Team:Tianjin/Project/Experiment/Endo-Induce

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<center><span style="font-family:Arial;font-size:46px;color:#000;"> Experiment</span></center>
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<center><span style="font-family:Arial;font-size:46px;color:#000;"><i> In vivo </i>alkane sensing test</span></center>
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= Endogenous Alkanes Induce=
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= Aim of this experiment=
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<p> To verify AlkSensor’s ability to distinguish different in vivo concentrations of alkane and different abilities of alkane biosynthesis. </p>
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=Experimental scheme=
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<div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/c/c3/Tju-endo1.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/c/c3/Tju-endo1.jpg" width="400px" /></a></div>
 
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<p> After systematic analysis of the microbial synthesis pathway from glucose to alkane in E.coli, we decide to choose the route from Fatty Acyl-ACP to alkane as the alkane producing module. Meanwhile, we conduct codon-optimization to the genes encoding NPDC and AAR based on the work done by Andreas Schirmer, making them more applicable to E.coli.</p>
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<p> We constructed three strains. All the three strains have the same alkane sensing module--AlkSensor with RFP as output. However, they have different abilities of alkane biosynthesis. Strain 1 has the strongest alkane producing ability. The alkane productivity of Strain 2 is less than Strain 1. Strain 3 does not have alkane biosynthesis module.</p>
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<p> After systematic analysis of the microbial synthesis pathway from glucose to alkane in <i>E.coli</i>, we decided to choose the pathway from fatty acyl-ACP to alkane as the alkane producing module. The alkane biosynthesis module transferred into <i>E. coli</i> is composed of two enzymes-aldehyde decarbonylase (NPDC) and acyl-ACP reductase (AAR). We conducted codon-optimization to the genes encoding NPDC and AAR based on the work done by Andreas Schirmer, making them more applicable to E.coli. The alkane biosynthesis module is constructed under different constitutive promoter so to generate different ability of alkane biosynthesis.</p>
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<p> Here, we want to test the response of Alk-Sensor to endogenous alkanes.
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<div style="text-align:center;vertical-align:middle;"><a href="#" target="_blank" ><img src="#" width="500px" /></a></div>
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Construct:</p>
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<p> We constructed 3 strains as shown in the figure below.</p>
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<b>Figure 1.</b>&nbsp; A pathway of alkane biosynthesis </div>
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<div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/d/d4/Tju-endo2.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/d/d4/Tju-endo2.jpg" width="700px" /></a></div>
 
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<p>strain1:No.53：producing module (constitutive promoter J23100)+ Alk-Sensor device</p>
 
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<p>strain2:No.54：producing module (constitutive promoter J23114) + Alk-Sensor device</p>
 
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<p>strain3:No.55：Null+ Alk-Sensor device</p>
 
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<p> The promoter strength of No.54 is stronger than that of No.53. Theorectically, alkane productivity of No.53 and No.54 will be different, and intracellular alkane concentration of the above 3 strains will be different-- No.54 the highest, No.53 lower and No.55 the lowest.</p>
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<p> Strain 1:producing module (constitutive promoter J23114) + AlkSensor </p>
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<p> Strain 2: producing module (constitutive promoter J23100)+ AlkSensor</p>
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<p> Strain 3:Null+ Alk-Sensor <p/>
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<p> The promoter strength of strain 1 is stronger than that of strain 2. Theocratically, alkane productivities and intracellular alkane concentrations of the above 3 strains will be different--strain 1 the highest, strain 2 lower and strain 3 the lowest.</p>
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<p> The 3 strains are cultured in 3ml M9 medium for about 24 hours.</p>
 
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<div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/0/08/Tju-Endo3.png" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/0/08/Tju-Endo3.png" width="706px" /></a></div>
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<div style="text-align:center;vertical-align:middle;"><a href="#" target="_blank" ><img src="#" width="500px" /></a></div>
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<div><center><b>Figure 1.</b> Cell Induced-Fluorescent Indensity </center></div>
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<b>Figure 2.</b>&nbsp; Construction scheme of three test strains </div>
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<p> <b>Result：</b>As shown in the diagram above, the fluorescent indensity of No.54 is the strongest, No.53 weaker and No.55 the weakest.</p>
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<p> The 3 strains are cultured in 3ml M9 medium for about 24 hours. And the result is in accordance with our expectation.</p>
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<b>Figure 3.</b>&nbsp; Cell Induced-Fluorescent Indensity </div>
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= Conclusion:=
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<p ><b>Conclusion:</b></p>
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<p>(1) AlkSensor can respond to the mixed intracellular alkanes, which can be used to detect alkanes inside the cell.</p>
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<p>(1) Alk-Sensor device can respond to the mixed intracellular alkanes, which can be used to detect alkanes inside the cell.</p>
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<p>(2) There is a positive correlation between the concentration of intracellular alkanes, and the expression level of the reporter.</p>
<p>(2) There is a positive correlation between the concentration of intracellular alkanes, and the expression level of the reporter.</p>

Revision as of 09:41, 17 October 2013

In vivo alkane sensing test

Aim of this experiment


To verify AlkSensor’s ability to distinguish different in vivo concentrations of alkane and different abilities of alkane biosynthesis.


Experimental scheme


We constructed three strains. All the three strains have the same alkane sensing module--AlkSensor with RFP as output. However, they have different abilities of alkane biosynthesis. Strain 1 has the strongest alkane producing ability. The alkane productivity of Strain 2 is less than Strain 1. Strain 3 does not have alkane biosynthesis module.

After systematic analysis of the microbial synthesis pathway from glucose to alkane in E.coli, we decided to choose the pathway from fatty acyl-ACP to alkane as the alkane producing module. The alkane biosynthesis module transferred into E. coli is composed of two enzymes-aldehyde decarbonylase (NPDC) and acyl-ACP reductase (AAR). We conducted codon-optimization to the genes encoding NPDC and AAR based on the work done by Andreas Schirmer, making them more applicable to E.coli. The alkane biosynthesis module is constructed under different constitutive promoter so to generate different ability of alkane biosynthesis.


Figure 1.  A pathway of alkane biosynthesis

Strain 1:producing module (constitutive promoter J23114) + AlkSensor

Strain 2: producing module (constitutive promoter J23100)+ AlkSensor

Strain 3:Null+ Alk-Sensor

The promoter strength of strain 1 is stronger than that of strain 2. Theocratically, alkane productivities and intracellular alkane concentrations of the above 3 strains will be different--strain 1 the highest, strain 2 lower and strain 3 the lowest.


Figure 2.  Construction scheme of three test strains

The 3 strains are cultured in 3ml M9 medium for about 24 hours. And the result is in accordance with our expectation.


Figure 3.  Cell Induced-Fluorescent Indensity

Conclusion:


(1) AlkSensor can respond to the mixed intracellular alkanes, which can be used to detect alkanes inside the cell.

(2) There is a positive correlation between the concentration of intracellular alkanes, and the expression level of the reporter.

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