Difference between revisions of "Team:Uppsala/Phage Display"

 
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                         Since the method of phage display was developed in 1985 [1] it has been applied for various purposes such as studying antibodies, observing immune system responses, and evolving antibodies for therapy purposes. It has also been used for whole cell recognition (often used for targeting cancer) and in-vivo screening of individual tissues for endothelial cell markers [2]
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                         Since the method of phage display was developed in 1985 it has been applied for various purposes such as studying antibodies, observing immune system responses, and evolving antibodies for therapy purposes [1]. It has also been used for whole cell recognition (often used for targeting cancer) and <i>in vivo</i> screening of individual tissues for endothelial cell markers [2].
 
                         More commonly, phage display is performed with a protein as the target, in order to find a ligand that would affect the function of the protein.  
 
                         More commonly, phage display is performed with a protein as the target, in order to find a ligand that would affect the function of the protein.  
 
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                             <img src="https://static.igem.org/mediawiki/2018/2/2a/T--Uppsala--Phage_Display_flowchart_New.svg" alt="Flowchart Phage Display" class="center" height="50%" width="50%">  
 
                             <img src="https://static.igem.org/mediawiki/2018/2/2a/T--Uppsala--Phage_Display_flowchart_New.svg" alt="Flowchart Phage Display" class="center" height="50%" width="50%">  
 
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                             <p><b>Figure 1.</b> Flowchart over the workflow of a typical phage display screening.</p>  
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                             <p><b>Figure 1.</b> Flowchart over the workflow of a typical phage display screening.</p>  
 
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                     <p>For the purpose of our project we need a way to detect the parasites. A peptide binding specifically to the exterior of the strongyle would fulfill this purpose. Since we are working with an under-researched organism, starting with a random peptide library seemed reasonable. The only similar organisms that have been used in this method were <i>C. elegens</i>, but as it is a free living nematode whereas strongyles are endoparasites we had reason to believe that the exterior of the membranes would differ greatly due to the different environments the different species inhabit.<br><br>
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                     <p>For the purpose of our project we need a way to detect the parasites. A peptide binding specifically to the exterior of the strongyle would fulfill this purpose. Since we are working with an under-researched organism, starting with a random peptide library seemed reasonable. The only similar organisms that have been used in this method were <i>C. elegens</i>, but as it is a free living nematode whereas strongyles are endoparasites. Due to this fact we had reason to believe that the exterior of the membranes would differ greatly due to the different environments the different species inhabit.<br><br>
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                         Using a random phage library streamlined our work. Phages ensure a physical link between the DNA sequence and its encoded peptides. Furthermore the indigenous behavior of lysogenic phages allowed visualizing and following the procedure as well as enabling amplification of the peptides upon infecting bacteria. Thus, in spite of lack of a premade protocol we decided to try and apply phage display for characterizing our nematodes.  
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                         Using a random phage library streamlined our work. Phages ensure a physical link between the DNA sequence and its encoded peptides. Furthermore the indigenous behavior of lysogenic phages allowed visualizing and following the procedure as well as enabling amplification of the peptides upon infecting bacteria. Thus, in spite the lack of a premade protocol we decided to try and apply phage display for characterizing our nematodes.  
 
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                         For a detailed description of our protocol click <a href="https://static.igem.org/mediawiki/2018/2/22/T--Uppsala--PhageDisplay_Panning_titering_amplification.pdf">here</a>. Our whole organism phage display experiments started with preparation of the containers that the whole procedure were performed in: microcentrifuge tubes with filter inserts. The preparation consisted of blocking the tubes with blocking buffer to prevent non-specific interactions.   
 
                         For a detailed description of our protocol click <a href="https://static.igem.org/mediawiki/2018/2/22/T--Uppsala--PhageDisplay_Panning_titering_amplification.pdf">here</a>. Our whole organism phage display experiments started with preparation of the containers that the whole procedure were performed in: microcentrifuge tubes with filter inserts. The preparation consisted of blocking the tubes with blocking buffer to prevent non-specific interactions.   
 
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                         To avoid selecting for phage with affinity for the plastic and not the organism, an affinity screening for the tubes was performed before introducing the target. After collecting the phage elute that doesn’t bind to the tubes, we performed our first affinity screening against the small strongyle. This was done by introducing the 12-mer peptides expressing phages to the strongyle placed in the filter tubes. Unbound phages were then washed away followed by elution and collection of the bound phages with an acidic buffer. All washing and elution steps were performed in the filter tubes, where liquid during centrifugation could pass through the filters, leaving the strongyle still on the top of the filter.
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                         To avoid selecting a phage with affinity for the plastic and not the organism, an affinity screening for the tubes was performed before introducing the target. After collecting the phage elute that doesn’t bind to the tubes, we performed our first affinity screening against the small strongyle. This was done by introducing the 12-mer peptides expressing phages to the strongyle placed in the filter tubes. Unbound phages were then washed away followed by elution and collection of the bound phages with an acidic buffer. All washing and elution steps were performed in the filter tubes, where liquid during centrifugation could pass through the filters, leaving the strongyle still on the top of the filter.
 
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                         Phage titering is done after every affinity screening to assess the amount of phages that bind to the target. By following the titering protocol consisting of plating phages together with mid-log phase bacteria visible blue plaques are formed on Xgal/IPTG plates. Xgal is a substrate for the enzyme β-galactosidase, which hydrolyzes the compound into a form that spontaneously dimerizes into an insoluble blue pigment. Our cell strain can only express the enzyme after phage infection and induction by IPTG. This makes it simple to distinguish infected from non infected colonies since only the infected ones will appear blue.  
 
                         Phage titering is done after every affinity screening to assess the amount of phages that bind to the target. By following the titering protocol consisting of plating phages together with mid-log phase bacteria visible blue plaques are formed on Xgal/IPTG plates. Xgal is a substrate for the enzyme β-galactosidase, which hydrolyzes the compound into a form that spontaneously dimerizes into an insoluble blue pigment. Our cell strain can only express the enzyme after phage infection and induction by IPTG. This makes it simple to distinguish infected from non infected colonies since only the infected ones will appear blue.  
 
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                         The goal with plating is to achieve plates with around 100 plaques, which is fulfilled by doing several dilution series of the infected bacteria. The reason for this is that plaques will only increase linearly with added phages when the multiplicity of infection (MOI) is much less than 1. Also low MOI result in one DNA sequence per plaque. To asses the titre, plaque forming unit (pfu) can be calculated by multiple numbers of plaques with the bacteria dilution.  
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                         The goal with plating is to achieve plates with around 100 plaques, which is fulfilled by doing several dilution series of the infected bacteria. The reason for this is that plaques will only increase linearly with added phages when the multiplicity of infection (MOI) is much less than 1. Also, low MOI result in one DNA sequence per plaque. To asses the titre, plaque forming unit (pfu) can be calculated by multiple numbers of plaques with the bacteria dilution.  
 
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                         So how do we prove that the selected phages clones really bind to our target? With the help of ELISA (enzyme-linked immunosorbent assay) single phage clones affinity to the target could be tested by screening against plastic binders. This allowed us to determine which samples are viable. The ELISA was performed in the centrifugal filter-tubes to expose the phages to the same environment as the panning stage of the process.  
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                         So how do we prove that the selected phages clones really bind to our target? With the help of ELISA (enzyme-linked immunosorbent assay) single phage clones, affinity to the target could be tested by screening against plastic binders. This allowed us to identify all the viable samples. The ELISA was performed in the centrifugal filter-tubes to expose the phages to the same environment as the panning stage of the process.  
 
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                     <h2>Titering Table</h2>
 
                     <h2>Titering Table</h2>
 
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                     <p><strong>Table 1:</strong> Titering results. *Negative Panning for Interactions with the Tube were Performed in Conjugate with Regular Panning.</p>
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                     <p><strong>Table 1:</strong> Titering results. *Negative panning for interactions with the tube were performed in conjugate with regular panning.</p>
 
                     <table class="pgrouptable tablesorter our-table" style="width: 100%;" cellspacing="0" cellpadding="0">
 
                     <table class="pgrouptable tablesorter our-table" style="width: 100%;" cellspacing="0" cellpadding="0">
 
                         <thead>
 
                         <thead>
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                                 <td>Series 1
 
                                 <td>Series 1
 
                                 </td>
 
                                 </td>
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                                 <td>2.3 x 10^7*
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                                 <td>\(2.3 \cdot 10^{7*}\)
 
                                 </td>
 
                                 </td>
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                                 <td>>10^15
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                                 <td>\(>10^{15}\)
 
                                 </td>
 
                                 </td>
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                                 <td>3.2 x 10^10
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                                 <td>\(3.2 \cdot 10^{10}\)
 
                                 </td>
 
                                 </td>
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                                 <td>4 x 10^12
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                                 <td>\(4 \cdot 10^{12}\)
 
                                 </td>
 
                                 </td>
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                                 <td>1 x 10^14
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                                 <td>\(1 \cdot 10^{14}\)
 
                                 </td>
 
                                 </td>
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                                 <td>6 x 10^12
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                                 <td>\(6 \cdot 10^{12}\)
 
                                 </td>
 
                                 </td>
 
                             </tr>
 
                             </tr>
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                                 <td>Faulty XGal
 
                                 <td>Faulty XGal
 
                                 </td>
 
                                 </td>
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                                 <td>1.8 x 10^10
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                                 <td>\(1.8 \cdot 10^{10}\)
 
                                 </td>
 
                                 </td>
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                                 <td>1.6 x 10^9
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                                 <td>\(1.6 \cdot 10^{9}\)
 
                                 </td>
 
                                 </td>
 
                                 <td>No Amp.
 
                                 <td>No Amp.
 
                                 </td>
 
                                 </td>
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                                 <td>7 x 10^5*
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                                 <td>\(7 \cdot 10^{5*}\)
 
                                 </td>
 
                                 </td>
 
                                 <td>-
 
                                 <td>-
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                                 <td>Series 3
 
                                 <td>Series 3
 
                                 </td>
 
                                 </td>
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                                 <td>2.9 x 10^7*
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                                 <td>\(2.9 \cdot 10^{7*}\)
 
                                 </td>
 
                                 </td>
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                                 <td>6.2 x 10^12
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                                 <td>\(6.2 \cdot 10^{12}\)
 
                                 </td>
 
                                 </td>
 
                                 <td>-
 
                                 <td>-
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                             <!-- Here goes the big image to the right -->  
 
                             <!-- Here goes the big image to the right -->  
 
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                             <img src=https://static.igem.org/mediawiki/2018/3/39/T--Uppsala--phageelisa_small.jpg>
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                             <p><strong>Figure 8.</strong> Negative control of phage ELISA, containing ONLY monoclonal M13 antibodies and NO phage.</p>   
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                             <p><strong>Figure 8.</strong> Negative control of phage ELISA, containing <b>only</b> monoclonal M13 antibodies and <b>no</b> phage.</p>   
 
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                         Five samples contain pure enough samples of sufficient concentration to satisfy the standard for third party sequencing. Series 1 produced a single sample of high enough quality, Series 2 were all deemed too low for accurate sequencing and Series 3 yieded four samples. The aligment used ClustalW with penalties 25 for gap-creation and 25 for gap-elongation to ensure strict alignments. No clear consensus motifs are distinguishable.
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                         Five samples contained pure enough samples of sufficient concentration to satisfy the standard for third party sequencing. Series 1 produced a single sample of high enough quality, Series 2 were all deemed too low for accurate sequencing and Series 3 yieded four samples. The aligment used ClustalW with penalties 25 for gap-creation and 25 for gap-elongation to ensure strict alignments. No clear consensus motifs were distinguishable.
  
 
                         Predictive analysis was performed with <a href="http://immunet.cn/sarotup/cgi-bin/TUPScan.pl">SAROTUP: Target-Unrelated Peptides Scanners</a>[3, 4, 5].
 
                         Predictive analysis was performed with <a href="http://immunet.cn/sarotup/cgi-bin/TUPScan.pl">SAROTUP: Target-Unrelated Peptides Scanners</a>[3, 4, 5].
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                     <h1 id="ConcPhage">Conclusion</h1>
 
                     <h1 id="ConcPhage">Conclusion</h1>
 
                     <p>
 
                     <p>
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                         Due to the fact that our ELISA-test was inconclusive we can not be sure that our peptides are specific binders. However, only one was predicted as a Polysterene binder and none contain any known TUP-sequences. A blastp on Biopanning Databank showed that EF01122224 showed some similarities with an antibody for a pork tapeworm epithelial protein and EF01122218 for an antibody for rabbit epithelia. The E-values were high, 185 and 10 respectively, but those two would be promising candidates for future studies.  
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                         Due to the fact that our ELISA-test was inconclusive we can not be sure that our peptides are specific binders. However, only one was predicted as a Polysterene binder and none contain any known TUP-sequences. A blastp on Biopanning Databank showed that EF01122224 showed some similarities with an antibody for a pork tapeworm epithelial protein and EF01122218 for an antibody for rabbit epithelia. The E-values were high, 211 and 10 respectively, but those two may be promising candidates for future studies.  
 
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Latest revision as of 13:49, 6 December 2018