Difference between revisions of "Team:RDFZ-China/Measurement"

 
(34 intermediate revisions by 3 users not shown)
Line 1: Line 1:
 
{{RDFZ-China}}
 
{{RDFZ-China}}
 +
 
<html>
 
<html>
 +
<body style="width:1263px; margin-left:auto;margin-right:auto;background-color:white" onscroll="scrollEvent()">
 +
    <div id="body">
 +
        <!--default for floating navigation-->
 +
        <div id="sidebar" class="paper-sidenav">
 +
            <ul>
 +
                <li>
 +
                    <ul>
 +
                        <li><a href="#section1">Expression Burden</a></li>
 +
                        <li><a href="#section2">Capacity Monitor</a></li>
 +
                        <li><a href="#section3">VioABDE</a></li>
 +
                        <li><a href="#section4">Alternative Device</a></li>
 +
                        <li><a href="#section5">References</a></li>
 +
                    </ul>
 +
                </li>
 +
            </ul>
 +
        </div>
 +
        <div id="comic">
 +
            <p>
 +
                <!-- <img src="https://static.igem.org/mediawiki/2018/thumb/1/18/T--RDFZ-China--PJDhead1263620.jpg/800px-T--RDFZ-China--PJDhead1263620.jpg" alt="pdcomic" /> -->
 +
            </p>
 +
        </div>
 +
        <div class="description">
 +
            <h1>Measurement</h1>
 +
            <div class="topic-title" id="section1">
 +
                <h3>Expression Burden</h3>
 +
                <p>In synthetic biology, a common issue in integrating exogenous sequences into host cells is that heterologous gene expression can cause significant metabolic burden. Typically, this gene expression burden is implied by a declining growth rate of bacteria, which is unfavorable for fermentation industry because it lowers yields and profits. According to Ceroni et al. (2015), a fluorescence-based method allows to measure gene expression capacity of cells, by inserting a “capacity monitor”.</p>
 +
            </div>
 +
            <div class="topic-title" id="section2">
 +
                <h3>Capacity Monitor</h3>
 +
                <p>The “Capacity Monitor” consists of a superfold GFP with a strong constitutive promoter, which is constantly expressed in the bacterial genome. Thus, any alternation in fluorescence intensity can reflect in overall resource availability in the cell. Particularly, with expression of synthesized genes, the expression of GFP will decrease, thus reporting a reallocation of cellular resources. </p>
 +
            </div>
 +
            <div class="topic-title" id="section3">
 +
                <h3>VioABDE</h3>
 +
                <p>After applying capacity monitor, we compared the fluorescence of E. coli with an empty vector and E. coli with VioABDE inserted and expressed. Both strains were incubated for 24 hours with fluorescence and OD constantly monitored. However, the OD of bacteria with VioABDE increased faster than the bacteria without Vio expression, which contradicts our initial expectation.</p>
 +
                <p>We co transformed the TlpA39-VioABDE into the capcatiy monitor component cell which was made by beyotime component cell kit. </p>
 +
                <p>Then the Capacity Monitor and Capacity Monitor-Vio were incubicated in microshaker in 39 degree Celsius, then they were added to the 96 well plates.
 +
                <img src="https://static.igem.org/mediawiki/2018/3/32/T--RDFZ-China--CPVIO.png",width=100%>
 +
                <img src="https://static.igem.org/mediawiki/2018/2/20/T--RDFZ-China--CM%2BVio.png",width=100% />"
 +
            </div>
 +
            <div class="topic-title" id="section4">
 +
                <h3>Alternative Device</h3>
 +
                <p>Since we target to modify bacteria used in fermentation industry, which primarily are used for material synthesis and production, our sequence inserts should occupy as limited resources in bacteria as possible to avoid significant expression burden. In order to reduce this expression stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking-S, 2011) and the “Cold Box” in 5’UTR region of CspA (Ionis Paris, 2017). With only one transcriptional regulator, less energy will be consumed, which is beneficial for fermentation industry.</p>
 +
            </div>
 +
        </div>
 +
        <div class="reference">
 +
            <div class="topic-title" id="section5">
 +
                <h3>References</h3>
 +
                <p>Ceroni, F., Algar, R., Stan, G. B., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature methods, 12(5), 415.</p>
 +
                <p>Ionis Paris. (2017). Biological thermo-responsive solution, that reduces the plant’s thermal stresses. Available from: https://2017.igem.org/Team:IONIS-PARIS.</p>
 +
                <p>Peking-S iGEM. (2011). A Chemical Wire Toolbox for Synthetic Microbial Consortia. Avaible from: https://2011.igem.org/Team:Peking_S.</p>
 +
            </div>
  
−
 
+
        </div>
−
 
+
</body>
−
 
+
−
 
+
−
<div class="clear"></div>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
<div class="column full_size">
+
−
<h1>Measurement</h1>
+
−
 
+
−
<p>There are a lot of exciting parts in the Registry, but many parts have still not been characterized. Synthetic Biology needs great measurement approaches for characterizing new parts, and efficient new methods for characterizing many parts at once. If you've done something exciting in the area of Measurement, describe it here!</p>
+
−
</div>
+
−
<div class="clear"></div>
+
−
 
+
−
<div class="column two_thirds_size">
+
−
<h3>Best Innovation in Measurement Special Prize</h3>
+
−
<p>If you've done excellent work in measurement, you should consider nominating your team for this special prize. Designing great measurement approaches for characterizing new parts or developing and implementing an efficient new method for characterizing thousands of parts are good examples.
+
−
<br><br>
+
−
To compete for the <a href="https://2018.igem.org/Judging/Awards">Best Innovation in Measurement 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>.
+
−
<br><br>
+
−
You must also delete the message box on the top of this page to be eligible for this prize.
+
−
 
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div class="column third_size">
+
−
<div class="highlight decoration_A_full">
+
−
<h3>Inspiration</h3>
+
−
<p>You can look at what other teams did to get some inspiration! <br />
+
−
Here are a few examples:</p>
+
−
<ul>
+
−
<li><a href="https://2016.igem.org/Team:Stanford-Brown">2016 Stanford-Brown</a></li>
+
−
<li><a href="https://2016.igem.org/Team:Genspace">2016 Genspace</a></li>
+
−
<li><a href="https://2015.igem.org/Team:William_and_Mary">2015 William and Mary</a></li>
+
−
<li><a href="https://2014.igem.org/Team:Aachen">2014 Aachen  </a></li>
+
−
</ul>
+
−
</div>
+
−
</div>
+
−
 
+
  
 
</html>
 
</html>
 +
{{RDFZ-China/footer}}

Latest revision as of 02:11, 18 October 2018

Measurement

Expression Burden

In synthetic biology, a common issue in integrating exogenous sequences into host cells is that heterologous gene expression can cause significant metabolic burden. Typically, this gene expression burden is implied by a declining growth rate of bacteria, which is unfavorable for fermentation industry because it lowers yields and profits. According to Ceroni et al. (2015), a fluorescence-based method allows to measure gene expression capacity of cells, by inserting a “capacity monitor”.

Capacity Monitor

The “Capacity Monitor” consists of a superfold GFP with a strong constitutive promoter, which is constantly expressed in the bacterial genome. Thus, any alternation in fluorescence intensity can reflect in overall resource availability in the cell. Particularly, with expression of synthesized genes, the expression of GFP will decrease, thus reporting a reallocation of cellular resources.

VioABDE

After applying capacity monitor, we compared the fluorescence of E. coli with an empty vector and E. coli with VioABDE inserted and expressed. Both strains were incubated for 24 hours with fluorescence and OD constantly monitored. However, the OD of bacteria with VioABDE increased faster than the bacteria without Vio expression, which contradicts our initial expectation.

We co transformed the TlpA39-VioABDE into the capcatiy monitor component cell which was made by beyotime component cell kit.

Then the Capacity Monitor and Capacity Monitor-Vio were incubicated in microshaker in 39 degree Celsius, then they were added to the 96 well plates. "

Alternative Device

Since we target to modify bacteria used in fermentation industry, which primarily are used for material synthesis and production, our sequence inserts should occupy as limited resources in bacteria as possible to avoid significant expression burden. In order to reduce this expression stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking-S, 2011) and the “Cold Box” in 5’UTR region of CspA (Ionis Paris, 2017). With only one transcriptional regulator, less energy will be consumed, which is beneficial for fermentation industry.

References

Ceroni, F., Algar, R., Stan, G. B., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature methods, 12(5), 415.

Ionis Paris. (2017). Biological thermo-responsive solution, that reduces the plant’s thermal stresses. Available from: https://2017.igem.org/Team:IONIS-PARIS.

Peking-S iGEM. (2011). A Chemical Wire Toolbox for Synthetic Microbial Consortia. Avaible from: https://2011.igem.org/Team:Peking_S.

footer