Difference between revisions of "Team:Wageningen UR/Results/Phage Display"

 
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                             <h4>Quorum sensing</h4>
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                             <h4>Phage display</h4>
 
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                             <li><a href="https://2017.igem.org/Team:Wageningen_UR">Home</a></li>
 
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                             <li>Quorum Sensing</li>
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                             <li>Phage Display</li>
 
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                             <h1>Quorum Sensing</h1> </div>
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                            <h2>Introduction</h2> </div>
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                             <p>Mantis is a modular bacterial diagnostic device that will generate a visible fluorescent signal upon detection of viral antigens in blood sample of viral-infected patients. When quantifying fluorescence, a low signal to noise ratio hinders a proper understanding of the imaging system. Therefore, it is necessary to both increase the sensitivity of the detection and decrease the background signal to allow for a higher accuracy and precision of the quantitative fluorescence measurements [1]. </p>
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                             <p>Phage display is a powerful laboratory technique used to characterize protein-ligand interactions and functions. Phage display can be used to select for proteins with the desired properties from large libraries. Using M13 phage display, we can select for the best affinity body against <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Viral_Antigens">Chikungunya virus (CHIKV)</a> and <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Trypanosoma">Human African Trypanosoma (HAT)</a> antigens using the <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Affinity_Bodies">affinity body library</a> we created. These affinity bodies can then be used to make the Mantis cells detect these diseases.
  
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                             <p>While the fast Mantis reporter module (<a href="https://2017.igem.org/Team:Wageningen_UR/Results/Cpx">Signal Transduction</a> and <a href="https://2017.igem.org/Team:Wageningen_UR/Results/SpecificVisualization">Specific Visualization</a>) focusses on generating a signal in the shortest possible time, in this project we focus on getting the most robust detection system. Due to the modular nature of Mantis, it is easy to swap out one system for the other, based on the requirements of the antigen we want to detect. For example, when highly contagious diseases are to be detected, a fast signal would be preferred. However, when the aim is to detect not diseases that are not very contagious, but pose a large health risk if untreated, the least amount of false negatives is desired. In order to achieve a robust signal generation system, a high signal-to-noise ratio is essential. Thus, a higher sensitivity of the fluorescent GFP detection system and a lower background signal are needed.
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                            <p>The robust mantis reporter module is based on a spatially separated two component system.. For that, two  bacterial cell populations (BCP) will be used, each expressing only one of the two components needed to generate the output signal. Both components will be isolated inside their own BCP so the fluorescent signal is not generated. The two components will only be able to interact with each other after both BCPs lyse, which will be biologically induced after sensing the antigen  (Figure 1). </p>
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                            <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/6/61/T--Wageningen_UR--Results_Quorum_Sensing_Figure_1.png" />
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                                <b>Figure 1:</b> Representation of an antigen-triggered generated output by a two interdependent components system.
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                                <b>Figure 1:</b> Representation of an antigen-triggered generated output by a two interdependent components system.
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Together with a reduction of the background signal, a higher sensitivity of the system is desired. For that a quorum sensing mechanism will be implemented in both BCPs so the signal output produced after the detection of the antigen is propagated to all the bacteria present, no matter if they are directly detecting the antigen or not (Figure 2).
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                                <b>Figure 2:</b> Representation of the antigen-triggered generated output amplified by the quorum sensing mechanism. The figure depicts the communication of a detected antigen from the directly sensing bacteria (green) to the non antigen-sensed ones (red).
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                        <p>In conclusion, when the antigen gets detected, a lytic mechanism triggers the lysis of both BCPs. This will allow the interaction of both interdependent components leading to the generation of a fluorescent signal. Provided that both components are separated by both bacterial cell membranes, the background signal is hypothesized to be reduced as the random interaction of those two components is expected to be lower than when present within the same BCP. Therefore, by reducing the background signal which could be generated by the random interaction of both components, the signal to noise ratio will be increased (Figure 3).
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                                <b>Figure 3:</b> Representation of the robust Mantis module.
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                             <h2>Quorum sensing mechanism</h2> </div>
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                             <h2>Generation of recombinant M13 phages</h2> </div>
 
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                                 Quorum sensing (QS) is a cell to cell communication system based on chemical molecules which allow bacteria to detect the presence of other bacteria. This mechanism allows bacteria to control gene expression in response to changes in bacteria concentration in the medium [1,4]. In many gram-negative bacteria quorum sensing is mainly controlled by production and diffusion of Acylated Homoserine Lactone (AHL). AHL is able to freely diffuse from cell to cell. Once AHL is detected, it is able to trigger other bacteria to generate more AHL molecules, creating a positive feedback loop. Our quorum sensing mechanism (originating from V. fischeri) is controlled by two proteins: LuxI (BBa_C0061) and LuxR (BBa_C0062). LuxR is a transcription factor that becomes active when complexed with AHL, leading to the transcription of genes placed under the pLux promoter (BBa_R0062) (Figure 4) [2].
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In M13 phage display, the DNA sequence encoding the protein of interest (in our case the affinity body library) is fused to a gene which codes for the M13 phage G3P minor coat protein. After correct assembly, the phage will display the affinity body on its surface, linking the genotype to the phenotype. To fuse the affinity body to the phage coat protein, two distinct strategies can be applied. The first method is to directly clone the affinity body sequence into the coding sequence of the coat protein in the phage genome. The other option is to use a small vector (phagemid vector) encoding for part of the phage G3P minor coat protein. The protein of interest can be cloned into the phagemid vector and will be expressed as a fusion with G3P (p III in Figure 1). The advantage of using phagemids is the ease of cloning into the smaller vector, as efficiency is important during library cloning. Furthermore, using a phagemid results in a monovalent display of the protein of interest compared to a polyvalent display with the phage vector [1]. This is an important difference since a polyvalent display could lead to the selection of weak-binding variants while this is circumvented with a monovalent display. However, the phagemid vector approach must be used in combination with a helper phage. In our case, we use the VCSM13 helper phage which is an M13-based phage and has a defective origin of replication. The phagemid vector has a working origin of replication and this leads to the preference for packing the phagemid vector in the phage particles. Taking this in consideration, we chose the phagemid vector approach.
  
 
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                                <b>Figure 4:</b> Gram-negative LuxIR-type quorum-sensing mechanism (AHL autoinducers are represented as red pentagons) Figure from (Ng & Bassler, 2009 [2].
 
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                                In nature, the LuxR-AHL complex controls transcription of LuxI via a positive feedback loop which increases the AHL production. When the AHL concentration is high in the cell, dimerization with LuxR protein takes place in higher amounts [3]. Both LuxI and LuxR are expressed under the pLux promoter. However, LuxI is under the Lux PR promoter (BBa_R0062) whose expression is upregulated by the LuxR complex and the LuxR gene is under the reverse Lux PL promoter (BBa_R0063)whose expression is downregulated by LuxR complex [4]. Both promoters are the same sequence but inverted, with one of them being up-regulated by the LuxR complex while the other one is downregulated.
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<b>Figure 1:</b> Schematic picture M13 phage, made by iGEM <a href="https://2011.igem.org/Team:Edinburgh/Phage_Display">Edinburgh 2011</a>.  
  
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Quorum sensing takes place in nature under high bacterial concentrations. However, in our case, quorum sensing mechanism is desired to only become active after the detection of the antigen. For that, the quorum sensing system needs to be repressed in order to prevent quorum sensing from happening unless the antigen is detected.
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                            <p>The robust Mantis reporter module implements a quorum sensing mechanism in such a way that after the binding of the antigen to one cell, AHL is produced and the detection signal is spread to other cells. In order to assess the expression of the quorum sensing mechanism, the quorum sensing plasmid (BBa_K1913005) which encodes for GFP under the pLux promoter was used (Figure 5). Therefore, in our system, when both AHL and LuxR are present in the cell, the dimer is formed and GFP is expressed as a reporter. Two negative controls were constructed to perform this experiment. Negative control 1 encodes only GFP under the pLux promoter and no LuxR nor LuxI (Figure 5). Provided that no AHL nor LuxR will be generated, the formation of the [AHL-LuxR] dimer will not take place and no fluorescence will be generated. Negative control 2 encodes for GFP and LuxR under the pLux promoter but no LuxI (Figure 5). In this case, as no AHL can be formed, the dimer wont be formed and therefore GFP can’t be generated.
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<div class="Title"><h3>Proof-of-principle</h3></div>
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Before generating a phage library we performed a proof-of-principle with the wild type (WT) affinity body sequence. This affinity body specifically binds Immunoglobulin G (IgG) antibodies. The WT affinity body sequence was ligated into the pComb3XSS phagemid. XL1-Blue cells were transformed with the phagemid and subsequently used to create recombinant phages containing the WT affinity body (Figure 2). The phages were amplified and titrated. In order to see whether the phagemid was preserved, we infected fresh XL1-Blue cells with the recombinant phages. The infected cell suspension was plated on LB agar plates with the appropriate antibiotic. Several colonies were picked, grown, plasmids were isolated and sent for sequencing.
  
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                                    <b>Figure 5:</b> Representation of the genetic encoding units from the Reporter quorum sensing casette, the Negative control 1 and the Negative control 2.
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<b>Figure 2:</b> Overview of the creation of the proof-of-principle recombinant M13 phages carrying the IgG affinity body sequence.
  
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                            <p>LuxI expression leads to the formation of an AHL synthase, producing AHL which can diffuse through the cell membrane from cell to cell. LuxR encodes for a protein which dimerizes with AHL. Once the dimer is formed, the genes under the pLux promoter become expressed, hence fluorescence is generated (Figure 6). It can be seen in the graph that negative control 1 is slightly more active, while the LuxR protein is not even in the cell. A possible explanation for this could be that the uncomplexed luxR has a certain repressing effect to some extent (Figure 6).
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                                    <b>Figure 6:</b> Assessment of the Reporter quorum sensing mechanism in DH5α. Negative control 1 encodes only GFP under the pLux promoter and no LuxR nor LuxI and Negative control 2 encodes for GFP and LuxR  under the pLux promoter but no LuxI (See Figure 5).
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The robust Mantis reporter module aims to change the spontaneously activating quorum sensing system (Figure) into a system with proper antigen-dependent signaling behavior by keeping under control the quorum sensing mechanism. To achieve this, the AiiA enzyme (BBa_K2387070) which catalyzes the degradation of N-acyl-homoserine lactones (AHLs), will be constitutively expressed. In order to test the mechanism, a strain containing a construct expressing GFP, LuxI and LuxR under the pLux promoter and the AiiA molecule under the pTet promoter, whose inhibitor TetR was produced constitutively under the pLac promoter, was constructed (Figure 7).
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                                        After ligation of the wild type affinity body sequence into the pComb3XSS phagemid, the mixture was electroporated into XL1-Blue <i>Escherichia coli</i> (<i>E. coli</i>) cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture, 250 mL of fresh medium was added and grown in 1L flasks until an OD<sub>600</sub> of ~0.6 was reached. VCSM13 helper phages were added at an MOI of 20 and incubated at 37°C without shaking. After incubation, the medium was changed to a 2xYT medium supplemented with 1% (w/v) glucose, 100 ug/mL ampicillin, 50 ug/mL kanamycin and 0.25mM IPTG and incubated overnight at 30°C. The next day the phages were precipitated and a titration was performed to assess infectivity.
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                                    <b>Figure 7:</b> Gene sequence depicting the Inducible quorum sensing construct whose decrease in fluorescence is induced under the addition of anhydrotetracycline (aTc), an analogue of tetracycline.
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                        </div>
  
−
                            <p>In this mechanism, the quorum sensing mechanism was hypothesized to be active under no induction. When induced by anhydrotetracycline (analogue of tetracycline) (aTc), the aiiA should be expressed as the inhibitor is captured by aTc leading to the expression of the gene downstream. Therefore, a decrease in fluorescence should be observed when aTc is added to the medium (Figure 8).</p>
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                            <div class "figure-fullwidth">
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<div class="Textbox Title">
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                                <div class="figure-center-imgagebox">
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/1/15/T--Wageningen_UR--Results_Quorum_Sensing_Figure_8.svg" <div class="figure-center-caption">
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                                    <b>Figure 8:</b> AiiA mechanism controlled under the pLac promoter. Left) No induction state, AiiA expression is inhibited by TetR, no AiiA expression can take place. In this state, the formation of the LuxR-AHL dimer is allowed which further binds to pLux leading to activation of the quorum sensing system and GFP generation. Right) aTc (Depicted in purple as Tetracyclin) captures the TetR inhibitor, leading to the activation of pTet which leads to the expression of AiiA, causing degradation of AHL and thus lowering the amount of active luxR-AHL.
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                                </div>
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                            </div>
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                            <p>The anhydrotetracycline inducible mechanism was tested together with the Reporter quorum sensing, Negative control 1 and Negative control 2 constructs. After triggering the system by adding aTc, a decrease in fluorescence was observed while comparing both induced and non induced aiiA expressing constructs (Figure 9). No difference was observed while comparing both induced and non induced non expressing AiiA (Reporter quorum sensing strain) (Figure 10).
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</p>
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                            <div class "figure-fullwidth">
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                                <div class="figure-center-imgagebox">
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/8/8e/T--Wageningen_UR--Results_Quorum_Sensing_Figure_9.png" <div class="figure-center-caption">
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                                    <b>Figure 9:</b> Comparison of fluorescence generation by a non induced and an induced aTc-Inducible Reporter quorum sensing mechanism in DH5α.
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                                </div>
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                            </div>
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                            <p>Fluorescence is modestly decreased when inducing the Inducible QS with high aTc concentrations in comparison to its non-induced state suggesting that the aiiA mechanism is working. This can indicate that AiiA works as expected and that AHL degradation might have been reduced as well leading to a recovery of fluorescence. However, the results seen do not show a fluorescence recovery comparable of the fluorescence level achieved with the Reporter QS which might imply that leaky expression of the pTet could be causing expression of AiiA also in the induced state (Figure 8).
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                            </p>
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<p>
 
<p>
−
Furthermore, it seems that overall fluorescence is still very high compared to the negative controls (Figure 10). Moreover, the Inducible QS strain showed a modest increase in fluorescence which is not in agreement of the expected decrease in fluorescence under induction. The reason for this is the low induction of the system, under higher aTc inductions, the decrease in fluorescence can be seen (Figure 9). However, higher aTc inductions are deadly for the cells and therefore the assessments cannot be performed. This suggested that it was needed to increase the repressing effect of AiiA gene in order to avoid the generation of a signal in the absence of the triggering signal. By doing so, the  sensitivity of the system could be increased. by allowing a base no signal level.
+
The resulting recombinant phage with wild type affinity body has an infective titer of 9.6*10<sup>11</sup> CFU/mL (colony forming units per mL). We visualized these phages using transmission electron microscopy (TEM) (Figure 3).  
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</p>
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</p></div>
  
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                            <div class "figure-fullwidth">
 
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                                <div class="figure-center-imgagebox">
 
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/e/e2/T--Wageningen_UR--Results_Quorum_Sensing_Figure_10.png" <div class="figure-center-caption">
 
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                                    <b>Figure 10:</b> Assessment of the aTc-Inducible Reporter quorum sensing mechanism in DH5α overtime. Negative control 1 encodes only GFP under the pLux promoter and no LuxR nor LuxI and Negative control 2 encodes for GFP and LuxR  under the pLux promoter but no LuxI (Figure 5).
 
  
  
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                                </div>
 
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                            </div>
 
  
−
                            <p>From the parallel modelling project (<a href="https://2017.igem.org/Team:Wageningen_UR/Model/QS">Quorum sensing and cell lysis</a>), it was suggested that either increasing LuxR degradation or increasing the AiiA concentration, which degrades AHL, will allow for a stronger control of the quorum sensing mechanism. Therefore, the AiiA gene was expressed in the pSB1C3 high copy number plasmid backbone and the quorum sensing mechanism was expressed in the pSB4K5 low copy number backbone. By doing so, the ratio of AiiA to LuxI and LuxR was aimed to be increased, hypothetically leading to a decreased autoinduction (Figure 13).
 
  
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                            </p>
 
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                            <div class "figure-fullwidth">
 
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                                <div class="figure-center-imgagebox">
 
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/8/86/T--Wageningen_UR--Results_Quorum_Sensing_Figure_11.svg" <div class="figure-center-caption">
 
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                                    <b>Figure 11:</b> AiiA mechanism controlled under the pBAD promoter. Left) No induction state, AiiA expression is triggered by pTet. Right) Arabinose activates the pBAD promoter leading to the expression of tetR which inhibits pTet, avoiding suppressing the expression of AiiA and leading to the formation of the LuxR-AHL dimer which further binds to pLux, leading to activation of the quorum sensing system and GFP generation.
 
  
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                                </div>
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<div class="figure-center">
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                            </div>
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<div class="figure-center-imagebox" >
 +
<img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/f/f8/T--Wageningen_UR--Results_PhageDisplay_TEM.jpg"/>
 +
</div>
 +
<div class="figure-center-caption">
 +
<b>Figure 3:</b> Transmission Electron Microscopy (TEM)  images of recombinant M13 phage expressing WT affinity body. Stained using phosphotungstic acid (PTA). Pictures were taken using JEOL JEM1400.
  
−
                            <p>The AiiA gene was then expressed under the pTet promoter (BBa_R0040) whose inhibition takes place when TetR (BBa_C0040) is expressed. TetR expression is in turn controlled by the pBAD promoter and therefore AiiA expression gets inhibited under the induction of arabinose (Figure 12).
 
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                            </p>
 
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                            <div class "figure-fullwidth">
 
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                                <div class="figure-center-imgagebox">
 
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/5/50/T--Wageningen_UR--Results_Quorum_Sensing_Figure_12.png" <div class="figure-center-caption">
 
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                                    <b>Figure 12:</b> Gene sequence depicting the Inducible quorum sensing construct whose increase in fluorescence is induced by the addition of arabinose.
 
  
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                                </div>
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</div>
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                            </div>
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</div>
  
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                            <p>When arabinose is added to the medium, pBAD triggers the expression of tetR which leads to the inhibition of pTet and hence represses the expression of aiiA which is under the control of pTet. Therefore, if unactivated, AiiA is constitutively expressed and a non-fluorescent state is expected.
 
−
                            </p>
 
  
−
                            <p>Fluorescence is decreased in comparison to both negative controls, suggesting that the QS system is repressed. The results seen imply that by inducing the system with arabinose, AHL is able to complex again LuxR triggering both the further expression of quorum sensing as well as the expression of GFP (Figure 13). The expression of AiiA gene in a higher copy number backbone (pSB1C3) resulted in a clearer change in fluorescence after inducing the system (Figure 13) when compared with the change caused when expressed in a low copy number backbone (pSB4K5) (Figure 9).
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<div class="figure-center">
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                            <div class "figure-fullwidth">
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                            <div class="figure-center-imagebox">
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                                <div class="figure-center-imgagebox">
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                                <img id="PosLuxR" class="figure-center-img" src="https://static.igem.org/mediawiki/2017/f/f2/T--Wageningen_UR--Results_Phage_Display_WTIgG.jpeg" />
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/7/76/T--Wageningen_UR--Results_Quorum_Sensing_Figure_13.png" <div class="figure-center-caption">
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                                <!-- The Modal -->
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                                    <b>Figure 13:</b> Assessment of the arabinose-Inducible Reporter quorum sensing mechanism in DH5α overtime. Negative control 1 encodes only GFP under the pLux promoter and no LuxR nor LuxI and Negative control 2 encodes for GFP and LuxR  under the pLux promoter but no LuxI (Figure 5).
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                                <div id="PosLuxR-Modal" class="modal">
  
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                                </div>
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                                    <!-- The Close Button -->
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                            </div>
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                                    <span class="close">&times;</span>
  
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                            <p>In the complete version of the robust Mantis reporter module, it will express a lytic mechanism under the pLux promoter which becomes activated by the LuxR-AHL dimer. After the detection of the antigen, the quorum sensing will be triggered, allowing both interdependent components to interact so the signal output gets generated. Moreover, AHL is able to diffuse from cell to cell, as soon as the QS gets activated in some of the bacteria, the lysis will be triggered in the rest of the cells via the diffusion of AHL from the directly QS activated to the non-directly activated ones [4].
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                            </p>
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                                     <img class="modal-content" src="https://static.igem.org/mediawiki/2017/f/f2/T--Wageningen_UR--Results_Phage_Display_WTIgG.jpeg" />
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                            <p>Therefore, this will allow the system to move from a state in which no lysis is generated to another one in which all the bacteria present in the medium will trigger lysis no matter if they are directly detecting the antigen or not. This means that the sensitivity of the signal will be increased as a result of the implementation of QS which allows the generation of an output signal by all the cells in the medium. In the future, LuxR degradation could be increased in order to achieve a better control of the system. As predicted by the parallel modelling part (https://2017.igem.org/Team:Wageningen_UR/Model/QS), either increasing LuxR degradation or increasing AiiA concentrations will keep quorum sensing under control in a non induced state. It has been already proven (Figure 13) that an increase in AiiA expression leads to a steeper change in fluorescence emission while comparing the non induced versus the induced state of the Inducible quorum system (when compared with the previous system (Figure 10) where no change can be observed).
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                            </p>
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                        </div>
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                    </section>
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                    <section id="TICS">
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                        <br>
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                        <div class="Title">
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                            <h2>Two interdependent components system</h2> </div>
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                        <div class="Textbox Title">
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                            <p>In order to generate a fluorescent output signal, three different systems were constructed and tested. The three proposed systems are composed by two components which do not generate the output signal independently unless they interact between each other.
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                            </p>
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                            <div class="Title">
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                                <h3>Bimolecular Fluorescence Complementation System</h3> </div>
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                            <p>This system is based on the generation of two BCPs each encoding for a complementary part of a fluorescent molecule. In this system, both BCPs will be combined in equal concentrations and a lytic process will be induced. This should lead to the fusion of both halves and hence generation of fluorescence. In order to confirm that both complementary fluorescent halves are able to fuse and lead to fluorescence, a third strain used as a positive control which was expressing both complementary halves in the same cell was also generated.
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                            </p>
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                            <p>The three BCPs were generated and the GFP fluorescence generated by combining BCP1 and BCP2 and inducing a lytic process was measured. This was compared with the fluorescence generated by the positive control consisting of both halves expressed under the same constitutive promoter. In parallel, a second positive control was created where an entire non split fluorescent protein was expressed. By comparing the fluorescence generated to  the two positive controls. Both the difference in fluorescence efficiency because of the in vitro reassembly and the decrease in efficiency caused by the split of the fluorescent protein can be evaluated.
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+
−
                            </p>
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                            <p>Two BCPs each encoding for half of the GFP molecule were generated (Constructs). Each BCP expresses half of the GFP protein with a Leucine Zipper attached to it (<a href="https://2017.igem.org/Team:Wageningen_UR/Results/Fluorescent">Choosing the best reporter</a>) under a constitutive promoter. A negative control encoding for both halves and second negative control encoding for the entire GFP were constructed under control of the same constitutive promoter were used as controls. In order to assess the recombination of both halves, all the BCPs were lysed using sonication (link to protocol) (Figure 14).
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                            </p>
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                            <div class "figure-fullwidth">
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                                <div class="figure-center-imgagebox">
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                                     <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/7/73/T--Wageningen_UR--Results_Quorum_Sensing_Figure_14.png" <div class="figure-center-caption">
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                                    <b>Figure 14:</b> Assessment of a bimolecular fluorescence complementation experiment triggered by sonication and performed with two BCPs. BCP1 encoding for the amino terminal half of GFP with leucine zippers (nGFP-LZ) and BCP2 encoding for the carboxi terminal half of GFP with leucine zippers (cGFP-LZ).Different volume ratios of BCPs 1 and 2 were used in the experiment.
+
  
 +
                                    <!-- Modal Caption (Image Text) -->
 +
                                    <div class="caption"><b>Figure 4:</b> Multiple sequence alignment of 3 random colonies after infection of fresh XL1-blue cells with the WT-IgG phages. Sequences align and proves the phagemid is preserved correctly. 
 +
                                    </div>
 
                                 </div>
 
                                 </div>
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                            </div>
 
  
−
                            <p>However, the results showed that bimolecular fluorescence complementation was not able to take place in vitro. Even though the recombination of both GFP halves was taking place in the in vivo positive control, when the BCPs expressing each of the GFP halves were mixed and sonicated no fluorescence could be detected (<a href="https://2017.igem.org/Team:Wageningen_UR/Results/Fluorescent">Choosing the best reporter</a>). The reason underlying the incapability of recombine in vitro is unknown.
 
−
                            </p>
 
−
                            <p>As a second attempt and in order to simulate biological conditions as much as possible, all the strains were co-transformed with the COLE7 lytic mechanism (BBa_K2387066). In this experiment, the lysis was induced biologically via the addition of L-arabinose (Figure 15).
 
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                            </p>
 
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                            <div class "figure-fullwidth">
 
−
                                <div class="figure-center-imgagebox">
 
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/f/f7/T--Wageningen_UR--Results_Quorum_Sensing_Figure_15.png" <div class="figure-center-caption">
 
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                                    <b>Figure 15:</b> Assessment of a bimolecular fluorescence complementation experiment triggered by a biological lysis and performed with two BCPs. BCP1 encoding for the amino terminal half of GFP with leucine zippers (nGFP-LZ) and BCP2 encoding for the carboxy terminal half of GFP with leucine zippers (cGFP-LZ). Different volume ratios of BCPs 1 and 2 were used in the experiment.
 
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−
                                </div>
 
 
                             </div>
 
                             </div>
  
−
                            <p>However, it was concluded that bimolecular fluorescence complementation neither took place in vitro when lysis was triggered biologically. The fusion of both halves took place in vivo as it can be seen for the strain expressing both halves of the GFP, suggesting that the bimolecular fluorescence complementation works but is prevented from happening when both halves are not contained within a cell.
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                             <div class="figure-center-caption">
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                            </p>
+
                                 <b>Figure 4:</b> Multiple sequence alignment of 3 random colonies after infection of fresh XL1-blue cells with the WT-IgG phages. Sequences align and prove the phagemid is preserved correctly.
−
                             <div class="Title">
+
−
                                 <h3>Proteolytic systems</h3> </div>
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                            <p>
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                            </p>
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                            <p>Because of the split-GFP system not working in vivo, an alternative system which has been reported to work in vitro was tested as well ([5]). Both systems are based on the generation of two BCPs, the first encoding for a dark quenched GFP molecule and the second one for a constitutively expressed TEV protease (BBa_K2387071).
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                            </p>
+
  
−
                            <div class="Title">
 
−
                                <h4>FRET (Förster Resonance Energy Transfer) system</h4> </div>
 
−
 
−
                            <p>In this approach, GFP was quenched by a REACh2 dark quencher (BBa_K1319004), the GFP quenching process takes place in this system via a FRET system. The REACh2 quenching protein (BBa_K1319002) fused to GFP reduces the observed fluorescence of GFP. This protein is a non-fluorescent but still absorbent version of EYFP [6]. GFP and REACh2 are fused with a linker (BBa_K1319016) which includes a specific TEV protease (BBa_K1319004) cleavage site. The fused protein brings GFP and REACh2 in proximity to each other which allows both GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is absorbed by REACh2 leading to a reduction in the GFP fluorescence.
 
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−
                            </p>
 
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                            <div class "figure-fullwidth">
 
−
                                <div class="figure-center-imgagebox">
 
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                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/0/04/T--Wageningen_UR--Results_Quorum_Sensing_Figure_16.png" <div class="figure-center-caption">
 
−
                                    <b>Figure 16:</b> Gene sequence depicting the GFP quenched by REACH2 construct.
 
−
 
−
                                </div>
 
 
                             </div>
 
                             </div>
 +
                        </div>
  
−
<div class="Title"><h4>Hydrophobic Quenching System</h4> </div>
+
<!HERE>
  
−
                            <p>In this second approach, the GFP molecule is quenched by a hydrophobic peptide from a mutated version of the M2 fragment of Influenza virus (Figure 17) and a second one able to constitutively express the TEV protease. The GFP is completely quenched by the binding of the hydrophobic peptide which tetramerizes the GFP disabling the maturation of the chromophore [3].
 
−
                            </p>
 
−
                           
 
−
<div class "figure-fullwidth">
 
−
                                <div class="figure-center-imgagebox">
 
−
                                    <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/a/a9/T--Wageningen_UR--Results_Quorum_Sensing_Figure_19.png" <div class="figure-center-caption">
 
−
                                    <b>Figure 17:</b> Gene sequence depicting the GFP quenched by M2 fragment construct.
 
  
−
                                </div>
 
−
                            </div>
 
  
−
                            <p>GFP fluorescence can be recovered after the removal of the quenching peptide leading to the generation of a robust reporter as a result of a proteolytic process. This proteolytic process is performed by the TEV protease which performs the cut of the linker fusing GFP to the hydrophobic peptide. This linker (BBa_K1319016) includes a specific TEV protease (BBa_K1319004) cleavage site.
 
−
                            </p>
 
  
−
<div class="Title"><h4>Assessment of REACH2 and mutated M2 fragment quenching systems</h4> </div>
 
  
−
<p>In both system, both BCPs were co-transformed with the COLE7 lytic mechanism to allow cell lysis after the induction of L-arabinose. Both BCPs were combined in equal concentrations and a lytic process was induced which should allow the TEV protease to cut the linker fusing both GFP to its quenching molecule (REACh2 or the mutated M2 fragment). Once both proteins are separated, the quenching interaction is prevented resulting in an increase in GFP fluorescence.
+
<div class="Title"><h3>Phage library</h3></div>
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                            </p>
+
<div class="Textbox Title">
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<p>
−
                            <p>
+
The proof-of-principle confirmed that infecting XL1-Blue cells carrying the pComb3XSS phagemid with helper phages results in recombinant phages. Therefore we proceeded with the creation of the phage library using the the affinity body library created <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Affinity_Bodies">here</a> (Figure 5).</p>
−
                                The induction of lysis via the addition of L-arabinose lead to the breakage of the bacterial cell membranes (Figure 21). Fluorescence is increased in comparison to the non cotransformed strains which are not able to lyse, suggesting that the the lytic process leads to the recovery of the quenched GFP. This can indicate that both REACH2 and the mutated M2 fragment work as expected and that its quenching effect might be inhibited after the linker binding it to the GFP is cut, allowing GFP to recover its fluorescence (Figure 17).
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−
                            </p>
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                                    <b>Figure 17:</b> Assessment of two proteolytic quenching mechanisms (GFP quenched by REACH2 or by a mutated version of M2 fragment). GFP-REACH2 stands for a strain expressing the GFP fluorophore quenched by REACH 2, GFP-M2 stands for a strain expressing the GFP fluorophore quenched by M2, TEV strain stands for a constitutively expressed TEV protease strain, TEV + GFP-REACH2 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains, TEV + GFP-M2 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains, GFP-REACH2 & COLE7 stands for a strain expressing the dark quenched GFP and co transformed with the COLE7 lytic mechanism,  GFP-M2 & COLE7 stands for a strain expressing the dark quenched GFP and co transformed with the COLE7 lytic mechanism, TEV + GFP-REACH2 & COLE7 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains both of them co transformed with the COLE7 mechanism and TEV + GFP-M2 & COLE7 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains both of them co transformed with the COLE7 mechanism.
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                            <p>In the case of GFP quenched by REACH2, the results seen do not show an increase in fluorescence when comparing the lysed cells with the non lysed ones as was seen in the mutated M2 approach (Figure 17). The reason for this is the already high fluorescence level of GFP when quenched by REACH2. The explanation for this comes from the fact that REACH2 is not preventing the maturation of the fluorophore as M2 was doing but is absorbing the emitted fluorescence of GFP. Therefore the REACH2 quencher emits fluorescence in a close emission range as GFP as it is a version of YFP. However, if the fluorescence is measured overtime, an increase in GFP fluorescence could be seen (Figure 18).
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                                    <b>Figure 18:</b> Assessment overtime of the dark GFP quenched by REACH2. GFP-REACH2 stands for a strain expressing the GFP fluorophore quenched by REACH 2, TEV strain stands for a constitutively expressed TEV protease strain, GFP-REACH2 & COLE7 stands for a strain expressing the dark quenched GFP and co transformed with the COLE7 lytic mechanism and TEV + GFP-REACH2 & COLE7 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains both of them co transformed with the COLE7 mechanism.
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<b>Figure 5:</b> Overview of the creation of the recombinant M13 phages carrying affinity body library.
  
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<p>The results seen might imply that by inducing the system with L-arabinose, both strains lyse and the linker connecting REACH2 to GFP is lost allowing GFP to recover its fluorescence. The fact that also GFP quenched by REACH2 in the absence of the TEV protease producing strains lead to fluorescence could be explained by a breakage of the linker connecting REACH2 to the GFP as a result of the lytic mechanism. (Figure 21)
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                            <p>When analyzing the GFP quenched by the mutated M2 fragment system, it was seen that both BCPs which were co transformed with the COLE7 lytic mechanism to allow bacterial cell membrane lysis after the induction of L-arabinose. Both BCPs were combined in equal concentrations and a lytic process was induced which theoretically allowed the TEV protease to cut the linker fusing both GFP and the hydrophobic peptide tetramerizing GFP chromophore. The GFP fluorescence can be fully restored leading to a strong and robust output fluorescence signal after the linker is cut.
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                            </p>
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<p>The induction of lysis via the addition of L-arabinose lead to the breakage of the bacterial cell membranes (Figure 21). Fluorescence is increased in comparison to the non cotransformed strains which are not able to lyse, suggesting that the the lytic process leads to the recovery of the quenched GFP. This can indicate that the mutated M2 fragment works as expected and that its hydrophobic quenching effect might be inhibited after the linker binding it to the GFP is cut, allowing GFP to recover its fluorescence. The results seen might imply that by inducing the system with L-arabinose, both strains lyse and the linker connecting M2 to GFP is lost allowing GFP to recover its fluorescence. The fact that also GFP quenched by M2 in the absence of the TEV protease producing strains lead to fluorescence could be explained by a breakage of the linker connecting M2 to the GFP as a result of the lytic mechanism (Figure 17).
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                            <section id="LM">
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                            <div class="Title">
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                                <h1>Lytic mechanisms</h1> </div>
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                            <p>Four different lytic mechanisms were designed to be triggered by the antigen, although for the ease of testing in this project L-arabinose is used to induce the signal in all constructs. In order to assess and evaluate these mechanisms, they were expressed under the inducible pBAD promoter (BBa_I0500) in two different backbones: a high copy number backbone (<a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a>) and a low copy number backbone (<a href="http://parts.igem.org/Part:pSB4K5">pSB4K5</a>).
 
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                            <p>Inducible versions of Colicin E7 (BBa_K2387066), T4 Holin (BBa_K2387068), T4 Endolysin (BBa_K2387069) and a double lytic mechanism (T4 Holin + T4 Endolysin) (BBa_K2387079) were tested in E. coli DH5α (Figure 20). Instead of directly quantifying cell lysis, the optical density at 600 nm was used.
 
  
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                                    <b>Figure 21:</b> Graphical representation of 4 lytic mechanisms layout.
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                            <p>Endolysins are phage-encoded peptidoglycan hydrolases (PGHs) which are used by bacteriophages to enzymatically degrade the peptidoglycan (PG) layer of the host bacterium from the inside at the end of their lytic cycle. By breaking the PG layer, an osmotic lysis can be favoured leading to cell death. However, host cell lysis is strictly regulated and controlled by the help and action of T4 Holin (BBa_K112000). T4 Holin constitutes a second component of a lysis cassette of phages. Holins oligomerize and create holes in the cytoplasmic membrane allowing the cytoplasm accumulated endolysins to degrade PG substrate [2].
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                            </p>
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                            <p>From the assessment it was concluded that T4 Holin and T4 Endolysin + T4 Holin were successfully achieving bacterial cell lysis.T4 Endolysin alone was not able to trigger the lysis of bacterial cells. However, when combined with T4 Holin, the lytic effect achieved was even higher than the one achieved with T4 Holin alone (Figure 21). The observed lytic function of T4 Endolysin when acting together with T4 Holin in the double lysis gene cassette is understandable as it requires the action of T4 Holin to make the initial holes in the membrane [7]. T4 Holin starts the membrane degradation process and after holes are made in the membrane, T4 Endolysin is able to further increase bacterial membrane lysis (Figure 21).
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                                    <b>Figure 22:</b> Assessment of four lytic mechanisms (Colicin E7 system, T4 Endolysin, T4 Holin and T4 Endolysin + T4 Holin cassette) in two backbones (pSB1C3 and pSB4K5). All the mechanisms are expressed under the pBAD promoter and therefore all the mechanisms were induced at different arabinose concentrations. The figure above depicts the OD after 20 hours of arabinose induction.
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<p>An SOS response operon regulates the expression of ColE7. This SOS response operon is formed by three genes (ceaE7, ceiE7 and celE7) whose products are ColE7, ImE7 and LysE7, respectively (Chak, Kuo, Lu, & James, 1991). ColE7 can be inhibited by complexing with the immunity protein ImE7. The inducible COLE7 system (BBa_K2387066) codes for an L-arabinose inducing lysis gene (BBa_K117000) encodes for the lysis protein of 4872 Da in molecular weight in those colicin-producing strains of bacteria. Once this gene becomes activated, two main functions of Colicin E7 system are covered by the lysis protein. The activation of the gene, leads to the partial lysis of the host cell membrane. This first function leads then to the lysis of host cell, uncovering the intracellular environment of the cell and thus allowing for ColE7 proteins free diffusion. Moreover, the lysis gene also removes the immunity protein from colicin protein, resulting in the activation of the endonuclease activity of colicin protein. In this case, the detachment of ColE7 from Immunity protein is triggered, activating this the ColE7 endonuclease activity.
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</p>
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<p>The BBa_K11700 codes for the lysis gene but not for the ColE7 or ImE7 proteins suggesting this that a specific E. coli strain (able to synthesize colicin) might be required. However, it was hypothesized that the only expression of E7 lysis gene (BBa_K117000) by E.coli DH5α , will cause partially lysis. The results indicated that E7 lysis protein might also have function in noncolicin-secreted strain of E.coli as the lysis was fully achieved after inducing the system (Figure 21) with L-arabinose (BBa_K2387066). Future approaches could study the differences in the lytic effect by comparing the lysis achieved when inserting the pK317 plasmid which contains the entire cole7 system and the inducible COLE7 system (BBa_K2387066).
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</p>
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<p>Based on the results, it was concluded that already low inductions of L-arabinose are able to induce a complete lysis of the bacterial cells (Figure 21). Either COLE7 system, T4 Endolysin + T4 Holin system or T4 Holin system alone could be implemented in the Robust Mantis module (Figure 3). Based on the simplicity of the Inducible COLE7 system (BBa_K2387066) which only requires one gene to work compared with the double lytic casette (BBa_K2387079), this system (BBa_K2387066) was the chosen one when co transforming strains in the proteolytic quenching experiments and the bimolecular fluorescence recombination.
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<section id="LM">
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                            <div class="Title">
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                                <h1>References</h1> </div>
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</section>
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<p>[1] M. Welvaert and Y. Rosseel, “On the definition of signal-to-noise ratio and contrast-to-noise ratio for FMRI data.,” PLoS One, vol. 8, no. 11, p. e77089, 2013.</p>
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<p>[2] W.-L. Ng and B. L. Bassler, “Bacterial quorum-sensing network architectures.,” Annu. Rev. Genet., vol. 43, pp. 197–222, 2009.</p>
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<p>[3] “Characterization of a quorum sensing device for synthetic biology design: Experimental and modeling validation,” Chem. Eng. Sci., vol. 103, pp. 91–99, Nov. 2013.</p>
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<p>[4] B. Pearson, K. H. Lau, W. DeLoache, P. Penumetcha, V. G. Rinker, A. Allen, R. Cool, E. Feeney, J. Igo, T. T. Eckdahl, X. Zhu, M. Parra, K. Muscalino, A. Lewis, A. Gordon, K. Davis, J. Barron, K. Roland, J. L. Poet, L. J. Heyer, A. Malcolm,Campbell, and A. M. Campbell, “Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter,” Interdiscip. Bio Cent., vol. 3, no. 3, pp. 1–8, Jul. 2011.</p>
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<p>[5] S. B. Nicholls, J. Chu, G. Abbruzzese, K. D. Tremblay, and J. A. Hardy, “Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity,” J. Biol. Chem., vol. 286, no. 28, pp. 24977–24986, Jul. 2011.</p>
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<p>[6] S. Ganesan, S. M. Ameer-Beg, T. T. C. Ng, B. Vojnovic, and F. S. Wouters, “A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP.,” Proc. Natl. Acad. Sci. U. S. A., vol. 103, no. 11, pp. 4089–94, Mar. 2006.</p>
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<p>[7] L. H. Burch, L. Zhang, F. G. Chao, H. Xu, and J. W. Drake, “The bacteriophage T4 rapid-lysis genes and their mutational proclivities.,” J. Bacteriol., vol. 193, no. 14, pp. 3537–45, Jul. 2011.</p>
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                                <b>Figure 1:</b> Caption of figure 1.
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                                        The ligation of the WT affinity body and library into the pComb3XSS vector has been described here. After ligation, the mixture was electroporated into XL1-Blue <i>E. coli</i> cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture, 250 mL of fresh medium was added and grown in 1L flasks until an OD<sub>600</sub> of ~0.6 was reached. VCSM13 helper phages were added at an MOI of 20 and incubated at 37°C without shaking. After incubation, the medium was changed to a 2xYT medium supplemented with 1% (w/v) glucose, 100 ug/mL ampicillin, 50 ug/mL kanamycin and 0.25mM IPTG and incubated overnight at 30°C. The next day the phages were precipitated and a titration was performed to assess infectivity. </p>
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<p>The resulting recombinant phage library has an infective titer of 5.7*10<sup>15</sup> CFU/mL (colony forming units per mL). This library can then be used to screen for the appropriate affibody against any antigen.
  
 
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                                                <b>Figure 2:</b> Caption of figure 2.
 
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                            <h2>Title 2</h2> </div>
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Antigens were produced for CHIKV, Zika virus (ZIKV) and Mayaro virus (MAYV) in the form of Virus-Like Particles (VLPs). Strep-tagged surface proteins were produced for CHIKV and HAT. In order to purify the VLPs and attach them to beads, antigens for the specific virus particles can be linked to protein-A resin beads, washed, blocked and used for phage display. However, due to the cost of the antigens and required resin beads, we chose to continue with only the strep-tagged surface proteins, attached to strep-tactin sepharose beads. </p>
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<p>The phage library was loaded on the beads, washed and then eluted. The eluted phage was then amplified and used again for a second and third round of biopanning, increasing the stringency of the wash with each step. In the last step, the eluted phage was used to infect XL1-Blue cells. Colony PCR is then used to amplify the phagemids and sent for sequencing to confirm the sequence of the best binding affinity body (Figure 6).</p></div>
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                                    <b>Figure 3:</b> Caption of figure 3.
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<b>Figure 6:</b> Overview of phage display biopanning including the library creation.
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The beads containing the CHIKV and HAT antigens were washed and subsequently blocked using PBS-TWEEN® with gelatin. Milk or BSA was not used for blocking due to the possible presence of immunoglobulins which is the natural target of our wild type affinity body. Next, the phage library was added and after incubation at room temperature, the unbound phages were washed away using PBS-TWEEN®. The phages that were still bound to the beads were eluted, titrated and amplified. After amplification, the phages were titrated again and the biopanning was repeated. Stringency was increased with each round of panning by increasing the TWEEN® concentration during the washing step. The TWEEN® concentrations in round 1, 2 and 3 were 0.1%, 0.3% and 0.6% respectively. The exact protocols used can be found <a href="https://2017.igem.org/Team:Wageningen_UR/Notebook/Protocols" target="_blank">here</a>.
  
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<p>
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After each round of biopanning, the eluted and amplified phages were titrated using the Colony Forming Units (CFU) protocol. In order to perform CFU, dilutions of the phage solution were made in 20 mM Tris-HCl from 10<sup>-8</sup> to 10<sup>-13</sup>. These phage dilutions were then plated together with XL1 cells. When a phage with our construct infects a healthy XL1 <i>E. coli</i>, it transfers the ampicillin (Amp) resistance gene that is present on the pComb3XSS vector. Therefore, the selection is possible on Amp plates.
 +
</p>
 +
<p>
 +
We found that during the first rounds of CFU titration, all plates showed similar amounts of colonies on each plate. As we previously had a contamination in our XL1 stock with an unknown Amp resistant bacteria we chose to repeat the CFU titration with fresh cells. However, due to time constraints, we had to continue with the biopanning rounds without knowing the titers of the elution and amplification fractions. During the second CFU titration, the results were similar to the first round. All plates showed a similar amount of colonies. We ruled out a contamination with an Amp-resistant bacteria. Taking into consideration that we loaded the initial biopanning round with 10<sup>15</sup> phages we hypothesized that we had to extend the dilution series from 10<sup>8</sup>-10<sup>13</sup> to 10<sup>10</sup>- 10<sup>20</sup>. The next day, the plates showed again the same results, with similar amounts of colonies on every plate. As the dilutions are extremely high it is very unlikely that these colonies represent the amount of phages in our fractions.
 +
</p>
 +
<p>
 +
As a troubleshooting step we performed a titration round with all buffers used in the protocol without adding our phage samples. Here, we found that we had a major contamination in one of the buffers. Due to the approaching deadlines, we could not repeat the entire biopanning experiment. The results are therefore inconclusive.
 +
</p>
 +
<p>
 +
What we would have expected is an enrichment after each biopanning step with the use of the titration data. After the final biopanning step we would expect a reasonable amount of candidates with high affinity for our antigens. In our phagemid, there is an amber stop codon present at the fusion site between the G3P protein and our affinity bodies. Using an amber stop codon non-suppressor strain our affinity body candidates could be expressed. As there is also a 6xHis-tag and HA-tag present on the affinity bodies, they could be purified and used for binding assays.
 +
</p>
 +
<p>
 +
Once the affinity body candidates were purified, a ForteBio Octet® could be used to determine the full kinetic profile of the biomolecular interactions of the candidate group to the purified antigens. These affinity measurements offer a real-time interaction of the molecular kinetics, better than the estimations which can be made by other methods such as ELISA. We could compare these kinetics to the antibodies commercially available for the antigens in order to select the best binding affinity body, candidate. The best affinity body that could be selected is used in the Mantis diagnostic.
 +
</p>
  
−
                    </section>
+
<p>
 +
Even though we were unable to select for an affinity body against the tested viruses, the library we have developed and the presented methodology will be of use for future iGEM teams and external researchers interested in creating diagnostics against particular pathogens.
 +
</p></div>
 +
</section>
 
                     <div class="Textbox Citations">
 
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References
 
References
−
</h3>
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</h2>
  
 
                         <ol>
 
                         <ol>
−
                             <li>Steens, Jurre. "Fantastic Phages and where to find them" Best journal ever 70.1 (2017): 2-11.</li>
+
                             <li>Paschke, Matthias. "Phage display systems and their applications." <i>Applied microbiology and biotechnology</i> 70.1 (2006): 2-11.</li>
  
 
                         </ol>
 
                         </ol>

Latest revision as of 22:34, 1 November 2017

Phage display

Phage Display

Phage display is a powerful laboratory technique used to characterize protein-ligand interactions and functions. Phage display can be used to select for proteins with the desired properties from large libraries. Using M13 phage display, we can select for the best affinity body against Chikungunya virus (CHIKV) and Human African Trypanosoma (HAT) antigens using the affinity body library we created. These affinity bodies can then be used to make the Mantis cells detect these diseases.


Generation of recombinant M13 phages

In M13 phage display, the DNA sequence encoding the protein of interest (in our case the affinity body library) is fused to a gene which codes for the M13 phage G3P minor coat protein. After correct assembly, the phage will display the affinity body on its surface, linking the genotype to the phenotype. To fuse the affinity body to the phage coat protein, two distinct strategies can be applied. The first method is to directly clone the affinity body sequence into the coding sequence of the coat protein in the phage genome. The other option is to use a small vector (phagemid vector) encoding for part of the phage G3P minor coat protein. The protein of interest can be cloned into the phagemid vector and will be expressed as a fusion with G3P (p III in Figure 1). The advantage of using phagemids is the ease of cloning into the smaller vector, as efficiency is important during library cloning. Furthermore, using a phagemid results in a monovalent display of the protein of interest compared to a polyvalent display with the phage vector [1]. This is an important difference since a polyvalent display could lead to the selection of weak-binding variants while this is circumvented with a monovalent display. However, the phagemid vector approach must be used in combination with a helper phage. In our case, we use the VCSM13 helper phage which is an M13-based phage and has a defective origin of replication. The phagemid vector has a working origin of replication and this leads to the preference for packing the phagemid vector in the phage particles. Taking this in consideration, we chose the phagemid vector approach.

Figure 1: Schematic picture M13 phage, made by iGEM Edinburgh 2011.

Proof-of-principle

Before generating a phage library we performed a proof-of-principle with the wild type (WT) affinity body sequence. This affinity body specifically binds Immunoglobulin G (IgG) antibodies. The WT affinity body sequence was ligated into the pComb3XSS phagemid. XL1-Blue cells were transformed with the phagemid and subsequently used to create recombinant phages containing the WT affinity body (Figure 2). The phages were amplified and titrated. In order to see whether the phagemid was preserved, we infected fresh XL1-Blue cells with the recombinant phages. The infected cell suspension was plated on LB agar plates with the appropriate antibiotic. Several colonies were picked, grown, plasmids were isolated and sent for sequencing.

Figure 2: Overview of the creation of the proof-of-principle recombinant M13 phages carrying the IgG affinity body sequence.

After ligation of the wild type affinity body sequence into the pComb3XSS phagemid, the mixture was electroporated into XL1-Blue Escherichia coli (E. coli) cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture, 250 mL of fresh medium was added and grown in 1L flasks until an OD600 of ~0.6 was reached. VCSM13 helper phages were added at an MOI of 20 and incubated at 37°C without shaking. After incubation, the medium was changed to a 2xYT medium supplemented with 1% (w/v) glucose, 100 ug/mL ampicillin, 50 ug/mL kanamycin and 0.25mM IPTG and incubated overnight at 30°C. The next day the phages were precipitated and a titration was performed to assess infectivity.

The resulting recombinant phage with wild type affinity body has an infective titer of 9.6*1011 CFU/mL (colony forming units per mL). We visualized these phages using transmission electron microscopy (TEM) (Figure 3).

Figure 3: Transmission Electron Microscopy (TEM) images of recombinant M13 phage expressing WT affinity body. Stained using phosphotungstic acid (PTA). Pictures were taken using JEOL JEM1400.
Figure 4: Multiple sequence alignment of 3 random colonies after infection of fresh XL1-blue cells with the WT-IgG phages. Sequences align and prove the phagemid is preserved correctly.

Phage library

The proof-of-principle confirmed that infecting XL1-Blue cells carrying the pComb3XSS phagemid with helper phages results in recombinant phages. Therefore we proceeded with the creation of the phage library using the the affinity body library created here (Figure 5).

Figure 5: Overview of the creation of the recombinant M13 phages carrying affinity body library.

The ligation of the WT affinity body and library into the pComb3XSS vector has been described here. After ligation, the mixture was electroporated into XL1-Blue E. coli cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture, 250 mL of fresh medium was added and grown in 1L flasks until an OD600 of ~0.6 was reached. VCSM13 helper phages were added at an MOI of 20 and incubated at 37°C without shaking. After incubation, the medium was changed to a 2xYT medium supplemented with 1% (w/v) glucose, 100 ug/mL ampicillin, 50 ug/mL kanamycin and 0.25mM IPTG and incubated overnight at 30°C. The next day the phages were precipitated and a titration was performed to assess infectivity.

The resulting recombinant phage library has an infective titer of 5.7*1015 CFU/mL (colony forming units per mL). This library can then be used to screen for the appropriate affibody against any antigen.


Selection

Antigens were produced for CHIKV, Zika virus (ZIKV) and Mayaro virus (MAYV) in the form of Virus-Like Particles (VLPs). Strep-tagged surface proteins were produced for CHIKV and HAT. In order to purify the VLPs and attach them to beads, antigens for the specific virus particles can be linked to protein-A resin beads, washed, blocked and used for phage display. However, due to the cost of the antigens and required resin beads, we chose to continue with only the strep-tagged surface proteins, attached to strep-tactin sepharose beads.

The phage library was loaded on the beads, washed and then eluted. The eluted phage was then amplified and used again for a second and third round of biopanning, increasing the stringency of the wash with each step. In the last step, the eluted phage was used to infect XL1-Blue cells. Colony PCR is then used to amplify the phagemids and sent for sequencing to confirm the sequence of the best binding affinity body (Figure 6).

Figure 6: Overview of phage display biopanning including the library creation.

The beads containing the CHIKV and HAT antigens were washed and subsequently blocked using PBS-TWEEN® with gelatin. Milk or BSA was not used for blocking due to the possible presence of immunoglobulins which is the natural target of our wild type affinity body. Next, the phage library was added and after incubation at room temperature, the unbound phages were washed away using PBS-TWEEN®. The phages that were still bound to the beads were eluted, titrated and amplified. After amplification, the phages were titrated again and the biopanning was repeated. Stringency was increased with each round of panning by increasing the TWEEN® concentration during the washing step. The TWEEN® concentrations in round 1, 2 and 3 were 0.1%, 0.3% and 0.6% respectively. The exact protocols used can be found here.

Results

After each round of biopanning, the eluted and amplified phages were titrated using the Colony Forming Units (CFU) protocol. In order to perform CFU, dilutions of the phage solution were made in 20 mM Tris-HCl from 10-8 to 10-13. These phage dilutions were then plated together with XL1 cells. When a phage with our construct infects a healthy XL1 E. coli, it transfers the ampicillin (Amp) resistance gene that is present on the pComb3XSS vector. Therefore, the selection is possible on Amp plates.

We found that during the first rounds of CFU titration, all plates showed similar amounts of colonies on each plate. As we previously had a contamination in our XL1 stock with an unknown Amp resistant bacteria we chose to repeat the CFU titration with fresh cells. However, due to time constraints, we had to continue with the biopanning rounds without knowing the titers of the elution and amplification fractions. During the second CFU titration, the results were similar to the first round. All plates showed a similar amount of colonies. We ruled out a contamination with an Amp-resistant bacteria. Taking into consideration that we loaded the initial biopanning round with 1015 phages we hypothesized that we had to extend the dilution series from 108-1013 to 1010- 1020. The next day, the plates showed again the same results, with similar amounts of colonies on every plate. As the dilutions are extremely high it is very unlikely that these colonies represent the amount of phages in our fractions.

As a troubleshooting step we performed a titration round with all buffers used in the protocol without adding our phage samples. Here, we found that we had a major contamination in one of the buffers. Due to the approaching deadlines, we could not repeat the entire biopanning experiment. The results are therefore inconclusive.

What we would have expected is an enrichment after each biopanning step with the use of the titration data. After the final biopanning step we would expect a reasonable amount of candidates with high affinity for our antigens. In our phagemid, there is an amber stop codon present at the fusion site between the G3P protein and our affinity bodies. Using an amber stop codon non-suppressor strain our affinity body candidates could be expressed. As there is also a 6xHis-tag and HA-tag present on the affinity bodies, they could be purified and used for binding assays.

Once the affinity body candidates were purified, a ForteBio Octet® could be used to determine the full kinetic profile of the biomolecular interactions of the candidate group to the purified antigens. These affinity measurements offer a real-time interaction of the molecular kinetics, better than the estimations which can be made by other methods such as ELISA. We could compare these kinetics to the antibodies commercially available for the antigens in order to select the best binding affinity body, candidate. The best affinity body that could be selected is used in the Mantis diagnostic.

Even though we were unable to select for an affinity body against the tested viruses, the library we have developed and the presented methodology will be of use for future iGEM teams and external researchers interested in creating diagnostics against particular pathogens.

References

  1. Paschke, Matthias. "Phage display systems and their applications." Applied microbiology and biotechnology 70.1 (2006): 2-11.