 +8.402 t 2 + 2.239 t + 20.74 R2= 0.9834
 +
<br>t=10 min Relative Fluorescence= 0.4742 t 3 -1.957 t 2 + 5.059 t +26.94 R2= 0.9544
 +
<br>t=20 min Relative Fluorescence= -3.069 t 3+17.46 t 2-25.51 t+ 21.98 R2= 0.9398
 +
<br>t=30 min Relative Fluorescence= -7.451 t 3 +36.49 t 2 -44.01 t + 39.25 R2= 0.9645
 +
<br>t=60 min Relative Fluorescence= -0.7971 t 3 +4.694 t 2 -13.22 t + 39.88 R2= 0.9761
  
−
</div>
+
<h3>
−
<div class="clear"></div>
+
    lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP：
 +
</h3>
  
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<div class="column full_size">
+
<br>t=0 min Relative Fluorescence= -9.425 t 3+43.48 t 2-48.63 t+ 44.85 R2= 0.9985
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<h3> Gold Medal Criterion #3</h3>
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<br>t=5 min Relative Fluorescence= 9.999 t 3 -56.32 t 2 +79.92 t + 12.47 R2= 0.9696
 +
<br>t=10 min Relative Fluorescence= 12.03 t^3 -62.83 * t^2 +86.66 * t+10.36 R2= 0.7222
 +
<br>t=20 min Relative Fluorescence= 4.578 t 3-32.79 t 2+59.31 t+9.459 R2= 0.898
 +
<br>t=30 min Relative Fluorescence= 6.297 t 3 -35.63 t 2 + 53.2 t + 9.274 R2= 0.8867
 +
<br>t=60 min Relative Fluorescence= 9.122 * t 3 - 51.26 * t 2 + 73.33 * t + 5.924 R2= 0.9866
 +
 
 +
<h3>
 +
    lldPRD operon promoter-Luxs × LsrA promoter-GFP
 +
</h3>
 +
 
 +
<br>t=0 min Relative Fluorescence= -11.78 t 3+46.85 t 2-28.71 t+ 10.35 R2= 0.9764
 +
<br>t=5 min Relative Fluorescence= -12.48 t 3 +45.65 t 2 -26.91 t + 18.86 R2= 0.9762
 +
<br>t=10 min Relative Fluorescence= -1.135 t 3 +5.337 t 2 -3.825 t + 23.12 R2= 0.511
 +
<br>t=20 min Relative Fluorescence= -10.64 t 3+35.98 t 2-12.38 t+ 9.098 R2= 0.9716
 +
<br>t=30 min Relative Fluorescence= 3.63 t 3 - 25.57 t 2 + 40.87 t + 14.54 R2= 0.893
 +
<br>t=60 min Relative Fluorescence= 9.562 t 3 - 53.63 t 2 + 74.38 t + 9.515 R2= 0.9907
 +
<h2>
 +
    Practical significance of model results
 +
</h2>
 +
<h3>
 +
    Figures
 +
</h3>
 +
 
 +
<h3>
 +
        T7- lldPRD operon promoter-GFP：
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/6/67/T--NEU_China_B--basepart0.png">
 +
<h3>
 +
    [IPTG]=1 mM
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/1/1b/T--NEU_China_B--basepart1.png">
 
<p>
 
<p>
−
Convince the judges that your project's design and/or implementation is based on insight you have gained from modeling. This could be either a new model you develop or the implementation of a model from a previous team. You must thoroughly document your model's contribution to your project on your team's wiki, including assumptions, relevant data, model results, and a clear explanation of your model that anyone can understand.
+
    When [IPTG]=1 mM , The concentration of fluorescence at each point of time shows a similar trend towards the change in the rate of lactate, which is increased by the increase in lactate concentration, and then the fluorescence starts to go up and then down, and then there's a tendency to increase the intensity of the fluorescence, at the end of the level of the lactic acid, which is at the peak of the lactic acid, which is at the level of 0.5 to the one, and the fluorescence is at a peak. According to actual production, the concentration of yogurt in fermentation cannot exceed 1mM[1]. Therefore, the engineered bacteria can monitor the lactic acid concentration of yogurt during fermentation. In terms of time, when IPTG was added with 1mm, the fluorescence value of 5-60 min was all higher than that of 0 min, indicating that the reaction of engineering bacteria to lactic acid was effective during this period. Therefore, 5 min was selected as the appropriate reaction time. Because the fluorescence intensity of fiber detection can be stable within 200 milliseconds, the actual production can be obtained, and the time for testing the lactic acid concentration by using this engineering bacteria is 5 min.  
−
<br><br>
+
−
The model should impact your project design in a meaningful way. Modeling may include, but is not limited to, deterministic, exploratory, molecular dynamic, and stochastic models. Teams may also explore the physical modeling of a single component within a system or utilize mathematical modeling for predicting function of a more complex device.
+
 
</p>
 
</p>
 +
<h3>
 +
    Lldr- T7-lldPRD operon promoter-GFP：
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/0/03/T--NEU_China_B--basepart2.png">
 +
<h3>
 +
    [IPTG]=1 mM
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/3/3a/T--NEU_China_B--basepart3.png">
 +
<p>
 +
    When [IPTG]=1 mM, with the increase of time, the intensity of fluorescence expressed by engineered bacteria all lower than the intensity at 0 min. This may be due to the fact that at 0 min, the expression of lldR has not yet started or the expression quantity is too low, so the opening of lldPRD operon promoter caused by the introduction of engineering bacteria is not prevented. However, as the reaction time increases, lldR gradually produces, and the GFP background expression decreases due to the opening of lldPRD operon promoter. From the perspective of time, when the reaction time was 5 min, the change trend of fluorescence intensity with the concentration of lactic acid was consistent with the initial reaction time, so the good reaction time of engineering bacteria could be achieved within 5 min.
 +
</p>
  
 +
<h3>
 +
    lldPRD operon promoter-GFP：
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/7/75/T--NEU_China_B--basepart4.png">
 +
<h3>
 +
    [IPTG]=0 mM
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/c/c1/T--NEU_China_B--basepart5.png">
 
<p>
 
<p>
−
Please see the <a href="https://2018.igem.org/Judging/Medals"> 2018
+
    When there is no lactose operon, different concentrations of lactic acid are added. Although the engineered bacteria will still react according to different concentrations of lactic acid, the curve of the function is obviously irregular with different reaction times.
−
Medals Page</a> for more information.  
+
</p>  
 +
<p>
 +
    lldPRD operon promoter-GFP & T7- lldPRD operon promoter-GFP& Lldr- T7-lldPRD operon promoter-GFP:
 +
    <br> We fitted the change of fluorescence intensity expression of the three engineered bacteria at the reaction time of 5 min, hoping to obtain the difference of the three engineered bacteria.
 
</p>
 
</p>
−
</div>
+
<img src="https://static.igem.org/mediawiki/2018/8/8c/T--NEU_China_B--basepart6.png">
−
 
+
<p>
−
<div class="column two_thirds_size">
+
    In the process of production of yogurt, the lactic acid content should not exceed 1 mM. So when the lactic acid content was lower than 1 mM, the lactose operon or lldR is added to the engineered bacteria and the lactic acid concentration is lower than 1 mM, indicating that the two engineered bacteria can achieve high sensitivity. Besides, when lactic acid content was lower than 1 mM, the lactose operon or lldR is added to the engineered bacteria is higher than those which only have lldPRD operon, indicating that both the lactose operon and lldR have an improved effect on the initial engineered bacteria.(BBa_K822000 ). Compared with the improvement of BBa_K822000 by lldR, it is obvious that the addition of lactose operon makes engineering bacteria more sensitive.
−
<h3>Best Model Special Prize</h3>
+
</p>
 +
<h3>
 +
    lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/a/ac/T--NEU_China_B--basepart7.png">
 +
×
 +
<img src="https://static.igem.org/mediawiki/2018/f/fa/T--NEU_China_B--basepart8.png">
 +
<img src="https://static.igem.org/mediawiki/2018/e/e9/T--NEU_China_B--basepart9.png">
 +
<p>
 +
    Except for the beginning of the reaction, The rest of the functional curves showed similar trends, especially when the reaction time was 5 min and 10 min, and the peak value was reached before the concentration of lactic acid was 1 mM. It proved that the engineering bacteria was best used to detect the lactic acid concentration in yogurt at the reaction time of 5 min and 10 min. In order to reduce the reaction time of engineered bacteria, the reaction time of 5 min was selected.
 +
</p>  
 +
<h3>
 +
    lldPRD operon promoter-Luxs × LsrA promoter-GFP
 +
</h3>
 +
<img src="https://static.igem.org/mediawiki/2018/1/19/T--NEU_China_B--basepart10.png">
 +
  ×
 +
<img src="https://static.igem.org/mediawiki/2018/b/b5/T--NEU_China_B--basepart11.png">
  
 +
<img src="https://static.igem.org/mediawiki/2018/2/26/T--NEU_China_B--basepart12.jpg">
 
<p>
 
<p>
−
To compete for the <a href="https://2018.igem.org/Judging/Awards">Best Model prize</a>, please describe your work on this page  and also fill out the description on the <a href="https://2018.igem.org/Judging/Judging_Form">judging form</a>. Please note you can compete for both the gold medal criterion #3 and the best model prize with this page.
+
    According to the result of fitting, after 30 min of reaction, the engineered bacteria were highly sensitive to different concentrations of lactic acid. However, the reacting time was to long, the increase of fluorescence value may be caused by the death of bacteria, which has no reference value.
−
<br><br>
+
−
You must also delete the message box on the top of this page to be eligible for the Best Model Prize.
+
 
</p>
 
</p>
−
 
−
</div>
 
−
 
−
 
−
<div class="column third_size">
 
−
<div class="highlight decoration_A_full">
 
−
<h3> Inspiration </h3>
 
 
<p>
 
<p>
−
Here are a few examples from previous teams:
+
    lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP & lldPRD operon promoter-Luxs × LsrA promoter-GFP& Lldr- T7-lldPRD operon promoter-GFP
 +
</p>
 +
<p>
 +
    We fitted the change of fluorescence intensity expression of the three engineered bacteria at the reaction time of 5 min, hoping to obtain the difference of the three engineering bacteria.
 
</p>
 
</p>
−
<ul>
+
<img src="https://static.igem.org/mediawiki/2018/e/e1/T--NEU_China_B--basepart13.jpg">
−
<li><a href="https://2016.igem.org/Team:Manchester/Model">2016 Manchester</a></li>
+
<p>
−
<li><a href="https://2016.igem.org/Team:TU_Delft/Model">2016 TU Delft</li>
+
    Apparently, when the lactic acid concentration is less than 1 mM, included lldPRD operon promoter - Luxs - Lldr x LsrA promoter - the GFP and Lldr - T7 has - lldPRD operon promoter - GFP, two kinds of engineering bacteria in the peak, and the sensitivity of the former than the latter. This indicates that when the lactic acid concentration of yogurt is not over the limit, these two types of engineered bacteria can produce higher sensitivity, and the addition of the swarm sensing system can improve the sensitivity of engineered bacteria to the lactic acid concentration. The engineering bacteria contains lldPRD operon promoter - Luxs x LsrA promoter - GFP in lactic acid concentration is lower than 2.5 mM, to produce higher sensitivity.
−
<li><a href="https://2014.igem.org/Team:ETH_Zurich/modeling/overview">2014 ETH Zurich</a></li>
+
</p>
−
<li><a href="https://2014.igem.org/Team:Waterloo/Math_Book">2014 Waterloo</a></li>
+
                   
−
</ul>
+
<p>
−
</div>
+
    All in all, from what has been discussed above, we can learn that contains lldPRD operon promoter - Luxs - Lldr x LsrA promoter - GFP engineering bacteria is applied to we have the best equipment engineering bacteria.
−
</div>
+
</p>                      
 +
<h2>
 +
    Experimental Purpose
 +
</h2>
 +
<p>
 +
    In order to quantify the relationship between fluorescence intensity and lactic acid concentration, the production of lactic acid can be controlled reasonably according to the real-time monitoring of fluorescence intensity, and the inhibitory effect of lactic acid on fermentation degree can be minimized and the fermentation degree can be maximized.
 +
</p>
 +
<p>
 +
    We can connect the function curve with the experimental purpose and further strengthen the experimental purpose that we have achieved through the experimental and modeling results
 +
    <br>(1) Determine whether the addition of lactose operon improves the effect of the original engineered bacteria
 +
    <br>(2) Determine whether the introduction of LLDR gene improves the effect of the original engineering bacteria
 +
    <br>(3) Determine whether the addition of group sense system improves the reaction of lactic acid manipulator promoter to lactic acid
 +
    <br>(4) Judge the actual reaction time of engineering bacteria applied in actual operation
 +
</p>
 +
<h3>
 +
    Reference:
 +
</h3>
 +
<q>
 +
    [1] Wanguang Li, Xinwen Wang, Yishun Ji. Comparative experiment of two lactic acid detection methods in yogurt [J]. Anhui agronomy bulletin, 2017, 23(21): 113-114
 +
</q>
 +
 
  
 
</html>
 
</html>

Revision as of 23:12, 15 October 2018

Ruby - Responsive Corporate Tempalte

Modeling Part Structure

Overview

In the following article, we describe the process of building our model. During the construction, we aim to build a model by combining the data fitting principle in mathematical modeling with the help of MATLAB through multiple experiments, and obtain a relatively successful fitting formula after multiple optimization, so as to reflect the data trend reasonably.

During the experiment, we first controlled the consistency of the concentration of the bacterial liquid so that it would not affect the experimental results. There are 3 groups of experimental statistic, they record 3 kinds of mutant statistic——The independent variable—concentration of lactic acid(lactate)，time of reaction(t), the dependent variable—intensity of fluorescence(Fluorescence). On the basis of reasonable hypothesis and correlation coefficient test, according to the fitting of known data, we get the function and function curve that can reflect data trend reasonably. This curve illustrates the functional relationship between the fluorescence intensity of the dependent variable and the independent variable.

How do we derive this model?

1: According to some reasonable assumptions given in professional knowledge, we believe that the linear function may be a non-linear function between the dependent variable and the independent variable, since the addition of lactic acid and IPTG will tend to influence the growth rate of the organism, and the lactic acid into the cell and the ai-2 generated by the Luxs catalysis also need to be of a certain and unequal time, so that the whole reaction process does not constitute a linear function requirement. So it's a logarithmic function or a polynomial function.
2: Under each reasonable hypothesis, MATLAB is used to perform corresponding fitting. According to the correlation coefficient R to test the fitting effect, the fitting function formula that can reasonably reflect the data trend can be obtained after continuous optimization.
T7- lldPRD operon promoter-GFP：
[IPTG]=1 mM：
t=0 min Relative Fluorescence= 1.496 t 3 -8.148 t 2 + 10.31 t + 8.919 R2= 0.1473
t=5 min Relative Fluorescence= 9.304 t 3 -49.68 t 2 + 63.56 t + 22.25 R2= 0.9464
t=10 min Relative Fluorescence= 1.286 t 3 -12.36 t 2 + 19.78 t + 29.03 R2= 0.9986
t=20 min Relative Fluorescence= 17.26 t 3 -83.37 t 2 + 96.5 t + 19.91 R2= 0.9957
t=30 min Relative Fluorescence= 9.669 t 3 - 48.15 t 2 + 58.23 t + 39.34 R2= 0.8645
t=60 min Relative Fluorescence= 20.96 t 3 - 106.2 t 2 + 134.9 t + 42.16 R2= 0.9482

Lldr- T7-lldPRD operon promoter-GFP：


[IPTG]=1 mM：
t=0 min Relative Fluorescence= 6.123 t 3-32.29 t 2+41.51 t+ 34.97 R2=0.4449
t=5 min Relative Fluorescence= 6.669 t 3 -38.13 t 2 + 55.27 t + 13.89 R2= 0.9808
t=10 min Relative Fluorescence= -3.422 t 3 +7.808 t 2 + 8.072 t + 11.18 R2=0.9946
t=20 min Relative Fluorescence= 10.74 t 3-48.53 t 2+57.9 t+ 9.947 R2= 0.9621
t=30 min Relative Fluorescence= 7.02 t 3 - 37.92 t 2 + 55.03 t + 9.921 R2= 0.9998
t=60 min Relative Fluorescence= 9.403 t 3 - 55.39 t 2 + 80.41 t + 8.451 R2= 0.9787

lldPRD operon promoter-GFP：


[IPTG]=0 mM：
t=0 min Relative Fluorescence= 2.27 t 3 - 12.95 t 2 +23.4 t+ 8.107 R2= 0.9883
t=5 min Relative Fluorescence= -2.332 t 3 +8.402 t 2 + 2.239 t + 20.74 R2= 0.9834
t=10 min Relative Fluorescence= 0.4742 t 3 -1.957 t 2 + 5.059 t +26.94 R2= 0.9544
t=20 min Relative Fluorescence= -3.069 t 3+17.46 t 2-25.51 t+ 21.98 R2= 0.9398
t=30 min Relative Fluorescence= -7.451 t 3 +36.49 t 2 -44.01 t + 39.25 R2= 0.9645
t=60 min Relative Fluorescence= -0.7971 t 3 +4.694 t 2 -13.22 t + 39.88 R2= 0.9761

lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP：


t=0 min Relative Fluorescence= -9.425 t 3+43.48 t 2-48.63 t+ 44.85 R2= 0.9985
t=5 min Relative Fluorescence= 9.999 t 3 -56.32 t 2 +79.92 t + 12.47 R2= 0.9696
t=10 min Relative Fluorescence= 12.03 t^3 -62.83 * t^2 +86.66 * t+10.36 R2= 0.7222
t=20 min Relative Fluorescence= 4.578 t 3-32.79 t 2+59.31 t+9.459 R2= 0.898
t=30 min Relative Fluorescence= 6.297 t 3 -35.63 t 2 + 53.2 t + 9.274 R2= 0.8867
t=60 min Relative Fluorescence= 9.122 * t 3 - 51.26 * t 2 + 73.33 * t + 5.924 R2= 0.9866

lldPRD operon promoter-Luxs × LsrA promoter-GFP


t=0 min Relative Fluorescence= -11.78 t 3+46.85 t 2-28.71 t+ 10.35 R2= 0.9764
t=5 min Relative Fluorescence= -12.48 t 3 +45.65 t 2 -26.91 t + 18.86 R2= 0.9762
t=10 min Relative Fluorescence= -1.135 t 3 +5.337 t 2 -3.825 t + 23.12 R2= 0.511
t=20 min Relative Fluorescence= -10.64 t 3+35.98 t 2-12.38 t+ 9.098 R2= 0.9716
t=30 min Relative Fluorescence= 3.63 t 3 - 25.57 t 2 + 40.87 t + 14.54 R2= 0.893
t=60 min Relative Fluorescence= 9.562 t 3 - 53.63 t 2 + 74.38 t + 9.515 R2= 0.9907

Practical significance of model results

Figures

T7- lldPRD operon promoter-GFP：

[IPTG]=1 mM

When [IPTG]=1 mM , The concentration of fluorescence at each point of time shows a similar trend towards the change in the rate of lactate, which is increased by the increase in lactate concentration, and then the fluorescence starts to go up and then down, and then there's a tendency to increase the intensity of the fluorescence, at the end of the level of the lactic acid, which is at the peak of the lactic acid, which is at the level of 0.5 to the one, and the fluorescence is at a peak. According to actual production, the concentration of yogurt in fermentation cannot exceed 1mM[1]. Therefore, the engineered bacteria can monitor the lactic acid concentration of yogurt during fermentation. In terms of time, when IPTG was added with 1mm, the fluorescence value of 5-60 min was all higher than that of 0 min, indicating that the reaction of engineering bacteria to lactic acid was effective during this period. Therefore, 5 min was selected as the appropriate reaction time. Because the fluorescence intensity of fiber detection can be stable within 200 milliseconds, the actual production can be obtained, and the time for testing the lactic acid concentration by using this engineering bacteria is 5 min.

Lldr- T7-lldPRD operon promoter-GFP：

[IPTG]=1 mM

When [IPTG]=1 mM, with the increase of time, the intensity of fluorescence expressed by engineered bacteria all lower than the intensity at 0 min. This may be due to the fact that at 0 min, the expression of lldR has not yet started or the expression quantity is too low, so the opening of lldPRD operon promoter caused by the introduction of engineering bacteria is not prevented. However, as the reaction time increases, lldR gradually produces, and the GFP background expression decreases due to the opening of lldPRD operon promoter. From the perspective of time, when the reaction time was 5 min, the change trend of fluorescence intensity with the concentration of lactic acid was consistent with the initial reaction time, so the good reaction time of engineering bacteria could be achieved within 5 min.

lldPRD operon promoter-GFP：

[IPTG]=0 mM

When there is no lactose operon, different concentrations of lactic acid are added. Although the engineered bacteria will still react according to different concentrations of lactic acid, the curve of the function is obviously irregular with different reaction times.

lldPRD operon promoter-GFP & T7- lldPRD operon promoter-GFP& Lldr- T7-lldPRD operon promoter-GFP:
We fitted the change of fluorescence intensity expression of the three engineered bacteria at the reaction time of 5 min, hoping to obtain the difference of the three engineered bacteria.

In the process of production of yogurt, the lactic acid content should not exceed 1 mM. So when the lactic acid content was lower than 1 mM, the lactose operon or lldR is added to the engineered bacteria and the lactic acid concentration is lower than 1 mM, indicating that the two engineered bacteria can achieve high sensitivity. Besides, when lactic acid content was lower than 1 mM, the lactose operon or lldR is added to the engineered bacteria is higher than those which only have lldPRD operon, indicating that both the lactose operon and lldR have an improved effect on the initial engineered bacteria.(BBa_K822000 ). Compared with the improvement of BBa_K822000 by lldR, it is obvious that the addition of lactose operon makes engineering bacteria more sensitive.

lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP

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Except for the beginning of the reaction, The rest of the functional curves showed similar trends, especially when the reaction time was 5 min and 10 min, and the peak value was reached before the concentration of lactic acid was 1 mM. It proved that the engineering bacteria was best used to detect the lactic acid concentration in yogurt at the reaction time of 5 min and 10 min. In order to reduce the reaction time of engineered bacteria, the reaction time of 5 min was selected.

lldPRD operon promoter-Luxs × LsrA promoter-GFP

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According to the result of fitting, after 30 min of reaction, the engineered bacteria were highly sensitive to different concentrations of lactic acid. However, the reacting time was to long, the increase of fluorescence value may be caused by the death of bacteria, which has no reference value.

lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP & lldPRD operon promoter-Luxs × LsrA promoter-GFP& Lldr- T7-lldPRD operon promoter-GFP

We fitted the change of fluorescence intensity expression of the three engineered bacteria at the reaction time of 5 min, hoping to obtain the difference of the three engineering bacteria.

Apparently, when the lactic acid concentration is less than 1 mM, included lldPRD operon promoter - Luxs - Lldr x LsrA promoter - the GFP and Lldr - T7 has - lldPRD operon promoter - GFP, two kinds of engineering bacteria in the peak, and the sensitivity of the former than the latter. This indicates that when the lactic acid concentration of yogurt is not over the limit, these two types of engineered bacteria can produce higher sensitivity, and the addition of the swarm sensing system can improve the sensitivity of engineered bacteria to the lactic acid concentration. The engineering bacteria contains lldPRD operon promoter - Luxs x LsrA promoter - GFP in lactic acid concentration is lower than 2.5 mM, to produce higher sensitivity.

All in all, from what has been discussed above, we can learn that contains lldPRD operon promoter - Luxs - Lldr x LsrA promoter - GFP engineering bacteria is applied to we have the best equipment engineering bacteria.

Experimental Purpose

In order to quantify the relationship between fluorescence intensity and lactic acid concentration, the production of lactic acid can be controlled reasonably according to the real-time monitoring of fluorescence intensity, and the inhibitory effect of lactic acid on fermentation degree can be minimized and the fermentation degree can be maximized.

We can connect the function curve with the experimental purpose and further strengthen the experimental purpose that we have achieved through the experimental and modeling results
(1) Determine whether the addition of lactose operon improves the effect of the original engineered bacteria
(2) Determine whether the introduction of LLDR gene improves the effect of the original engineering bacteria
(3) Determine whether the addition of group sense system improves the reaction of lactic acid manipulator promoter to lactic acid
(4) Judge the actual reaction time of engineering bacteria applied in actual operation

Reference:

[1] Wanguang Li, Xinwen Wang, Yishun Ji. Comparative experiment of two lactic acid detection methods in yogurt [J]. Anhui agronomy bulletin, 2017, 23(21): 113-114