Difference between revisions of "Team:Mingdao/Composite Part"

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             <h1 id = "d-introduction">Composite Part</h1>
 
             <h1 id = "d-introduction">Composite Part</h1>
 
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             <h2>Introduction</h2>
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             <h2>Composite Part</h2>
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<p>In our project, we want to calculate the bacteria concentration in the testers.
 
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<p>
 
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<p>
 
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However, our devices can only detect GFP intensity, so we can only utilize GFP intensity to calculate bacteria concentration.
 
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<p>
 
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What’s more, with the view to making sure our system works successfully, we need to make sure that testers can detect GFP in our devices. Since the GFP in mosquitoes take some time to be synthesized, we can detect the green fluorescence only few hours after the mosquitoes take in the tester’s blood. To prevent from the misleading of our devices and system, we should calculate the very beginning time that the testers can detect the green fluorescence in the devices.
 
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<p>
 
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<h3>Guiding Questions</h3>
 
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<p>
 
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<p>
 
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<p>1. How many bacteria can be tested in our model ? (Model 1)
 
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2. How long do our devices take to send out signal ? (Model 2)</p>
 
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<h3>Focus on Our Model</h3>
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/1f/T--Mingdao--project_mos3.png" alt="" style="width: 50%; margin-bottom: 20px;">
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<p>
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            <h3>Ac5-hCD4-dToll-polyA / pSB1C3</h3>
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<p>
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            <h3>Part: BBa_K2543010</h3>
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<p style="text-indent:2em">
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Ac5 is a strong and constitutive promoter from Drosophila actin 5c gene and commonly used in insect expression system. <br />
  
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                <p>Since our devices can only detect the GFP intensity, we can only utilize GFP intensity to calculate E.coli concentration. After obtaining E.coli concentration, we will utilize it to calculate the very beginning time that testers can detect GFP. Finally, the two parameters will be demonstrated on our devices for the testers to take as reference.  
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Human CD4 (hCD4) is a cell marker expressed on the subtype of  T helper cell. CD4 acts as a coreceptor to help T cell development and cell function. CD4 plays an important role in T cell activation and immune signaling. The extracellular domain of hCD4 (1-396 aa) can form dimer and regulate the function of T cell activation. <br />
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<img class="center" src="https://static.igem.org/mediawiki/2018/f/fa/T--Mingdao--Modeling001.jpg" alt=" " style="width:70%" >
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Toll is a transmembrane protein involved in insect immune defense system to recognize pathogens like bacteria, viruses and fungi. Toll activated by pathogens transmits the signaling to express anti-microbial peptide (AMP) to kill the pathogens. Drosophila transmembrane domain (808-828 aa) and intracellular domain (829-1097 aa) of Toll (dToll) play an important roll in regulating the immune signaling.<br />
  
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            <img class="center" src="https://static.igem.org/mediawiki/2017/a/a8/T--CSMU_NCHU_Taiwan--safety-line.png" alt="" style="width:100%">
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Polyadenylation (polyA) is one of the process of eukaryotic mRNA translation. It adds a poly(A) tail to protect mRNA from enzymatic degradation and aid in transcription termination. Polyadenylation signal  of simian virus 40 (SV40 poly A) has been used widely in mammalian and many eukaryotic gene expression system.<br />
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This construct creates a synthetic human CD4-Drosophila Toll chimera receptor system. The system  functions not only in response to human HIV virus and also transmit Toll signaling to activate the expression of anti-microbial peptide (AMP).<br />
 
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            <h2 id = "d-model1">Model 1: Calculating E.coli Concentration by GFP Intensity</h2>
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<p style="text-indent:2em">  
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HIV is a huge epidemic around the world which can cause AIDS in infected people. To identify HIV is very difficult for people in limited-resource countries and individuals who wants privacy. An easy-to-use, cheap and portable testing device is urgently need around the world.  
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</p>
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<h3>Method</h3>
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<p>
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<p>
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<p>To find the mathematical relationship between GFP and E.coli concentration, we measure the GFP growing curve with different MOI value every two hours. Then, perform a series of calculation and finally arrive at the mathematical relationship between GFP and E.coli concentration.</p>
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<p>
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<br />
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<h3>Obtaining the Mathematical Relationship</h3>
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<p>
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<p>Table 1.0 shows the corresponding Fluorescence intensity with different MOI value. However, the units of E.coli we want should be transformed into another form.</p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/f/fb/T--Mingdao--Modeling019.jpg"alt=" " style="width:35%" >
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/12/T--Mingdao--Modeling017.jpg"alt=" " style="width:35%" >
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<br />
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<h3>Conversion of MOI to E.coli density</h3>
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<p>
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<p>The equation of E.coli density is shown below:
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/10/T--Mingdao--Modeling02.jpg"alt=" " style="width:70%" >
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<p>
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Since the MOI value refers to the ratio of E.coli cells to mosquito cells, we can use the density of mosquito cells to calculate the E.coli density. Plus, the mosquito cells are seeded at the density of 1.8×〖10〗^5 cells/well, and the volume of each well is 100μL.
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<p>
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Thus, the equation become
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/a/ac/T--Mingdao--Modeling03.jpg"alt=" " style="width:70%" >
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<p>
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<p>Then, we will turn the MOI in Table 1.0 into E.coli density to form Table 1.1. Next, we will use Table 1.1 to keep figuring out the mathematical relationship between E.coli concentration and GFP intensity.</p>
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<h3>Forming the mathematical expression</h3>
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<p style="text-indent:2em">
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<p>
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To further engineer the mosquito to recognize HIV, we designed and created a synthetic HIV-specific receptor composed of human CD4 extracellular domain (1-396 aa) and drosophila transmembrane and intracellular domains (808-828 aa and 829-1097 aa, respectively) based on UniProt protein database.  
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<p>
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<p>Before we keep working on our calculation, it’s worth noticed that the GFP intensity has already existed while there is no E.coli . Thus, the intensity of GFP with no E.coli should be eliminated as discussing the relationship between E.coli concentration and GFP intensity, which means [GFP] should minus the [〖GFP〗_0] with no E.coli, and add it back in our final result.
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<p>
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With that in mind, we form the Table 1.2
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/3/36/T--Mingdao--Modeling019r8.jpg"alt=" " style="width:35%">
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<p>
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Now we can begin with our data analyzing.
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</p>
 
</p>
 
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<h3>Data Analyzing</h3>
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<p style="text-indent:2em">
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<p>
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The DNA sequences of human CD4 and Drosophila Toll domains were synthesized by Integrated DNA Technologies, Inc. (IDT). The DNAs were cloned onto pSB1C3 and confirmed by sequencing. The fusion protein of CD4-Toll was further assembled with polyA and driven by Ac5 promoter.
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<p>
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<P>Figure 2.0 shows the graphic expression between the [E.coli] and GFP intensity, the Exponential Function is shown below:<p>
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</p>
 
</p>
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<p>
 
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<p>
 
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<img class="center" src="https://static.igem.org/mediawiki/2018/7/71/T--Mingdao--Modeling07.jpg"alt=" " style="width:70%" >
 
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<p>
 
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<p>
 
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<p>Next, we will bring in that [〖GFP〗_0 ]=813 to the Exponential Function and obtain the final graphic expression and function.</p>
 
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<p>
 
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<img class="center" src="https://static.igem.org/mediawiki/2018/0/0a/T--Mingdao--Modeling08.jpg"  alt=" " style="width:70%">
 
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<p>
 
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<p> Combining the constants, we arrive at</p>
 
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<p>
 
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<img class="center" src="https://static.igem.org/mediawiki/2018/7/7e/T--Mingdao--Modeling10.jpg" alt=" " style="width:70%">
 
 
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<h2><strong>Conclusion</strong></h2>
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/1f/T--Mingdao--project_mos3.png" alt="" style="width: 50%; margin-bottom: 20px;">
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<h3>Application</h3>
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<p>
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<p>
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<p>With the formula, we can now build a calculator to calculate the [E.coli] by GFP Intensity, and apply the formula to our devices to from a well-designed prototype. The devices will calculate the [E.coli] automatically based on the GFP intensity they detect. As a result, the testers will be able to know the [E.coli] in their blood through our devices.</p>
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<h3>Limitation</h3>
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<p>
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<p>
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<p>However, there are some limitations to our Model 1. Not knowing when all the E.coli cells bind to the GAM 1 promoter, we can’t make sure when we can utilize the formula to conduct calculation.
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<p>
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Consequently, we will conduct Model 2 to figure out the limitation.</p>
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<p>
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      <img class="center" src="https://static.igem.org/mediawiki/2017/a/a8/T--CSMU_NCHU_Taiwan--safety-line.png" style="width:100%">
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<p style="text-indent:2em">
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To test the expression vector driven by Ac5 promoter, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA. GFP positive cells and intensity were analyzed 2 days after transfection.
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</p>
 
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<h2 id="d-model2">Model 2: Number of E.coli Cells Binding to The GAM 1 Promoter Increase With Time</h2>
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            <h3>EXPERIMENT</h3>
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    <p style="text-indent:2em">
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C6/36 cells (1.8 x 105 cells/well in a 96-well plate)<br />
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Liposome-mediated transfection and culture for 2 more days<br />
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Read fluorescence intensity at Ex/Em = 480/520 nm with a microplate reader<br />
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Observe GFP+ cells under a fluorescence microscope<br />
 
<br />
 
<br />
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<h3>Method</h3>
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/1f/T--Mingdao--project_mos3.png" alt="" style="width: 50%; margin-bottom: 20px;">
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<p>
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              <h3>RESULT</h3>
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<p>
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<p style="text-indent:2em">
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<p>To know when all the E.coli cells bind to GAM 1 promoter, we measure the GFP growing curves with different MOI value every two hours, and differentiate the growing curves to find the time that the instant GFP transcription rate reaches the maximum, which is the time all the E.coli cells bind to GAM 1 promoter.
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As data shown here, Ac5 is a strong and constitutive promoter which can drive GFP to high expression level in mosquito cells. And we can transfect more than 50% of GFP positive cell with liposome-mediated DNA delivery.  
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<p>
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After the differentiation, we will be able to obtain the graphic expression between the binding time and the [E.coli]. By analyzing the graphic expression, the very beginning time that the testers can detect GFP can be calculated.
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<p>
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In addition, the limitation mentioned in Model 1 can also be quantified via the graphic expression we obtained in Model 2.
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<p>
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</p>
 
</p>
 
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<h3>Standardization of GFP Growing Curve</h3>
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<p style="text-indent:2em">
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<p>
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To test feasibility of fusion CD4-Toll chimera, we acquired the plasmid of Drosomycin promoter-luciferase from world-renowned insect geneticist, Dr. Jean-Luc Imler and conducted the luc reporter assay with Ac5-CD4-Toll-polyA in the mosquito cells.
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<p>
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</p>
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<p>Since we need to differentiate the GFP growing curve, the standardization is essential. Thus, we will digitize the GFP growing curve so that we can conduct the differentiation successfully. </p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/1f/T--Mingdao--project_mos3.png" alt="" style="width: 50%; margin-bottom: 20px;">
 
<br />
 
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<h3>Raw Data</h3>
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            <h3>EXPERIMENT</h3>
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<p>
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    <p style="text-indent:2em">
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<p>
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C6/36 cells (1.8 x 105 cells/well in a 96-well plate)<br />
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<p>
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Liposome-mediated transfection and culture for 2 more days<br />
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We perform two experiments respectively with different purposes.
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Add gp120 of HIV (1 μg/ml*) or not and incubate for 24 hours<br />
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<p>  
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Cell lysis and luciferase assay<br />
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The first one is to made for the blank so there is no DNA inside the well and no inducement of GAM 1 promoter, since the cells itself contribute to some absorbance, too.
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*The concentration of gp120 in the serum of HIV-infected people is between 0.12~1 μg/ml.
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<p>  
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The second one consists of E.coli with induced GAM 1 promoter. Both of the experiments are measured by our plate reader every two hours.
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<p>  
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The Table 2.0 and Table 2.1 are shown below</p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/b/b2/T--Mingdao--Modeling11.jpg" alt=" " style="width:100%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/3/37/T--Mingdao--Modeling12.jpg" alt=" " style="width:100%">
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<h3>Absorbance of green fluorescence protein</h3>
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<img class="center" src="https://static.igem.org/mediawiki/2018/1/1f/T--Mingdao--project_mos3.png" alt="" style="width: 50%; margin-bottom: 20px;">
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<p>
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              <h3>RESULT</h3>
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<p>
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<p style="text-indent:2em">
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<p>The actual absorbance of GFP is the absorbance of the E.coli cells with induced GAM 1 promoter minus the absorbance of E.coli cells without DNA and no inducement of GAM 1 promoter. After the calculation, the result are shown as Table 2.2</p>
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The result indicated that luciferase activity driven by Drosomycin promoter can be triggered by CD4-Toll chimera. The activity was decreased in the presence of gp120 of HIV. The finding demonstrates the possibility that GE mosquito created by our project could be applied to detect HIV virus in infected human blood.
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/2/22/T--Mingdao--Modeling13.jpg" alt=" " style="width:100%">
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<br />
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<h3>Standardization</h3>
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<p>
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<p>
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<p>Then, we will illustrate the growing curves and find the formula of each growing curve so that we can differentiate them. It’s noticed that we don’t adopt the GFP growing curve without E.coli, since its GAM 1 promoter isn’t induced.
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<p>
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After analyzing, we find that cubic equation perfectly fits to our experiment data. Those cubic equations are shown as Figure 3.0 to Figure 3.4</p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/c/cb/T--Mingdao--Modeling002.jpg" alt=" " style="width:70%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/4/47/T--Mingdao--Modeling003.jpg" alt=" " style="width:70%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/4/41/T--Mingdao--Modeling004.jpg" alt=" " style="width:70%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/9/9c/T--Mingdao--Modeling005.jpg" alt=" " style="width:70%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/6/6b/T--Mingdao--Modeling006.jpg" alt=" " style="width:70%">
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<p>
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<p>
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<p>Also, the mathematical expressions of these cubic equations are shown as Table 3.0 and the graphic expressions are shown as Figure 4.0</p>
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<p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/a/a8/T--Mingdao--Modeling007.jpg" alt=" " style="width:70%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/4/46/T--Mingdao--Modeling008.jpg" alt=" " style="width:70%">
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<h3><strong>Number of E.coli Cells Binding to GAM 1 Promoter</strong></h3>
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<br />
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<h3>Derivative of the green fluorescence growing curve<h3>
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<p>
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<p>
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<p>When all the E.coli cells bind to GAM 1 promoter, the instant GFP transcription rate will reach the maximum value. As a consequence, what we need to do is to differentiate the GFP growing curves at different MOI value, and find the maximum of the differentiated formulas as well as the corresponding time.
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<p>
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With that in mind, we conduct the derivative of the mathematical expressions in Table 3.0 and form Table 3.1
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</p>
 
</p>
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<p>
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<p>
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          <video playinline controls="true">
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<img class="center" src="https://static.igem.org/mediawiki/2018/9/99/T--Mingdao--Modeling009.jpg" alt=" " style="width:70%">
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<source src="https://static.igem.org/mediawiki/2017/7/71/T--CSMU_NCHU_Taiwan--ProjectDescription.mp4" type="video/mp4" >
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<br />
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</video>
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<h3>Obtaining The Maximum and The Corresponding Time</h3>
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              <h3>Reference</h3>
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<p>
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<p>
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<p>To calculate the maximum of the derivative of the green fluorescence growing curve, we need to conduct the second derivative and find the maximum and corresponding time. The result is shown as Table 3.2</p>
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<p>
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<p>
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<img class="center" src="https://2018.igem.org/File:T--Mingdao--Modeling010.jpg"alt=" " style="width:70%">
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<p>
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<p>
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<p>Then, we will turn the MOI value into E.coli density to form Table 3.3
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Also, the graphic expression of the relationship between time and E.coli density is shown as Figure 5.0</p>
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<p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/7/74/T--Mingdao--Modeling011.jpg" alt=" " style="width:70%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/d/d7/T--Mingdao--Modeling013.jpg" alt=" " style="width:70%">
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<p>
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<p>
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<p>We also arrive at the equation between time and E.coli concentration</p>
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<p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/d/d2/T--Mingdao--Modeling014.jpg" alt=" " style="width:70%">
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<br />
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<h3><strong>Conclusion</strong></h3>
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<br />
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<h3>Application</h3>
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<p>
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<p>
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<p>With the formula, we can utilize the [E.coli] calculated in Model 1 to calculate how long the testers should wait to detect green fluorescence in our devices and demonstrate it on our devices to inform the testers. Then, the formula will also be applied to our calculator as well.</p>
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<p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2017/a/a8/T--CSMU_NCHU_Taiwan--safety-line.png" alt="" style="width:100%">
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                                        <div style = "border-style:solid; text-align:center; padding:20px" class="col-sm-12">
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                                    <div class="row">
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                                    <h2 style = "padding:0">CALCULATOR</h2>
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                                        <div class="col-sm-6">
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                                        <h3 style="padding:0"> E.coli Concentration Calculator</h3>
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                                        <script>
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                                            function round(value, decimals) {
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                                                return Number(Math.round(value+'e'+decimals)+'e-'+decimals);
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                                            }
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                                            calculateee = function(){
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                                                var XInputLOCSP = document.getElementById("inputLOCS").value;
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                                                var XCrystDamP = 54.163*(2.7182^(0.0024*(XInputLOCSP)));
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                                                XInputLOCSPid.innerHTML = round(XInputLOCSP,1);
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                                                XCrysDamPid.innerHTML = round(XCrystDamP,4);
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                                                XGSRPid.innerHTML = round(XGSRP,2);
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                                                XNPConcPid.innerHTML = round(XNPConcP,2);
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                                                XEyedropPid.innerHTML = round(XEyedropP,2);
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                                                XResultPid.innerHTML = round(XResultP,2);
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                                            }
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                                        </script>
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                                        <span style="width:70%">Type in the value:</span><input id = "inputLOCS" type="text" style="width:30%">
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                                        <br><span style="font-size:13px">We guarentee that by applying this prevention eyedrop daily, your LOCS score will remain below your threshold for 50 years.</span>
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                                        <br><button onclick = "calculateee()">Calculate!</button><br>
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                                        <br>
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                                        <span>Prevention Results </span>
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                                        <table class="table table-hover fixed" style="font:16px"> 
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                                            <col width="150px" />
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                                            <col width="150px" />
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                                            <col width="100px" />
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                                            <thead>
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                                                <tr>
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                                                    <th>Variable</th>
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                                                    <th>Value</th>
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                                                    <th>Source</th>
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                                                </tr>
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                                            </thead>
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                                            <tbody align="right">
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                                                <tr>
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                                                    <td>The Value You Imput</td>
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                                                    <td> <span id="XInputLOCSPid">&nbsp;&nbsp;&nbsp;</span></td>
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                                                    <td></td>
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                                                </tr>
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                                                <tr>
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                                                    <td>The Value You Get</td>
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                                                    <td> <span id="XCrysDamPid">&nbsp;&nbsp;&nbsp; </span>&nbsp;Density</td>
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                                                    <td>Model 1</td>
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                                                </tr>
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                                            </tbody>
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                                        </table>
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                                    </div>
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                                        <div class="col-sm-6">
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                                        <h3 style="padding:0">Responding Time Calculator</h3>
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                                        <script>
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                                            function round(value, decimals) {
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                                                return Number(Math.round(value+'e'+decimals)+'e-'+decimals);
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                                            }
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                                            calculateeeT = function(){
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                                                var XInputLOCST = document.getElementById("inputLOCSTget").value;
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                                                var XCrysDamT = 0.0002*(XInputLOCST)+6.0864;
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                                                var XAbsorbanceT = XCrysDamT/9.276;
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                                                var XCH25HT = XAbsorbanceT/0.228;
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                                                var XEyedropT = XCH25HT/14.04/0.001;
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                                                var XResultT = XEyedropT*50/1000;
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                                                var XNumofEyedropT = Math.ceil(XResultT/0.75);
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                                                XInputLOCSTid.innerHTML = round(XInputLOCST,1);
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                                                XCrysDamTid.innerHTML = round(XCrysDamT,4);
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                                                XAbsorbanceTid.innerHTML = round(XAbsorbanceT,3)
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                                                XCH25HTid.innerHTML = round(XCH25HT,2);
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                                                XEyedropTid.innerHTML = round(XEyedropT,2);
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                                                XResultTid.innerHTML = round(XResultT,2);
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                                                XNumofEyeDropTid.innerHTML = XNumofEyedropT;
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                                            }
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                                        </script>
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                                        <span style="width:70%">Enter value:</span><input id = "inputLOCSTget" type="text" style="width:30%">
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                                        <br><span style="font-size:13px">By applying the following treatment, leaving an hour before each dose of eyedrops, we guarentee that it will lower your LOCS score to essentially 0.</span>
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                                        <br><button onclick = "calculateeeT()">Calculate!</button><br>
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                                        <br>
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                                        <span>Treatment Results </span>
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                                        <table class="table table-hover fixed" style="font:16px"> 
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                                            <col width="150px" />
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                                            <col width="150px" />
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                                            <col width="100px" />
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                                            <thead>
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                                                <tr>
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                                                    <th>Variable</th>
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                                                    <th>Value</th>
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                                                    <th>Source</th>
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                                                </tr>
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                                            </thead>
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                                            <tbody align="right">
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                                                <tr>
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                                                    <td>The value you imput</td>
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                                                    <td> <span id="XInputLOCSTid">&nbsp;&nbsp;&nbsp;</span></td>
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                                                    <td></td>
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                                                </tr>
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                                                <tr>
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                                                    <td>The value you get</td>
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                                                    <td> <span id="XCrysDamTid">&nbsp;&nbsp;&nbsp; </span>&nbsp; Hr </td>
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                                                    <td>Model 2</td>
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                                                </tr>
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                                                <tr>
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                                            </tbody>
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                                        </table>
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                                    </div>
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                                  </div>
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                            </div>
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<img class="center" src="https://static.igem.org/mediawiki/2017/a/a8/T--CSMU_NCHU_Taiwan--safety-line.png" alt="" style="width:100%">
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<br />
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<h2 id="d-conclusion">Conclusion</h2>
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<br />
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<p>Our model not only help to build the formulaic system which applied to our devices, but also make us better understand our project. Because our devices can only detect the GFP intensity, our model is required to build a well-designed devices and system.
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In our model 1, we obtain the formula which allows us to calculate [E.coli] from GFP intensity. While in model 2, we obtain the formula which allows us to calculate how long the testers should wait to get the result of the test based on the [E.coli] calculated in mode 1. For [E.coli] and the time interval, they will be demonstrated on our devices to show them to the testers.
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To sum up, Our model act as a bridge between our devices and the testers, and quantifies the significant parameters in our project, which allow the masses to simply get the result of the test without complex calculations.</p>
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           </div>
 
           </div>
 
         </div>
 
         </div>

Revision as of 04:13, 11 October 2018

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Description

Composite Part


Composite Part


Ac5-hCD4-dToll-polyA / pSB1C3

Part: BBa_K2543010

Ac5 is a strong and constitutive promoter from Drosophila actin 5c gene and commonly used in insect expression system.
Human CD4 (hCD4) is a cell marker expressed on the subtype of T helper cell. CD4 acts as a coreceptor to help T cell development and cell function. CD4 plays an important role in T cell activation and immune signaling. The extracellular domain of hCD4 (1-396 aa) can form dimer and regulate the function of T cell activation.
Toll is a transmembrane protein involved in insect immune defense system to recognize pathogens like bacteria, viruses and fungi. Toll activated by pathogens transmits the signaling to express anti-microbial peptide (AMP) to kill the pathogens. Drosophila transmembrane domain (808-828 aa) and intracellular domain (829-1097 aa) of Toll (dToll) play an important roll in regulating the immune signaling.
Polyadenylation (polyA) is one of the process of eukaryotic mRNA translation. It adds a poly(A) tail to protect mRNA from enzymatic degradation and aid in transcription termination. Polyadenylation signal of simian virus 40 (SV40 poly A) has been used widely in mammalian and many eukaryotic gene expression system.
This construct creates a synthetic human CD4-Drosophila Toll chimera receptor system. The system functions not only in response to human HIV virus and also transmit Toll signaling to activate the expression of anti-microbial peptide (AMP).

HIV is a huge epidemic around the world which can cause AIDS in infected people. To identify HIV is very difficult for people in limited-resource countries and individuals who wants privacy. An easy-to-use, cheap and portable testing device is urgently need around the world.


To further engineer the mosquito to recognize HIV, we designed and created a synthetic HIV-specific receptor composed of human CD4 extracellular domain (1-396 aa) and drosophila transmembrane and intracellular domains (808-828 aa and 829-1097 aa, respectively) based on UniProt protein database.


The DNA sequences of human CD4 and Drosophila Toll domains were synthesized by Integrated DNA Technologies, Inc. (IDT). The DNAs were cloned onto pSB1C3 and confirmed by sequencing. The fusion protein of CD4-Toll was further assembled with polyA and driven by Ac5 promoter.


To test the expression vector driven by Ac5 promoter, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA. GFP positive cells and intensity were analyzed 2 days after transfection.


EXPERIMENT

C6/36 cells (1.8 x 105 cells/well in a 96-well plate)
Liposome-mediated transfection and culture for 2 more days
Read fluorescence intensity at Ex/Em = 480/520 nm with a microplate reader
Observe GFP+ cells under a fluorescence microscope

RESULT

As data shown here, Ac5 is a strong and constitutive promoter which can drive GFP to high expression level in mosquito cells. And we can transfect more than 50% of GFP positive cell with liposome-mediated DNA delivery.


To test feasibility of fusion CD4-Toll chimera, we acquired the plasmid of Drosomycin promoter-luciferase from world-renowned insect geneticist, Dr. Jean-Luc Imler and conducted the luc reporter assay with Ac5-CD4-Toll-polyA in the mosquito cells.


EXPERIMENT

C6/36 cells (1.8 x 105 cells/well in a 96-well plate)
Liposome-mediated transfection and culture for 2 more days
Add gp120 of HIV (1 μg/ml*) or not and incubate for 24 hours
Cell lysis and luciferase assay
*The concentration of gp120 in the serum of HIV-infected people is between 0.12~1 μg/ml.

RESULT

The result indicated that luciferase activity driven by Drosomycin promoter can be triggered by CD4-Toll chimera. The activity was decreased in the presence of gp120 of HIV. The finding demonstrates the possibility that GE mosquito created by our project could be applied to detect HIV virus in infected human blood.


Reference

Introduction

Model 1

Model 2

Conclusion