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

 
(24 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
<html>
 
<html>
  
−
 
+
<div class="center-block" style="max-width:800px;">
−
<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.
+
−
 
+
−
<br>T7- lldPRD operon promoter-GFP：
+
−
 
+
−
<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
+
−
 
+
−
<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
+
−
 
+
−
<h3>
+
−
    lldPRD operon promoter-GFP：
+
−
</h3>
+
−
 
+
−
<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
+
−
 
+
−
<h3>
+
−
    lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP：
+
−
</h3>
+
−
 
+
−
<br>t=0 min Relative Fluorescence= -9.425 t 3+43.48 t 2-48.63 t+ 44.85 R2= 0.9985
+
−
<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>
+
−
    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.
+
−
</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>
+
−
    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.
+
−
</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>
+
−
<img src="https://static.igem.org/mediawiki/2018/8/8c/T--NEU_China_B--basepart6.png">
+
−
<p>
+
−
    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.
+
−
</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>
+
−
    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.
+
−
</p>
+
−
<p>
+
−
    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>
+
−
<img src="https://static.igem.org/mediawiki/2018/e/e1/T--NEU_China_B--basepart13.jpg">
+
−
<p>
+
−
    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.
+
−
</p>
+
−
                   
+
−
<p>
+
−
    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.
+
−
</p> 
+
−
 
+
−
 
+
−
 
+
−
<html>
+
−
<script>
+
−
////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
+
−
 
+
−
$(document).ready(function () {
+
−
 
+
−
$("#HQ_page").attr('id', '');
+
−
 
+
−
 
+
−
//highlight current page on the menu
+
−
highlight_current_page_menu();
+
−
 
+
−
//accessing submenus
+
−
$(".menu_item").click(function () {
+
−
$(".submenu_control_icon", this).toggleClass("open");
+
−
$(this).next(".submenu").fadeToggle(400);
+
−
});
+
−
 
+
−
//mobile menu access
+
−
$(".igem_2018_team_mobile_bar").click(function () {
+
−
$(this).next().toggleClass("displaying_menu");
+
−
});
+
−
 
+
−
});
+
−
 
+
−
 
+
−
function highlight_current_page_menu() {
+
−
 
+
−
var page_url = "https://2018.igem.org/";
+
−
page_url = page_url + wgPageName;
+
−
$("a[href$='" + page_url + "']").children().addClass("current_page");
+
−
 
+
−
//if the page is in a submenu, open the submenu and make the appropiate changes
+
−
if ($(".current_page").hasClass("submenu_item")) {
+
−
 
+
−
$(".current_page").parent().parent().fadeToggle(400);
+
−
$(".current_page").parent().parent().prev().addClass("current_page");
+
−
$(".menu_item.current_page > .submenu_control_icon").toggleClass("open");
+
−
 
+
−
}
+
−
}
+
−
 
+
−
 
+
−
////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
+
−
</script>
+
−
 
+
−
 
+
−
<style>
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
 
+
−
 
+
−
/**************************************************************************************************************************************************************************************************/
+
−
/* DEFAULT WIKI SETTINGS */
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
 
+
−
#home_logo, #sideMenu { display:none; }
+
−
#sideMenu, #top_title, .patrollink  {display:none;}
+
−
#content { margin-left:0px; margin-top:-7px; padding:0px; width:100%;}
+
−
body {background-color:white; }
+
−
#bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
+
−
 
+
−
.judges-will-not-evaluate { border: 4px solid #e4dede; padding: 2% !important; width: 92%!important;}
+
−
 
+
−
 
+
−
/**************************************************************************************************************************************************************************************************/
+
−
/* MENU */
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
/*this wraps the whole of the menu*/
+
−
.igem_2018_team_menu {
+
−
background-color:#c4baba;
+
−
border-left: 1px solid #c4baba;
+
−
display:block;
+
−
float:right;
+
−
height:100vh;
+
−
max-width: 270px;
+
−
overflow-y: auto;
+
−
overflow-x: hidden;
+
−
padding:0px;
+
−
position:fixed;
+
−
right:0%;
+
−
text-align:left;
+
−
width: 15%;
+
−
}
+
−
 
+
−
 
+
−
.igem_2018_team_menu.displaying_menu{
+
−
display:block;
+
−
}
+
−
 
+
−
.igem_2018_team_menu  a {
+
−
color: #484848;
+
−
text-decoration:none;
+
−
}
+
−
 
+
−
.igem_2018_team_menu img {
+
−
width:100%;
+
−
}
+
−
 
+
−
 
+
−
.igem_2018_team_menu .menu_item {
+
−
    background-color: #c4baba;
+
−
    border-bottom: 1px solid #928b8b;
+
−
    clear: both;
+
−
color: #484848;
+
−
    cursor: pointer;
+
−
float: left;
+
−
    font-size: 120%;
+
−
    font-weight: bold;
+
−
    padding: 15px 0px 15px 5%;
+
−
  width: 100%;
+
−
}
+
−
 
+
−
.igem_2018_team_menu .menu_item.direct_link {
+
−
color: #484848;
+
−
padding-left: 15%;
+
−
}
+
−
.igem_2018_team_menu .menu_item:hover {
+
−
background-color: #ecb656;
+
−
}
+
−
 
+
−
 
+
−
.igem_2018_team_menu .menu_item .submenu_control_icon {
+
−
color: #484848;
+
−
float: left;
+
−
width: 10%;
+
−
}
+
−
 
+
−
/* submenu icon "+"  "-"*/
+
−
.igem_2018_team_menu .menu_item .submenu_control_icon::before {
+
−
content: "+";
+
−
}
+
−
 
+
−
/* submenu icon "+"  "-"*/
+
−
.igem_2018_team_menu .menu_item .submenu_control_icon.open::before {
+
−
content: "-";
+
−
}
+
−
 
+
−
/*submenu wrapper*/
+
−
.igem_2018_team_menu .submenu{
+
−
background-color: #e4dede;
+
−
clear:both;
+
−
display:none;
+
−
float: left; 
+
−
width:100%;
+
−
}
+
−
 
+
−
/*styling for a submenu item*/
+
−
.igem_2018_team_menu .submenu .submenu_item {
+
−
border-bottom: 1px solid #c4baba;
+
−
  color: #635d5d;
+
−
    height: 30px;
+
−
  float: left;
+
−
    font-size: 110%;
+
−
font-weight: bold;
+
−
    padding: 12px 0px 0px 15%;
+
−
    width: 100%;
+
−
}
+
−
 
+
−
.igem_2018_team_menu .submenu .submenu_item:hover {
+
−
background-color: #f3bd5d;
+
−
}
+
−
 
+
−
 
+
−
.igem_2018_team_menu .submenu .submenu_item.current_page,
+
−
.igem_2018_team_menu .menu_item.current_page,
+
−
.igem_2018_team_menu .menu_item.direct_link.current_page {
+
−
background-color:#7acbd8;
+
−
}
+
−
 
+
−
 
+
−
 
+
−
/*mobile menu bar styling*/
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
 
+
−
.igem_2018_team_mobile_bar {
+
−
background-color:#e4dede;
+
−
border-bottom: 1px solid #c4baba;
+
−
cursor:pointer;
+
−
display:none;
+
−
float:left;
+
−
margin-top: 0;
+
−
padding: 5px 0;
+
−
position:fixed;
+
−
width:100%;
+
−
}
+
−
 
+
−
.igem_logo_mobile img {
+
−
width:70px;
+
−
}
+
−
 
+
−
.igem_logo_mobile {
+
−
float:left;
+
−
padding-left: 5%;
+
−
width: 30%;
+
−
}
+
−
 
+
−
.igem_menu_control_mobile img {
+
−
width:25px;
+
−
}
+
−
 
+
−
.igem_menu_control_mobile {
+
−
float:right;
+
−
padding-right:5%;
+
−
padding-top:5px;
+
−
text-align:right;
+
−
width: 30%;
+
−
}
+
−
 
+
−
 
+
−
 
+
−
/**************************************************************************************************************************************************************************************************/
+
−
/* CONTENT OF THE PAGE */
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
/* general wrapper for the content */
+
−
.igem_2018_team_content {
+
−
background-color:white;
+
−
display:block;
+
−
width: 87%;
+
−
}
+
−
 
+
−
/* subwrapper to center content */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper {
+
−
margin:auto;
+
−
max-width: 1400px;
+
−
width:90%;
+
−
}
+
−
 
+
−
 
+
−
 
+
−
/*general styling*/
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h1 { font-size: 210%;}
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h2 { font-size: 190%;}
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h3 { font-size: 170%;}
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h4 { font-size: 150%;}
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h5 { font-size: 140%;}
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h6 { font-size: 130%;}
+
−
 
+
−
 
+
−
/* styling for the titles h1, h2*/
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h1, .igem_2018_team_content .igem_2018_team_column_wrapper h2 {
+
−
border-bottom:0px;
+
−
color: #635d5d;
+
−
font-family: "Arial Black", Gadget, sans-serif;
+
−
padding: 10px 0px;
+
−
}
+
−
 
+
−
/* styling for the titles h3, h3, h5, h6 */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h3,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h4,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h5,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper h6 {
+
−
border-bottom:0px;
+
−
color: #928b8b; 
+
−
font-family: "Arial Black", Gadget, sans-serif;
+
−
padding: 5px 0px;
+
−
}
+
−
 
+
−
/* text */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper p {
+
−
font-size: 130%;
+
−
font-family: Arial, Helvetica, sans-serif;
+
−
padding: 5px 0px;
+
−
text-align: left;
+
−
color: #484848;
+
−
}
+
−
 
+
−
/* Links */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper a {
+
−
color: #5bc7d8;
+
−
font-weight: bold;
+
−
text-decoration: underline;
+
−
text-decoration-color:#5bc7d8;
+
−
transition: all 0.4s ease;
+
−
-webkit-transition: all 0.4s ease;
+
−
-moz-transition: all 0.4s ease;
+
−
-ms-transition: all 0.4s ease;
+
−
-o-transition: all 0.4s ease;
+
−
}
+
−
 
+
−
/* hover for the links */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper a:hover {
+
−
color: #f8b732;
+
−
text-decoration:none;
+
−
}
+
−
 
+
−
 
+
−
/* Table */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper table {
+
−
border: 1px solid #928b8b;
+
−
border-collapse: collapse;
+
−
font-size: 130%;
+
−
width: 100%;
+
−
}
+
−
 
+
−
/* table cells */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper td {
+
−
border: 1px solid #c4baba;
+
−
border-collapse: collapse;
+
−
font-size: 105%;
+
−
padding: 10px;
+
−
vertical-align: text-top;
+
−
}
+
−
 
+
−
/* table headers */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper th {
+
−
background-color:#c4baba;
+
−
border: 1px solid #928b8b;
+
−
border-collapse: collapse;
+
−
font-size: 110%;
+
−
padding: 10px;
+
−
vertical-align: text-top;
+
−
}
+
−
 
+
−
 
+
−
 
+
−
/* non numbered lists */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ul, .igem_2018_team_content .igem_2018_team_column_wrapper ol {
+
−
font-size: 130%;
+
−
font-family: Arial, Helvetica, sans-serif;
+
−
padding:0px 20px;
+
−
}
+
−
 
+
−
 
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ul ul li, .igem_2018_team_content .igem_2018_team_column_wrapper ul ul ul li,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ul ol li, .igem_2018_team_content .igem_2018_team_column_wrapper ul ul ol li,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ol ol li, .igem_2018_team_content .igem_2018_team_column_wrapper ul ol ul li,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ol ul li, .igem_2018_team_content .igem_2018_team_column_wrapper ul ol ol li,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ol ul ul li, .igem_2018_team_content .igem_2018_team_column_wrapper ol ol ul li,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper ol ol ol li, .igem_2018_team_content .igem_2018_team_column_wrapper ol ul ol li{ font-size: 76%; }
+
−
 
+
−
 
+
−
 
+
−
/*layout classes*/
+
−
/**************************************************************************************************************************************************************************************************/
+
−
 
+
−
/*main layout class */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column  {
+
−
float:left;
+
−
margin: 1% 2%;
+
−
padding: 0px;
+
−
}
+
−
 
+
−
/* 100% */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.full_size { width:96%; }
+
−
 
+
−
/* 66% */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.two_thirds_size { width: 62.6%; }
+
−
 
+
−
/* 33% */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.third_size { width: 29.3%; }
+
−
 
+
−
 
+
−
 
+
−
 
+
−
/*styling for all images*/
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.full_size img,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.two_thirds_size img,
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.third_size img {
+
−
margin-bottom: 15px;
+
−
width: 100%;
+
−
}
+
−
 
+
−
 
+
−
/* page break */
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .clear {
+
−
clear:both;
+
−
}
+
−
/*add extra space to page break with clear class*/
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .clear.extra_space {
+
−
height: 30px;
+
−
}
+
−
 
+
−
/* horizontal line to divide the page*/
+
−
.igem_2018_team_content .igem_2018_team_column_wrapper .line_divider {
+
−
    border-top: 1px solid #c4baba;
+
−
  margin: auto;
+
−
  width: 98%;
+
−
}
+
−
 
+
−
 
+
−
 
+
 
 
−
/*support classes*/
+
<h1>
−
/**************************************************************************************************************************************************************************************************/
+
Modeling
−
 
+
</h1>
−
 
+
<h2>
−
/*Button  */
+
Mathematical Model
−
/************************************************/
+
</h2>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .button_link {
+
<h2>
−
font-size: 130%;
+
Overview
−
margin: 30px auto;
+
</h2>
−
text-align: center;
+
<div class="p">
−
}
+
In the following article, we described the process of building our model. During the construction, we aimed 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 optimizations, so as to reflect the data trend reasonably.
−
 
+
</div>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .button_link a  {
+
<div class="p">
−
background-color: #5bc7d8;
+
During the experiment, we firstly controlled the consistency of the concentration of the bacterial liquid to reduce the experimental bias. There were 3 groups of experimental statistic, we recorded three kinds of variables: 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 correlative coefficient test, according to the fitting of known data, we got the functions and function curves that could reflect data trend reasonably. These curves illustrated the functional relationship between the fluorescence intensity of the dependent variable and the independent variable.
−
color: #635d5d !important;
+
</div>
−
font-weight: bold;
+
<h2>
−
margin: auto;
+
How do we derive this model?
−
text-decoration: none !important;
+
</h2>
−
padding: 10px 15px;
+
<div class="p">
−
}
+
(1) According to some reasonable assumptions given in professional knowledge, we believed that function got according to data may be a non-linear function between the dependent variable and the independent variable, since the addition of lactate and IPTG would tend to influence the growth rate of the organism, and the lactate 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 did not constitute linear function requirements. So we hypothesized that they are polynomial functions.
−
 
+
</div>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .button_link a:hover {
+
<div class="p">
−
background-color: #f8b732 !important; 
+
(2) Under each reasonable hypothesis, MATLAB was used to perform corresponding fitting. According to the correlative coefficient R to test the fitting effect, the fitting function formula that reasonably reflected the data trend can be obtained after continuous optimization.
−
}
+
</div>
−
 
+
<h2>
−
 
+
T7- lldPRD operon promoter-GFP
−
 
+
</h2>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight {
+
<h2>
−
padding: 15px 20px;
+
[IPTG]=1 mM
−
}
+
</h2>
−
 
+
<div class="p">
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight p,
+
<br>t=0 min Relative Fluorescence= 1.496 t^3 - 8.148 t^2 + 10.31 t + 8.919 R<sup>2</sup>= 0.1473
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight h1,
+
<br>t=5 min Relative Fluorescence= 9.304 t^3 - 49.68 t^2 + 63.56 t + 22.25 R<sup>2</sup>= 0.9464
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight h2,
+
<br>t=10 min Relative Fluorescence= 1.286 t^3 - 12.36 t^2 + 19.78 t + 29.03 R<sup>2</sup>= 0.9986
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight h3,
+
<br>t=20 min Relative Fluorescence= 17.26 t^3 - 83.37 t^2 + 96.5 t + 19.91 R<sup>2</sup>= 0.9957
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight h4,
+
<br>t=30 min Relative Fluorescence= 9.669 t^3 - 48.15 t^2 + 58.23 t + 39.34 R<sup>2</sup>= 0.8645
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight h5,
+
<br>t=60 min Relative Fluorescence= 20.96 t^3 - 106.2 t^2 + 134.9 t + 42.16 R<sup>2</sup>= 0.9482
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight h6 {
+
</div>
−
padding: 5px 15px;
+
−
}
+
<h2>
−
 
+
Lldr- T7-lldPRD operon promoter-GFP
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight.decoration_background {
+
</h2>
−
background-color: #e4dede;
+
<h2>
−
}
+
[IPTG]=1 mM
−
 
+
</h2>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight.decoration_A_top {
+
<div class="p">
−
    border-top: 4px solid #5bc7d8;
+
<br>t=0 min Relative Fluorescence= 6.123 t^3 - 32.29 t^2 + 41.51 t+ 34.97 R<sup>2</sup>=0.4449
−
}
+
<br>t=5 min Relative Fluorescence= 6.669 t^3 - 38.13 t^2 + 55.27 t + 13.89 R<sup>2</sup>= 0.9808
−
 
+
<br>t=10 min Relative Fluorescence= -3.422 ^3 + 7.808 t^2 + 8.072 t + 11.18 R<sup>2</sup>=0.9946
−
 
+
<br>t=20 min Relative Fluorescence= 10.74 t^3 - 48.53 t^2 + 57.9 t+ 9.947 R<sup>2</sup>= 0.9621
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight.decoration_A_full {
+
<br>t=30 min Relative Fluorescence= 7.02 t^3 - 37.92 t^2 + 55.03 t + 9.921 R<sup>2</sup>= 0.9998
−
    border: 4px solid #5bc7d8;
+
<br>t=60 min Relative Fluorescence= 9.403 t^3 - 55.39 t^2 + 80.41 t + 8.451 R<sup>2</sup>= 0.9787
−
}
+
</div>
−
 
+
<h2>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight.decoration_B_top {
+
lldPRD operon promoter-GFP
−
    border-top: 4px solid #f8b732
+
</h2>
−
}
+
<h2>
−
 
+
[IPTG]=0 mM
−
 
+
</h2>
−
.igem_2018_team_content .igem_2018_team_column_wrapper .highlight.decoration_B_full {
+
<div class="p">
−
    border: 4px solid #f8b732;
+
<br>t=0 min Relative Fluorescence= 2.27 t^3 - 12.95 t^2 + 23.4 t+ 8.107 R<sup>2</sup>= 0.9883
−
}
+
<br>t=5 min Relative Fluorescence= -2.332 t^3 + 8.402 t^2 + 2.239 t + 20.74 R<sup>2</sup>= 0.9834
−
 
+
<br>t=10 min Relative Fluorescence= 0.4742 t^3 - 1.957 t^2 + 5.059 t +26.94 R<sup>2</sup>= 0.9544
−
 
+
<br>t=20 min Relative Fluorescence= -3.069 t^3 + 17.46 t^2 - 25.51 t+ 21.98 R<sup>2</sup>= 0.9398
−
 
+
<br>t=30 min Relative Fluorescence= -7.451 t^3 + 36.49 t^2 - 44.01 t + 39.25 R<sup>2</sup>= 0.9645
−
 
+
<br>t=60 min Relative Fluorescence= -0.7971 t^3 + 4.694 t^2 - 13.22 t + 39.88 R<sup>2</sup>= 0.9761
−
/*mobile*/
+
</div>
−
/**************************************************************************************************************************************************************************************************/
+
<h2>
−
 
+
lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP
−
 
+
</h2>
−
/* 1800px  */
+
<div class="p">
−
/************************************************/
+
<br>t=0 min Relative Fluorescence= -9.425 t^3 + 43.48 t^2 - 48.63 t+ 44.85 R<sup>2</sup>= 0.9985
−
@media only screen and (max-width: 1800px) {
+
<br>t=5 min Relative Fluorescence= 9.999 t^3 - 56.32 t^2 + 79.92 t + 12.47 R<sup>2</sup>= 0.9696
−
.igem_2018_team_content { width: 85%;}
+
<br>t=10 min Relative Fluorescence= 12.03 t^3 - 62.83 t^2 + 86.66 t+ 10.36 R<sup>2</sup>= 0.7222
−
.igem_2018_team_menu {display:block;}
+
<br>t=20 min Relative Fluorescence= 4.578 t^3 - 32.79 t^2 + 59.31 t+ 9.459 R<sup>2</sup>= 0.898
−
}
+
<br>t=30 min Relative Fluorescence= 6.297 t^3 - 35.63 t^2 + 53.2 t + 9.274 R<sup>2</sup>= 0.8867
−
 
+
<br>t=60 min Relative Fluorescence= 9.122 t^3 - 51.26 t^2 + 73.33 t + 5.924 R<sup>2</sup>= 0.9866
−
/* 1400px  */
+
</div>
−
/************************************************/
+
<div class="p">
−
@media only screen and (max-width: 1400px) {
+
lldPRD operon promoter-Luxs × LsrA promoter-GFP
−
.igem_2018_team_menu .menu_item { font-size:100%;}
+
</div>
−
.igem_2018_team_menu .submenu .submenu_item { font-size:90%;}
+
<div class="p">
−
.igem_2018_team_menu {display:block;}
+
<br>t=0 min Relative Fluorescence= -11.78 t^3 + 46.85 t^2 - 28.71 t+ 10.35 R<sup>2</sup>= 0.9764
−
}
+
<br>t=5 min Relative Fluorescence= -12.48 t^3 + 45.65 t^2 - 26.91 t + 18.86 R<sup>2</sup>= 0.9762
−
 
+
<br>t=10 min Relative Fluorescence= -1.135 t^3 + 5.337 t^2 - 3.825 t + 23.12 R<sup>2</sup>= 0.511
−
@media only screen and (max-width: 1001px) {
+
<br>t=20 min Relative Fluorescence= -10.64 t^3 + 35.98 t^2 - 12.38 t+ 9.098 R<sup>2</sup>= 0.9716
−
.igem_2018_team_menu {display:block;}
+
<br>t=30 min Relative Fluorescence= 3.63 t^3 - 25.57 t^2 + 40.87 t + 14.54 R<sup>2</sup>= 0.893
−
}
+
<br>t=60 min Relative Fluorescence= 9.562 t^3 - 53.63 t^2 + 74.38 t + 9.515 R<sup>2</sup>= 0.9907
−
 
+
</div>
−
/* 1000px  */
+
<h2>
−
/************************************************/
+
Practical significance of model results
−
@media only screen and (max-width: 1000px) {
+
</h2>
−
.igem_2018_team_content {width:100%; margin-left:0px;}
+
Figures
−
.igem_2018_team_menu {display:none; margin-top: 45px; min-width:50%; width:50%;}
+
<h3>
−
.igem_2018_team_mobile_bar {display:block;}
+
T7- lldPRD operon promoter-GFP
−
.igem_2018_team_content .igem_2018_team_column_wrapper .column.full_size, .igem_2018_team_content .igem_2018_team_column_wrapper .column.two_thirds_size,.igem_2018_team_content .igem_2018_team_column_wrapper .column.third_size {width:96%; }
+
</h3>
−
 
+
<div>
−
}
+
<div>
−
 
+
<img src="https://static.igem.org/mediawiki/2018/2/23/T--NEU_China_B--m0.png" alt="">
−
 
+
</div>
−
@media only screen and (max-width: 500px) {
+
Figure 1. Part Constitution of “T7- lldPRD operon promoter-GFP” (BBa_K2824006)
−
.igem_2018_team_menu {min-width:100%; width:100%; }
+
</div>
−
}
+
<div>
−
 
+
<div>
−
 
+
<img src="https://static.igem.org/mediawiki/2018/thumb/e/e1/T--NEU_China_B--m1.png/800px-T--NEU_China_B--m1.png.jpeg" alt="">
−
/**************************************************************************************************************************************************************************************************/
+
</div>
−
 
+
Figure 2. Relative fluorescence intensity under series of lactate concentration and time. IPTG concentration =1 mM.
−
 
+
</div>
−
 
+
<div class="p">
−
 
+
When [IPTG]=1 mM, fluorescence intensity at each point of time showed a similar trend towards the concentration change of lactate, meaning that fluorescence intensity firstly increases along with the higher lactate concentration and then goes down. The fluorescence intensity reached to the peak under applying lactate concentration from 0.5 mM to 1 mM. According to yogurt fermentation in reality, the concentration of lactate could not exceed 1 mM<sup>[1]</sup>. Therefore, this engineered <i>E.coli</i> could monitor the lactate concentration of yogurt during fermentation. In terms of time, the fluorescence value of 5-60 min was all higher than that of 0 min, indicating that the reaction of engineering <i>E.coli</i> to lactate was effective during this period. Therefore, 5 min was selected as the appropriate reaction time. Also, the fluorescence intensity of optical fiber detection could be stable within 200 milliseconds, so the time for testing the lactate concentration by using this engineering <i>E.coli</i> was 5 min.  
−
 
+
</div>
−
</style>
+
<h2>
−
 
+
Lldr- T7-lldPRD operon promoter-GFP
−
 
+
</h2>
−
<!------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------->
+
<div>
−
<!--- THIS IS WHERE THE HTML BEGINS --->
+
<div>
−
<!------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------->
+
<img src="https://static.igem.org/mediawiki/2018/d/d3/T--NEU_China_B--m2.png" alt="">
−
 
+
</div>
−
<head>
+
Figure 3. Part Constitution of “Lldr- T7-lldPRD operon promoter-GFP” (BBa_K2824008)
−
 
+
</div>
−
<meta charset="utf-8">
+
−
<meta http-equiv="X-UA-Compatible" content="IE=edge">
+
<h2>
−
<meta name="viewport" content="width=device-width, initial-scale=1">
+
[IPTG]=1 mM
−
<title>Title Page</title>
+
</h2>
−
<!-- Bootstrap CSS -->
+
<div>
−
<link href="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.7/css/bootstrap.min.css" rel="stylesheet">
+
<div>
−
<script src="https://cdn.bootcss.com/jquery/2.2.1/jquery.min.js"></script>
+
<img src="https://static.igem.org/mediawiki/2018/thumb/0/0c/T--NEU_China_B--m3.png/800px-T--NEU_China_B--m3.png.jpeg" alt="">
−
<script src="https://cdn.bootcss.com/bootstrap/3.3.7/js/bootstrap.min.js"></script>
+
</div>
−
</head>
+
Figure 4. Relative Fluorescence Intensity under series of lactate concentration and time. IPTG concentration =1 mM.
−
 
+
</div>
−
 
+
<div class="p">
−
 
+
When [IPTG]=1 mM, with the increase of time, fluorescence intensity expressed by engineered <i>E.coli</i> is all lower than at 0 min. This might be due to the fact that at 0 min, the expression of lldR had not yet started or the expression quantity is too low, so the opening of lldPRD operon promoter caused by the introduction of engineering <i>E.coli</i> was not prevented. However, as the reaction time increases, lldR gradually produced so that the primitive expression of GFP decreases. From the perspective of time, when the reaction time was at 5 min, the change trend of fluorescence intensity with the concentration of lactate was consistent with the initial reaction time, so the good reaction time of engineering E.coli could be achieved within 5 min.
−
<!------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------->
+
</div>
−
<!--- Menu --->
+
<h2>
−
<!------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------->
+
lldPRD operon promoter-GFP
−
 
+
</h2>
−
<div class="igem_2018_team_mobile_bar">
+
<div>
−
 
+
<div>
−
<div class="igem_logo_mobile">
+
<img src="https://static.igem.org/mediawiki/2018/6/61/T--NEU_China_B--m4.png" alt="">
−
<img src="https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg">
+
</div>
−
</div>
+
Figure 5. Part Constitution of “lldPRD operon promoter-GFP” (BBa_K2824004)
−
 
+
</div>
−
 
+
<h2>
−
<div class="igem_menu_control_mobile">
+
[IPTG]=0 mM
−
<img id="menu_control_image" src="https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg">
+
</h2>
−
</div>
+
<div>
−
 
+
<div>
−
</div>
+
<img src="https://static.igem.org/mediawiki/2018/thumb/b/bb/T--NEU_China_B--m5.png/800px-T--NEU_China_B--m5.png.jpeg" alt="">
−
 
+
</div>
−
<!------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------->
+
Figure 6. Relative Fluorescence Intensity under series of lactate concentration and time. IPTG concentration =0 mM.
−
<!--- Content of the page  --->
+
</div>
−
<!------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------->
+
<div class="p">
−
<style>
+
When there was no lactose operon and adding different concentrations of lactate, although the engineered <i>E.coli</i> still reacted with different concentrations of lactate, curves of the function were obviously irregular with different reaction times.
−
.bs-docs-nav {
+
</div>
−
    background-color: #38c5e5;
+
<div class="p">
−
    border-color: #e7e7e7;
+
lldPRD operon promoter-GFP & T7- lldPRD operon promoter-GFP& Lldr- T7-lldPRD operon promoter-GFP
−
    }
+
</div>
−
    .nav .navbar-nav>li .dropdown-menu {margin:0;}
+
<div class="p">
−
    .nav .navbar-nav>li:hover .dropdown-menu{display:block;}
+
We fitted the change of fluorescence intensity of the three engineered <i>E.coli</i> above at the reaction time of 5 min, aiming at obtaining the differences of the three engineered <i>E.coli</i>.
−
    .navbar .nav > li .dropdown-menu {margin: 0;} 
+
</div>
−
    .navbar .nav > li:hover .dropdown-menu {
+
<div>
−
display: block;
+
<img src="https://static.igem.org/mediawiki/2018/thumb/4/48/T--NEU_China_B--m6.png/800px-T--NEU_China_B--m6.png.jpeg" alt="">
−
background-color: #38c5e5;
+
Figure 7. Comparison of Relative fluorescence intensity under series of lactate concentration and time.
−
}
+
</div>
−
    .rightbottom{position:fixed;bottom:50px;right:50px;}
+
<div class="p">
−
.nav>li>a {
+
In the process of production of yogurt in reality, the lactate content should not exceed 1 mM. So, when the lactate content was lower than 1 mM as well as adding the lactose operon or lldR to the engineered <i>E.coli</i>, their lactate concentration was lower than 1 mM, indicating that these two engineered <i>E.coli</i> can detect lactate in yogurt with high sensitivity. Besides, when lactate content was lower than 1 mM, engineered <i>E.coli</i> with the lactose operon or lldR was higher than those which only contains lldPRD operon, indicating that both the lactose operon and lldR had an improved effect on the initial engineered <i>E.coli</i> (BBa_K822000). Compared with the improvement of BBa_K822000 by lldR and lactose operon, it was obvious that the addition of lactose operon makes engineering E.coli was more sensitive to normal yogurt lactate concentration.
−
    position: relative;
+
</div>
−
    display: block;
+
<h2>
−
    color:#ffffff;
+
lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP
−
}
+
</h2>
−
</style>
+
<div>
−
<header class="navbar navbar-static-top bs-docs-nav" style="margin-top: 15px" id="top">
+
<img src="https://static.igem.org/mediawiki/2018/thumb/a/aa/T--NEU_China_B--m7.png/797px-T--NEU_China_B--m7.png" alt="">
−
<div class="container">
+
Figure 8. Part Constitution of “lldPRD operon promoter-Luxs-Lldr” (BBa_K2824007) and “LsrA promoter-GFP” (BBa_K2824009)
−
<div class="navbar-header">
+
</div>
−
<button class="navbar-toggle collapsed" type="button" data-toggle="collapse" data-target="#bs-navbar" aria-controls="bs-navbar"
+
<div class="p">
−
aria-expanded="false">
+
<img src="https://static.igem.org/mediawiki/2018/thumb/6/6b/T--NEU_China_B--m8.png/800px-T--NEU_China_B--m8.png.jpeg" alt="">
−
<span class="sr-only">Toggle navigation</span>
+
Figure 9. Relative Fluorescence Intensity under series of lactate concentration and time.
−
<span class="icon-bar"></span>
+
</div>
−
<span class="icon-bar"></span>
+
−
<span class="icon-bar"></span>
+
<div class="p">
−
</button>
+
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 lactate is 1 mM. It demonstrated that the engineered <i>E.coli</i> is best used to detect the lactate concentration in yogurt at 5 min and 10 min. In order to reduce the reaction time of engineered bacteria, the reaction time of 5 min was selected.
−
<a href="https://2018.igem.org/Team:NEU_China_B" class="navbar-brand">LED</a>
+
</div>
−
</div>
+
<H2>
−
<nav id="bs-navbar" class="collapse navbar-collapse">
+
lldPRD operon promoter-Luxs × LsrA promoter-GFP
−
<ul class="nav navbar-nav navbar-right">
+
</H2>
−
<li class="dropdown">
+
<div>
−
<a href="#">Project<span class="caret"></span></a>
+
<div>
−
<ul class="dropdown-menu">
+
<img src="https://static.igem.org/mediawiki/2018/c/c3/T--NEU_China_B--m9.png" alt="">
−
<li><a href="https://2018.igem.org/Team:NEU_China_B/background">Background</a></li>
+
</div>
−
<li>
+
Figure 10. Part Constitution of “lldPRD operon promoter-Luxs” (BBa_K2824005) and “LsrA promoter-GFP” (BBa_K2824009)
−
<a href="https://2018.igem.org/Team:NEU_China_B/Description">
+
</div>
−
Description
+
<div>
−
</a>
+
<img src="https://static.igem.org/mediawiki/2018/thumb/0/0e/T--NEU_China_B--m10.png/800px-T--NEU_China_B--m10.png.jpeg" alt="">
−
</li>
+
Figure 11. Relative Fluorescence Intensity under series of lactate concentration and time.
−
<li>
+
</div>
−
<a href="https://2018.igem.org/Team:NEU_China_B/Design">
+
<div class="p">According to the result of fitting, after 30 min of reaction, the engineered <i>E.coli</i> was highly sensitive to different concentrations of lactate. However, the reacting time was too long, the increase of fluorescence intensity might be caused by the death of bacteria.</div>
−
Applied Design
+
<div class="p">lldPRD operon promoter-Luxs-Lldr × LsrA promoter-GFP & lldPRD operon promoter-Luxs × LsrA promoter-GFP& Lldr- T7-lldPRD operon promoter-GFP</div>
−
</a>
+
<div class="p">We fitted the change of fluorescence intensity expression of the three engineered <i>E.coli</i> at the reaction time of 5 min, hoping to obtain the difference of the three engineering <i>E.coli</i>.</div>
−
</li>
+
<div>
−
<li>
+
<img src="https://static.igem.org/mediawiki/2018/thumb/0/07/T--NEU_China_B--m11.png/800px-T--NEU_China_B--m11.png.jpeg" alt="">
−
<a href="https://2018.igem.org/Team:NEU_China_B/Improve">
+
<div class="p">Figure 12. Comparison of Relative fluorescence intensity under series of lactate concentration and time.</div>
−
Improve
+
</div>
−
</a>
+
<div class="p">
−
</li>
+
Similarly, when the lactate concentration was less than 1 mM, included “lldPRD operon promoter - Luxs – Lldr” combined with “LsrA promoter” as well as “Lldr - T7 - lldPRD operon promoter – GFP”, these two kinds of engineered <i>E.coli</i> showed the peak value, and the sensitivity of the former was higher than the latter. This indicated that when the lactate concentration of yogurt was not over the limitation, these two types of engineered E.coli could produce higher sensitivity, and the addition of the QS system could improve the sensitivity of engineered <i>E.coli</i> to the lactate concentration. The engineered <i>E.coli</i> containing “lldPRD operon promoter – Luxs” and “LsrA promoter – GFP”, in lactate concentration was lower than 2.5 mM, could sense with higher sensitivity.
−
<li>
+
</div>
−
<a href="https://2018.igem.org/Team:NEU_China_B/Model">
+
<div class="p">
−
Models
+
In summary, from what had been discussed above, we can learn that the engineered <i>E.coli</i> containing “lldPRD operon promoter - Luxs - Lldr x LsrA promoter – GFP” was selected as our best engineered E.coli in applying device.
−
</a>
+
</div>                  
−
</li>
+
<h2>
−
</ul>
+
Experimental Purpose
−
</li>
+
</h2>
−
<li class="dropdown">
+
<div class="p">
−
<a href="#">Lab<span class="caret"></span></a>
+
In order to quantify the relationship between fluorescence intensity and lactate concentration, lactate concentration could be monitored reasonably according to the real-time fluorescence intensity. The inhibited effect of lactate on yogurt fermentation could be minimized.
−
<ul class="dropdown-menu">
+
</div>
−
<li>
+
<div class="p">
−
<a href="https://2018.igem.org/Team:NEU_China_B/InterLab">
+
We can connect the function curves with the experimental purposes and further strengthen the experimental purposes that we have achieved through modeling results:
−
InterLab
+
</div>
−
</a>
+
<div class="p">
−
</li>
+
<br>(1) Determine whether the addition of lactose operon improves the effect of the original engineered <i>E.coli</i>.
−
<li><a href="https://2018.igem.org/Team:NEU_China_B/protocols">Protocols</a></li>
+
<br>(2) Determine whether the introduction of lldR gene improves the effect of the original engineered <i>E.coli</i>.
−
<li>
+
<br>(3) Determine whether the addition of QS system improves the reaction of lactate operon promoter to lactate.
−
<a href="https://2018.igem.org/Team:NEU_China_B/Notebook">
+
<br>(4) Judge the actual reaction time of engineered <i>E.coli</i> applied in actual operation
−
Notebook
+
</div>
−
</a>
+
<h2>
−
</li>
+
Physical Model
−
<li><a href="https://2018.igem.org/Team:NEU_China_B/Safety">Safety</a></li>
+
</h2>
−
 
+
<h2>
−
</ul>
+
3D model
−
</li>
+
</h2>
−
<li class="dropdown">
+
<div class="p">
−
<a href="#">Parts<span class="caret"></span></a>
+
We used 3D printing technology in constructing container of our device, and selected photosensitive resin as printing materials. The main reason for this is that photosensitive resin has a certain light-fixation quality for this material contain a certain ultraviolet initiator or photosensitive agent. When exposed to a certain wavelength of ultraviolet irradiation immediately caused by polymerization reaction, it could complete fixation to achieve the entire process of product modeling<sup>[2]</sup>. It is common that ultraviolet radiation in the field of medicine has a good anti-bacterial effect. The use of photosensitive resin materials can greatly guarantee the sterile conditions of the experimental device.
−
<ul class="dropdown-menu">
+
</div>
−
<li>
+
<div class="p">
−
<a href="https://2018.igem.org/Team:NEU_China_B/Basic_Part">
+
In addition, photosensitive resin materials have high printing precision, great product details with light feature. Also, they can withstand 120 degrees Celsius<sup>[2]</sup>, indicating that they can be sterilized through physical method, resulting in less effect other microbe to our device.  
−
Basic Parts
+
</div>
−
</a>
+
<div class="p">
−
</li>
+
After a thorough discussion and research, we finally adopted the drop-shaped mold type by using 3D modeling software. The entire experimental device reserved a 2 mm diameters hole above the water droplets for inserting the optical fibers, left one side of each 0.5mm hole for adding the sample. The whole device is sprayed with paint to ensure the opacity of the whole decoration and avoid the early excitation of green fluorescent protein.
−
<li>
+
</div>
−
<a href="https://2018.igem.org/Team:NEU_China_B/Composite_Part">
+
<div>
−
Composite Parts
+
<img src="https://static.igem.org/mediawiki/2018/thumb/f/fe/T--NEU_China_B--m12.png/800px-T--NEU_China_B--m12.png.jpeg" alt="">
−
</a>
+
Figure 13. Design of 3D model. (a) design draft; (b) real object
−
</li>
+
</div>
−
</ul>
+
<div class="p" style="font-style:italic;font-face:"Roboto";font-size:"3";"  >
−
</li>
+
<br><b>Reference:
−
<li class="dropdown">
+
<br>1. Wanguang Li, Xinwen Wang, Yishun Ji. Comparative experiment of two lactate detection methods in yogurt [J]. Anhui agronomy bulletin, 2017, 23(21): 113-114.
−
<a href="#">Human practice<span class="caret"></span></a>
+
<br>2. Biwu Huang, Wangfu Xie, Zhihong Yang. Preparation and Properties of a 3D Printed Stereolithography Rapid Prototyping Photosensitive Resin [J]. Functional Materials, 2014, 45 (24): 24100- 24104.</b>
−
<ul class="dropdown-menu">
+
</div>
−
<li>
+
−
<a href="https://2018.igem.org/Team:NEU_China_B/Human_Practices">
+
−
Human Practices
+
−
</a>
+
−
</li>
+
−
<li>
+
−
<a href="https://2018.igem.org/Team:NEU_China_B/Collaborations">
+
−
Collaborations
+
−
</a>
+
−
</li>
+
−
</ul>
+
−
</li>
+
−
<li class="dropdown">
+
−
<a href="#">Team<span class="caret"></span></a>
+
−
<ul class="dropdown-menu">
+
−
<li>
+
−
<a href="https://2018.igem.org/Team:NEU_China_B/Team">
+
−
Team Members
+
−
</a>
+
−
</li>
+
−
<li>
+
−
<a href="https://2018.igem.org/Team:NEU_China_B/Attributions">
+
−
Attributions
+
−
</a>
+
−
</li>
+
−
</ul>
+
−
</li>
+
−
</ul>
+
−
<ul class="nav navbar-nav navbar-right">
+
−
</ul>
+
−
</nav>
+
−
</div>
+
−
</header>
+
−
 
+
−
<div class="rightbottom">
+
−
<a class="btn btn-default" href="#top" role="button">got to top</a>
+
−
</div>
+
−
<div class="container" style="margin-top: 20px">
+
−
 
+
−
<h2>
+
−
Physical Model
+
−
</h2>
+
−
<p>
+
−
<br>We use the technology of 3d printing in the construction of the whole model of our product. The selected material is photosensitive resin material.  
+
−
 
+
−
<br>The main reason is that the photosensitive resin has a certain light-solid, which added a certain ultraviolet initiator or photosensitive agent, in a certain wavelength of ultraviolet irradiation immediately caused by polymerization reaction, complete curing, to achieve the entire process of product modeling. As we all know, ultraviolet radiation in the field of medicine has a good bactericidal effect, the use of photosensitive resin materials, can greatly guarantee the sterile conditions of the experimental device.
+
−
+
−
<br>Except for these, Printing precision of photosensitive resin material is very high, the printing finished product details are very good, the products are light, can withstand 120 degrees of high temperature, good chemical means to sterilize, no adverse effects on fungus growth, suitable for fungus growth.
+
−
+
−
<br>After a thorough discussion and research, we use 3D modeling software to build the device model, we finally adopt the drop-shaped mold Type. The entire experimental device reserves a 2mm diameter hole above the water droplets to insert the fiber, leaving the left and right sides of each 0.5mm hole to add the sample. The whole device is sprayed with paint to ensure the opacity of the whole decoration and avoid the early excitation of GFP fluorescent protein.  
+
−
+
−
</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>
+

Latest revision as of 03:52, 18 October 2018

Ruby - Responsive Corporate Tempalte

Modeling

Mathematical Model

Overview

In the following article, we described the process of building our model. During the construction, we aimed 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 optimizations, so as to reflect the data trend reasonably.
During the experiment, we firstly controlled the consistency of the concentration of the bacterial liquid to reduce the experimental bias. There were 3 groups of experimental statistic, we recorded three kinds of variables: 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 correlative coefficient test, according to the fitting of known data, we got the functions and function curves that could reflect data trend reasonably. These curves illustrated 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 believed that function got according to data may be a non-linear function between the dependent variable and the independent variable, since the addition of lactate and IPTG would tend to influence the growth rate of the organism, and the lactate 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 did not constitute linear function requirements. So we hypothesized that they are polynomial functions.
(2) Under each reasonable hypothesis, MATLAB was used to perform corresponding fitting. According to the correlative coefficient R to test the fitting effect, the fitting function formula that reasonably reflected 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 ^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

Figure 1. Part Constitution of “T7- lldPRD operon promoter-GFP” (BBa_K2824006)
Figure 2. Relative fluorescence intensity under series of lactate concentration and time. IPTG concentration =1 mM.
When [IPTG]=1 mM, fluorescence intensity at each point of time showed a similar trend towards the concentration change of lactate, meaning that fluorescence intensity firstly increases along with the higher lactate concentration and then goes down. The fluorescence intensity reached to the peak under applying lactate concentration from 0.5 mM to 1 mM. According to yogurt fermentation in reality, the concentration of lactate could not exceed 1 mM[1]. Therefore, this engineered E.coli could monitor the lactate concentration of yogurt during fermentation. In terms of time, the fluorescence value of 5-60 min was all higher than that of 0 min, indicating that the reaction of engineering E.coli to lactate was effective during this period. Therefore, 5 min was selected as the appropriate reaction time. Also, the fluorescence intensity of optical fiber detection could be stable within 200 milliseconds, so the time for testing the lactate concentration by using this engineering E.coli was 5 min.

Lldr- T7-lldPRD operon promoter-GFP

Figure 3. Part Constitution of “Lldr- T7-lldPRD operon promoter-GFP” (BBa_K2824008)

[IPTG]=1 mM

Figure 4. Relative Fluorescence Intensity under series of lactate concentration and time. IPTG concentration =1 mM.
When [IPTG]=1 mM, with the increase of time, fluorescence intensity expressed by engineered E.coli is all lower than at 0 min. This might be due to the fact that at 0 min, the expression of lldR had not yet started or the expression quantity is too low, so the opening of lldPRD operon promoter caused by the introduction of engineering E.coli was not prevented. However, as the reaction time increases, lldR gradually produced so that the primitive expression of GFP decreases. From the perspective of time, when the reaction time was at 5 min, the change trend of fluorescence intensity with the concentration of lactate was consistent with the initial reaction time, so the good reaction time of engineering E.coli could be achieved within 5 min.

lldPRD operon promoter-GFP

Figure 5. Part Constitution of “lldPRD operon promoter-GFP” (BBa_K2824004)

[IPTG]=0 mM

Figure 6. Relative Fluorescence Intensity under series of lactate concentration and time. IPTG concentration =0 mM.
When there was no lactose operon and adding different concentrations of lactate, although the engineered E.coli still reacted with different concentrations of lactate, curves of the function were 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 of the three engineered E.coli above at the reaction time of 5 min, aiming at obtaining the differences of the three engineered E.coli.
Figure 7. Comparison of Relative fluorescence intensity under series of lactate concentration and time.
In the process of production of yogurt in reality, the lactate content should not exceed 1 mM. So, when the lactate content was lower than 1 mM as well as adding the lactose operon or lldR to the engineered E.coli, their lactate concentration was lower than 1 mM, indicating that these two engineered E.coli can detect lactate in yogurt with high sensitivity. Besides, when lactate content was lower than 1 mM, engineered E.coli with the lactose operon or lldR was higher than those which only contains lldPRD operon, indicating that both the lactose operon and lldR had an improved effect on the initial engineered E.coli (BBa_K822000). Compared with the improvement of BBa_K822000 by lldR and lactose operon, it was obvious that the addition of lactose operon makes engineering E.coli was more sensitive to normal yogurt lactate concentration.

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

Figure 8. Part Constitution of “lldPRD operon promoter-Luxs-Lldr” (BBa_K2824007) and “LsrA promoter-GFP” (BBa_K2824009)
Figure 9. Relative Fluorescence Intensity under series of lactate concentration and time.
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 lactate is 1 mM. It demonstrated that the engineered E.coli is best used to detect the lactate concentration in yogurt at 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

Figure 10. Part Constitution of “lldPRD operon promoter-Luxs” (BBa_K2824005) and “LsrA promoter-GFP” (BBa_K2824009)
Figure 11. Relative Fluorescence Intensity under series of lactate concentration and time.
According to the result of fitting, after 30 min of reaction, the engineered E.coli was highly sensitive to different concentrations of lactate. However, the reacting time was too long, the increase of fluorescence intensity might be caused by the death of bacteria.
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 E.coli at the reaction time of 5 min, hoping to obtain the difference of the three engineering E.coli.
Figure 12. Comparison of Relative fluorescence intensity under series of lactate concentration and time.
Similarly, when the lactate concentration was less than 1 mM, included “lldPRD operon promoter - Luxs – Lldr” combined with “LsrA promoter” as well as “Lldr - T7 - lldPRD operon promoter – GFP”, these two kinds of engineered E.coli showed the peak value, and the sensitivity of the former was higher than the latter. This indicated that when the lactate concentration of yogurt was not over the limitation, these two types of engineered E.coli could produce higher sensitivity, and the addition of the QS system could improve the sensitivity of engineered E.coli to the lactate concentration. The engineered E.coli containing “lldPRD operon promoter – Luxs” and “LsrA promoter – GFP”, in lactate concentration was lower than 2.5 mM, could sense with higher sensitivity.
In summary, from what had been discussed above, we can learn that the engineered E.coli containing “lldPRD operon promoter - Luxs - Lldr x LsrA promoter – GFP” was selected as our best engineered E.coli in applying device.

Experimental Purpose

In order to quantify the relationship between fluorescence intensity and lactate concentration, lactate concentration could be monitored reasonably according to the real-time fluorescence intensity. The inhibited effect of lactate on yogurt fermentation could be minimized.
We can connect the function curves with the experimental purposes and further strengthen the experimental purposes that we have achieved through modeling results:

(1) Determine whether the addition of lactose operon improves the effect of the original engineered E.coli.
(2) Determine whether the introduction of lldR gene improves the effect of the original engineered E.coli.
(3) Determine whether the addition of QS system improves the reaction of lactate operon promoter to lactate.
(4) Judge the actual reaction time of engineered E.coli applied in actual operation

Physical Model

3D model

We used 3D printing technology in constructing container of our device, and selected photosensitive resin as printing materials. The main reason for this is that photosensitive resin has a certain light-fixation quality for this material contain a certain ultraviolet initiator or photosensitive agent. When exposed to a certain wavelength of ultraviolet irradiation immediately caused by polymerization reaction, it could complete fixation to achieve the entire process of product modeling[2]. It is common that ultraviolet radiation in the field of medicine has a good anti-bacterial effect. The use of photosensitive resin materials can greatly guarantee the sterile conditions of the experimental device.
In addition, photosensitive resin materials have high printing precision, great product details with light feature. Also, they can withstand 120 degrees Celsius[2], indicating that they can be sterilized through physical method, resulting in less effect other microbe to our device.
After a thorough discussion and research, we finally adopted the drop-shaped mold type by using 3D modeling software. The entire experimental device reserved a 2 mm diameters hole above the water droplets for inserting the optical fibers, left one side of each 0.5mm hole for adding the sample. The whole device is sprayed with paint to ensure the opacity of the whole decoration and avoid the early excitation of green fluorescent protein.
Figure 13. Design of 3D model. (a) design draft; (b) real object

Reference:
1. Wanguang Li, Xinwen Wang, Yishun Ji. Comparative experiment of two lactate detection methods in yogurt [J]. Anhui agronomy bulletin, 2017, 23(21): 113-114.
2. Biwu Huang, Wangfu Xie, Zhihong Yang. Preparation and Properties of a 3D Printed Stereolithography Rapid Prototyping Photosensitive Resin [J]. Functional Materials, 2014, 45 (24): 24100- 24104.