Difference between revisions of "Team:Wageningen UR/Results/Fluorescent"

(Created page with "{{Wageningen_UR/Menuv2}} {{Wageningen_UR/StyleCSSv2}} {{Wageningen_UR/MobileMenu}} <html> <div class="container-fluid OurContent"> <div class="row"> <div class="c...")
 
Line 25: Line 25:
 
<section class="TrypIntro">
 
<section class="TrypIntro">
 
                             <div class="Title">
 
                             <div class="Title">
                                 <h1>Parasitic antigens</h1> </div>
+
                                 <h1>Choosing the best reporter</h1> </div>
  
 
                             <div class="Textbox Results-Desc">
 
                             <div class="Textbox Results-Desc">
 
     <!--Introduction-->
 
     <!--Introduction-->
 
<p>
 
<p>
                                   The aim of this project is to express African Sleeping Sickness &#40;<i>Trypanosoma brucei gambiense</i>&#41; antigens in <i>E. coli</i>, and to purify these antigens using the Strep-II tag. These are then used for the selection of Affinity Bodies using phage display. In this way, the detection module of the diagnostic device is made to specifically detect surface antigens of Trypanosomes, thus HAT. Hence, this part functions as a proof-of-concept that, besides viral infections, Mantis can detect parasitic infections as well. </p>
+
                                   The aim of this project is to analyze different reporter proteins to choose the best. As our project signal is based on bimolecular complementation, the reporter proteins will be split and analyzed under reassembly directed through synthetic leucine zippers. The best reporter will have to fulfill a set of characteristics. First, it has to show a bright signal, so the device can detect easily if the system has been activated. Second, the maturation of the reporter must be a fast as possible, to be able to detect a signal in a short timeframe. Last, our device will be used in tropical areas, where temperatures can be really high. Therefore, the reporter must also be able to produce a strong signal and mature fast at high temperatures. </p>
 
 
 
                             </div></section>
 
                             </div></section>
Line 39: Line 39:
 
                                 <div class="panel panel-default">
 
                                 <div class="panel panel-default">
 
                                     <div class="panel-heading" role="tab" id="headingOne">
 
                                     <div class="panel-heading" role="tab" id="headingOne">
<a data-toggle="collapse" data-parent="#accordion" href="#Approach" aria-expanded="true" aria-controls="Approach">
+
<a data-toggle="collapse" data-parent="#accordion" href="#CApproach" aria-expanded="true" aria-controls="CApproach">
 
                                         <h4 class="panel-title">
 
                                         <h4 class="panel-title">
 
<div class="col-xs-11">
 
<div class="col-xs-11">
Approach  
+
Approach: Chromoproteins
 
</div><div class="col-xs-1"><i class="fa fa-arrow-down" aria-hidden="true"></i></div></a></h4>
 
</div><div class="col-xs-1"><i class="fa fa-arrow-down" aria-hidden="true"></i></div></a></h4>
 
</div>
 
</div>
 
 
                                     <div id="Approach" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne">
+
                                     <div id="CApproach" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne">
 
                                         <div class="panel-body bg-primary">
 
                                         <div class="panel-body bg-primary">
 
     <!--Text for Approach-->                                         
 
     <!--Text for Approach-->                                         
 
 
<h4>Introduction</h4><p>
+
The proteins that are selected to be used for the phage display are surface proteins from Trypanosomes. The selection was made based on the titer in the blood and the reactivity to IgG in earlier findings. The surface of Trypanosomes is mostly covered in the Lille Trypanosoma Antigen Type Variant Surface Glycoprotein &#40;LiTat VSG&#41;. This makes it unsuitable to be used as the protein to which our test is based upon. The Invariant Surface Glycoprotein &#40;ISG&#41; is also a surface antigen of Trypanosomes. There are several forms, of different sizes. Of those, ISG64 &#40;64 kDa&#41; and ISG65 &#40;65 kDa&#41; are the most reactive towards antibodies for both forms of HAT, followed by the 75 kDa ISG75 &#91;1&#93;. By removing the signal peptide on the N-terminal, and the transmembrane domain on the C-terminal, a soluble protein is created &#91;2&#93;. This will simplify the expression and purification process but keep the immunogenicity. Although there are a 100 VSG for every ISG, its genetic stability is adventurous &#91;3&#93;. </p>
+
+
<h4>Construct</h4>
+
<p>The three ISG antigens suitable as a biomarker for HAT are PCR amplified from genomic Trypanosoma brucei DNA. Genomic DNA from Trypanosoma brucei gambiense is made available by the WHO Collaborating Center for Research and Training on Human African Trypanosomiasis Diagnostics in Antwerp. The extracellular domains of ISG64, ISG65 and ISG75 are PCR amplified using gene-specific primers extended with a 5&rsquo; KpnI and SacI restriction sites.
+
This is followed by cloning into the Multiple Cloning Site (MSC) of the <i>E. coli</i> expression vector pET52b via restriction digestion with KpnI and SacI. This resulted in a recombinant gene &#40;rISG&#41; with a 5&rsquo; Strep-tag II and a 3&rsquo; 10x HIS-tag. The construct is present under an IPTG-inducible promoter, see figure 1.</p>
+
+
 
 
<div class="figure-fullwidth">                  
+
</div> </div></div></div>
                <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/5/5a/T--Wageningen_UR--Results_Tryps_Construct_Map.png"/>
+
 
                    </div>
+
<!--Accordion of FApproach-->
<div class="figure-center">
+
<div class="figure-center-caption">
+
                            <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
<b>Figure 1:</b> Map of the recombinant ISG genes after clonation into the pET52b expression vector. 
+
                                <div class="panel panel-default">
</div></div>
+
                                    <div class="panel-heading" role="tab" id="headingOne">
+
<a data-toggle="collapse" data-parent="#accordion" href="#FApproach" aria-expanded="true" aria-controls="FApproach">
+
                                        <h4 class="panel-title">
<p>This construct is transformed to <i>E. coli DH5&alpha;</i>. The constructs were checked with colony PCR and sequencing. The sequence of the construct was compared to the sequence of the original template, as well as the reference sequence from online databases. Whereas the sequence of rISG64 and rISG65 could be validated, the one of rISG75 could not. Too many unexplainable mismatches were found to continue with protein expression. One explanation could be that ISG75 is part of a gene family, and a family member has been amplified. Because the correctness of the sequence could not be verified, this construct was not further used. </p>
+
<div class="col-xs-11">
+
Approach: Fluorescent Proteins
<p>After this validation step, the two remaining plasmids were transformed to <i>E. coli Rosetta</i> for protein expression. This strain contains the pRARE plasmid, having extra tRNA genes compromising for the rare codons present in the parasitic genome. </p>
+
</div><div class="col-xs-1"><i class="fa fa-arrow-down" aria-hidden="true"></i></div></a></h4>
+
</div>
<h4>Protein expression</h4>
+
<p>The induction of protein expression of the pET52b-ISG constructs was tested, as well as the solubility of the recombinant proteins, see figure 2. </p>
+
 
 
 +
                                    <div id="FApproach" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne">
 +
                                        <div class="panel-body bg-primary">
 +
    <!--Text for FApproach-->                                       
 
 
<div class="figure-center">
+
<div class="figure-center-imagebox" >
+
<img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/b/b4/T--Wageningen_UR--Results_Tryps_SDS_Test_Expression.jpg"/>
+
</div>
+
<div class="figure-center-caption">
+
<b>Figure 2:</b> SDS Gel of cell lysate before and after IPTG induction, as well as the soluble and insoluble fraction hereof. The assumed bands for rISG64 and rISG65 are indicated with the red box.
+
</div></div>
+
 
+
+
<p>As seen, protein expression could be induced, where the protein is present in the soluble fraction as expected. </p>
+
+
<h4>Protein purification</h4>
+
<p>Next, 200 ml cultures were grown, following by induction with 0.5 mM IPTG. Protein purification was conducted by affinity purification in gravity columns using strep-tactin, making use of the StrepII-tag. Purity was checked on SDS gel, and protein concentration in the eluted fractions was measured using a protein quantitation assay. More can be read <a href="https://2017.igem.org/Team:Wageningen_UR/Notebook" target="_blank">here</a>. </p>
+
 
 
 
</div> </div></div></div>
 
</div> </div></div></div>

Revision as of 08:46, 25 October 2017

Choosing the best reporter

The aim of this project is to analyze different reporter proteins to choose the best. As our project signal is based on bimolecular complementation, the reporter proteins will be split and analyzed under reassembly directed through synthetic leucine zippers. The best reporter will have to fulfill a set of characteristics. First, it has to show a bright signal, so the device can detect easily if the system has been activated. Second, the maturation of the reporter must be a fast as possible, to be able to detect a signal in a short timeframe. Last, our device will be used in tropical areas, where temperatures can be really high. Therefore, the reporter must also be able to produce a strong signal and mature fast at high temperatures.

The extracellular domain of the Invariant Surface Glycoprotein 64 and 65, fused to both a StrepII-tag and 10x HIS-tag has successfully been purified using strep-tactin gravity column, see figure 3.

Figure 3: SDS gel of the protein fractions eluted from the strep-tactin column, both the flowthrough after loading the cell lysis onto the column, a few washing steps and the elution fractions.

The final 50 μl elution fraction (Elute 4) contains 283 μg/ml protein for rISG64, whereas the elution for rISG65 just contains 63 μg/ml protein. As seen from the high amounts of protein in the flowthrough, the column has reached its saturation point.

These tagged proteins, bound to the strep-tactin beads, are used for phage display selection.

Moreover, two biobricks were created of these constructs: BBa_K2387060 and BBa_K2387061. For this, the recombinant ISG gene, including the two tags, was cloned into the linearized pSB1C3 vector using biobrick assembly.

References

  1. Biéler, Sylvain, et al. "Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis." PloS one 11.12 (2016): e0168074.
  2. Sullivan, Lauren, et al. "Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device." PLoS neglected tropical diseases 7.2 (2013): e2087.
  3. Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.