Difference between revisions of "Team:HBUT-China/Demonstrate"

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              <h1>Demonstrate</h1>
 
              <h1>Demonstrate</h1>
 
            </div>
 
            </div>
            <h5>We analyzed the experimental data through modeling.</h5>
+
          <p>We analyzed the experimental data through modeling.</p>
<small class="bigtitle">1.Assumptions:</small>
+
<small class="bigtitle">1.Assumptions:</small>
<h5>We made four assumptions in order to simplify some of the aspects of the model. Many similar assumptions have been made in the literature.</h5>
+
<p>We made four assumptions in order to simplify some of the aspects of the model. Many similar assumptions have been made in the literature.</p>
<p>The degradation of  and other substances are linear.</p>
+
<p>The degradation of  and other substances are linear.</p>
<p>Each cell has the same state.</p>
+
<p>Each cell has the same state.</p>
<p>The reaction in the process satisfies the first-order kinetic reaction.</p>
+
<p>The reaction in the process satisfies the first-order kinetic reaction.</p>
<small class="bigtitle">2. Transcription Repression and Release Repression</small>
+
<small class="bigtitle">2. Transcription Repression and Release Repression</small>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/5/5f/Hbut-demon-1.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/5/5f/Hbut-demon-1.png" alt="this is a photo">
</div>
+
</div>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/f/fd/Hbut-demon-2.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/f/fd/Hbut-demon-2.png" alt="this is a photo">
</div>
+
</div>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/d/db/Hbut-demon-3.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/d/db/Hbut-demon-3.png" alt="this is a photo">
</div>
+
</div>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/a/a7/Hbut-demon-4.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/a/a7/Hbut-demon-4.png" alt="this is a photo">
</div>
+
</div>
<h5>Using the assumption about the first-order kinetic reaction,</h5>
+
<p>Using the assumption about the first-order kinetic reaction,</p>
<h5>Give:</h5>
+
<p>Give:</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/b/b3/Hbut-demon-5.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/b/b3/Hbut-demon-5.png" alt="this is a photo">
</div>
+
</div>
+
<small class="bigtitle">3. Quasi-steady-state Approximation (QSSA)</small>
+
<small class="bigtitle">3. Quasi-steady-state Approximation (QSSA)</small>
<h5>Ordinary differential equations are too difficult to be solved, in order to get the solution of the equations, quasi-steady-state approximation is necessary.</h5>
+
<p>Ordinary differential equations are too difficult to be solved, in order to get the solution of the equations, quasi-steady-state approximation is necessary.</p>
<h5>We use the approximate conditions as little as possible and try to make the quasi steady state of matter consistent with the actual situation.</h5>
+
<p>We use the approximate conditions as little as possible and try to make the quasi steady state of matter consistent with the actual situation.</p>
+
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/4/4a/Hbut-demon-6.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/4/4a/Hbut-demon-6.png" alt="this is a photo">
</div>
+
</div>
+
+
<h5>According to the formula’s deformation and derivation, </h5>
+
<p>According to the formula’s deformation and derivation, </p>
<h5>gives:</h5>
+
<p>gives:</p>
  
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/5/5c/Hbut-demon-7.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/5/5c/Hbut-demon-7.png" alt="this is a photo">
</div>
+
</div>
+
  
<small class="bigtitle">4. Incomplete Repression</small>
+
<small class="bigtitle">4. Incomplete Repression</small>
<h5>Under incomplete repression, the bacteria express red fluorescent proteins without the presence of nickel ions.</h5>
+
<p>Under incomplete repression, the bacteria express red fluorescent proteins without the presence of nickel ions.</p>
<h5>The incomplete repression of promoters is a major issue in our fluorescence system. This issue was particularly observed in following diagram.</h5>
+
<p>The incomplete repression of promoters is a major issue in our fluorescence system. This issue was particularly observed in following diagram.</p>
<h5>The common formulation of the Hill equation is as follows:</h5>
+
<p>The common formulation of the Hill equation is as follows:</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/3/30/Hbut-demon-8.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/3/30/Hbut-demon-8.png" alt="this is a photo">
</div>
+
</div>
<h5>Consequently, we modeled incomplete repression by using the ratio of occupied promoter concentration to total promoter concentration.</h5>
+
<p>Consequently, we modeled incomplete repression by using the ratio of occupied promoter concentration to total promoter concentration.</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/a/a9/Hbut-demon-9.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/a/a9/Hbut-demon-9.png" alt="this is a photo">
</div>
+
</div>
  
<h5>In view of the fact that is difficult to be measured and have same trend to , we used the concentration of nickel ions to approximate the equation.</h5>
+
<p>In view of the fact that is difficult to be measured and have same trend to , we used the concentration of nickel ions to approximate the equation.</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
  <img src="https://static.igem.org/mediawiki/2017/9/95/Hbut-demon-10.png" alt="this is a photo">
+
  <img src="https://static.igem.org/mediawiki/2017/9/95/Hbut-demon-10.png" alt="this is a photo">
    </div>
+
    </div>
<h5>So, the fluorescence intensity can be obtained without considering the cytotoxicity of Nickel Ions:</h5>
+
<p>So, the fluorescence intensity can be obtained without considering the cytotoxicity of Nickel Ions:</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/9/9d/Hbut-demon-11.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/9/9d/Hbut-demon-11.png" alt="this is a photo">
</div>
+
</div>
<h5>The para meters for the production function are:</h5>
+
<p>The para meters for the production function are:</p>
<h5><i>A</i>-basal expression level of promoter</h5>
+
<p><i>A</i>-basal expression level of promoter</p>
<h5><i>B</i>-maximal expression level of promoter</h5>
+
<p><i>B</i>-maximal expression level of promoter</p>
<h5><i>K<sub>d</sub></i>-half maximal effective concentration of N-Ni</h5>
+
<p><i>K<sub>d</sub></i>-half maximal effective concentration of N-Ni</p>
<h5><i>n</i>-Hill coefficient for induction. </h5>
+
<p><i>n</i>-Hill coefficient for induction. </p>
<h5>Added Variable, The differential equation of was updated by:</h5>
+
<p>Added Variable, The differential equation of was updated by:</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/3/3b/Hbut-demon-12.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/3/3b/Hbut-demon-12.png" alt="this is a photo">
</div>
+
</div>
<h5>The solution of this differential equation is as follows:</h5>
+
<p>The solution of this differential equation is as follows:</p>
<div align="center" style="margin:10px 0;">
+
<div align="center" style="margin:10px 0;">
<img src="https://static.igem.org/mediawiki/2017/2/22/Hbut-demon-13.png" alt="this is a photo">
+
<img src="https://static.igem.org/mediawiki/2017/2/22/Hbut-demon-13.png" alt="this is a photo">
</div>
+
</div>
 +
<small class="bigtitle">5.Application </small>
 +
<p>We designed a kit, including 10ml centrifuge tubes, filter columns (containing 0.22 M filter membrane), the national standard concentration of nickel ion solution 50ml(3μg/L), pure water 20ml, engineering bacteria 5ml.</p>
 +
<p>Instructions:
 +
<p>1. Add 3ml chloramphenicol containing LB medium and 30μL of original bacteria to the test tube. Incubate at 37°C 200rpm for overnight, and dilute the cultures to a target OD600 of 0.8;</p>
 +
<p>2.Take out 10ml centrifuge tubes for each sample besides one for blank one for stdrand. Add 2.7ml chloramphenicol containing LB medium and 30μL overnignt bacteria into each tube;</p>
 +
<p>3.Place the filter columns into the centrifuge tube. Add 300μL pure water into one column,300μL nickel ion standard solution into another column, and 300μL sample solution into other columns;</p>
 +
<p>4.Detect the fluorescence after incubate at 37°C 200rpm for 4hr.</p>
 
        </div>
 
        </div>
 
         <div class="col-lg-2"></div>
 
         <div class="col-lg-2"></div>

Revision as of 14:58, 27 October 2017

  • \

We analyzed the experimental data through modeling.

1.Assumptions:

We made four assumptions in order to simplify some of the aspects of the model. Many similar assumptions have been made in the literature.

The degradation of and other substances are linear.

Each cell has the same state.

The reaction in the process satisfies the first-order kinetic reaction.

2. Transcription Repression and Release Repression
this is a photo
this is a photo
this is a photo
this is a photo

Using the assumption about the first-order kinetic reaction,

Give:

this is a photo
3. Quasi-steady-state Approximation (QSSA)

Ordinary differential equations are too difficult to be solved, in order to get the solution of the equations, quasi-steady-state approximation is necessary.

We use the approximate conditions as little as possible and try to make the quasi steady state of matter consistent with the actual situation.

this is a photo

According to the formula’s deformation and derivation,

gives:

this is a photo
4. Incomplete Repression

Under incomplete repression, the bacteria express red fluorescent proteins without the presence of nickel ions.

The incomplete repression of promoters is a major issue in our fluorescence system. This issue was particularly observed in following diagram.

The common formulation of the Hill equation is as follows:

this is a photo

Consequently, we modeled incomplete repression by using the ratio of occupied promoter concentration to total promoter concentration.

this is a photo

In view of the fact that is difficult to be measured and have same trend to , we used the concentration of nickel ions to approximate the equation.

this is a photo

So, the fluorescence intensity can be obtained without considering the cytotoxicity of Nickel Ions:

this is a photo

The para meters for the production function are:

A-basal expression level of promoter

B-maximal expression level of promoter

Kd-half maximal effective concentration of N-Ni

n-Hill coefficient for induction.

Added Variable, The differential equation of was updated by:

this is a photo

The solution of this differential equation is as follows:

this is a photo
5.Application

We designed a kit, including 10ml centrifuge tubes, filter columns (containing 0.22 M filter membrane), the national standard concentration of nickel ion solution 50ml(3μg/L), pure water 20ml, engineering bacteria 5ml.

Instructions:

1. Add 3ml chloramphenicol containing LB medium and 30μL of original bacteria to the test tube. Incubate at 37°C 200rpm for overnight, and dilute the cultures to a target OD600 of 0.8;

2.Take out 10ml centrifuge tubes for each sample besides one for blank one for stdrand. Add 2.7ml chloramphenicol containing LB medium and 30μL overnignt bacteria into each tube;

3.Place the filter columns into the centrifuge tube. Add 300μL pure water into one column,300μL nickel ion standard solution into another column, and 300μL sample solution into other columns;

4.Detect the fluorescence after incubate at 37°C 200rpm for 4hr.