Team:Imperial College London/Modules/Detection

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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Detection Module
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Detection Module
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|'''The Detection Module: Based on a Cleavable Surface Protein'''
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| Having discussed with experts if ''Schistosoma'' cercaria would be the most useful lifecycle stage of ''Schistosoma'' to detect, we developed a surface protein that allows detection of ''Schistosoma'' in water. The key motivation for targeting the parasite larvae, called cercaria, is prevention of the disease altogether. The cercaria are often found in water, but their location as well as time of their appearance is unpredictable, putting the population in endemic areas at risk of infection. By providing a fast and reliable test, non-profit organisations  would be able to regularly check the water for the presence of cercaria and warn people of the danger. Furthermore we are exploiting an essential aspect of the cercarias’ biochemistry without which they are unable to progress in their life cycle. To learn more about the parasite life cycle follow this link to our section on [https://2010.igem.org/Team:Imperial_College_London/Schistosoma ''' ''Schistosoma'' '''].
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|<div ALIGN=CENTER>
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|-
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|[[Image:IC_Detection2.JPG|350px]]
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|-
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|Overview of the detection module.
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|}
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</div>
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__NOTOC__
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|-
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|colspan="2"|
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'''Design'''
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In order to set off the fast response module in the presence of the schistosoma cercaria, we  designed a protein that carries our signal peptide ComC out of the cell where the protease has access to it. The protease can then proceed to cleave ComC off the protein, allowing quorum sensing via the the ComCDE system to take place. One big problem we have to overcome is the cell wall that will obstruct the protease’s access to the protein carrying the signal peptide.  
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We make use of a cercarial protease, called elastase [http://www.uniprot.org/uniprot/P12546 '''(UniProt,'''] [http://merops.sanger.ac.uk/cgi-bin/pepsum?id=S01.144 '''MEROPS)'''], which is necessary for cercaria to penetrate human. The surface our ''B. subtilis'' is coated with a surface protein which is design to release a sequestered quorum sensing signal upon cleavage by the elastase.
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The Detection module consists of a cell wall anchor, a signal peptide called ComC, and a linker that connects the two and is specifically designed to be cleaved by the protease we want to detect.
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|-
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Candidate Proteins: CwlB and CwlC
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|
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'''The AIP activates signal transduction'''
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'''What do we have to consider?'''
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We used the autoinducing peptide (AIP) from the ComCDE system of ''S. Pneumoniae'' because it is linear and lacks posttranslational modifications. Using a system foreign to ''B. subtilis'' also reduces false activation and the noise our system has to deal with, making it more robust. Because of the nature of the AIP receptor ComD, in order to activate signal transduction the AIP must be in the cleaved form, i.e. not attached to the surface protein anymore. This adds an additional level of robustness to the system.
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|-
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|[[Image:IC_Detection.JPG|350px]]
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|-
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|The detection module consists of three section: Cell Wall anchor, Linker and AIP
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|}
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</div>
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|-
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'''The Linker is a substrate for the cercarial elastase'''
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There are several restrictions to the choice of surface proteins we have to consider. First of all the use of peptides as quorum sensing signals is mainly restricted to gram positive bacteria such as B. subtilis. Additionally we had to overcome the problem of false positive activation of our system that could be caused when ComC comes in close proximity to the membrane bound receptor ComD. Therefore we decided to choose a cell wall bound protein rather than a membrane bound proteins as originally planned. Finally, because of the nature of the protease and it cleavage specificty we have to attach ComC to the C-terminus of our protein in order to preserve the original ComC sequence fter cleavge has occurred.
+
The AIP is connected to a cell wall anchor via a linker. It is this linker that confers specificity to our surface protein which is to be cleaved by the elastase. The elastase itself recognises a four amino acid sequence, which we included in our linkers. In order to test the optimal design for the linker, we created 6 different linkers which vary in length and flexibility. Furthermore we created a do-it-yourself [https://2010.igem.org/Team:Imperial_College_London/Software_Tool '''Software Tool'''] which enables the custom design of the surface protein sequence to include a protease cleavage site of choice. This allows the detection of any protease by incorporating such new surface proteins into our system.
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'''What are our candidate genes? '''
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'''Detection of Parasite proteases'''
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The two most promising proteins we have identified are CwlB and CwlC, both of which found naturally on the cell wall of Bacillus subtilis. CwlB, also known as lytC, is a major autolysin involved in cell wall turn-over and more specifically responsible for breaking down peptidoglycans (N-acetylmuramoyl-L-alanine amidase). Cells producing lytC are uniformly coated with lytC after the middle of the exponential phase. CwlC is also a hydrolase involved in peptidoglycan break down, however it role is less well understood although it too acts as N-acetylmuramoyl-L-alanine amidase playing an important role in mother-cell lysis during sporulation Yamamoto et al.. Both proteins have in common that they use a cell wall binding domain (CWB) consisting of several repeats to bind non-covalently to the bacterial cell wall using ionic interactions. The CWBs themselves do not show sequence homology and are of very different sizes, but as they function in a similar manner, both can be eluded from the cell wall by solution with a high osmolarity. Mishima ''et al''.  
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In order to detect ''Schistosoma'' in water they have to release their elastase. This does not usually occur in the absence of a stimulus however it is easy to trigger this behaviour ''in vivo''. Upon detection of human (or mouse) skin lipids and temperatures close to 37°C, the invasive behaviour is triggered. Several proteases are released from pre- and postacetabular glands or the cercaria at the leading edge of the invading parasite. While multiple enzymes are released, only one protease activity was found to be necessary for invasion in ''S. mansoni'', and ''S. haematobium'' [http://books.google.co.uk/books?id=JL7qs-O3z1MC&pg=PA299&lpg=PA299&dq=advances+in+parasitology+london&source=bl&ots=b8mvkz6zyC&sig=t7Pwy0egEDQ9dW-GIZEK7Pb6xxs&hl=en&ei=n05ATKLnJsqNjAePyOAG&sa=X&oi=book_result&ct=result&resnum=3&ved=0CCEQ6AEwAg#v=onepage&q=advances%20in%20parasitology%20london&f=false (Kasny ''et al.'' 2009)]: Schistosoma elastase 2a and b (SmCE-1a/b) [http://www.jbc.org/content/277/27/24618.abstract (Salter ''et al.'' 2002)]. SmCE are trypsin family serine proteases the specificity of which has been investigated by various studies. It appears that the following cleavage sites are preferred:
 +
* P4 - S,T
 +
* P3 - S,W,Y
 +
* P2 - P
 +
* P1 - L
 +
Subtle differences exist between SmCE-2a and b as far as their P4 and P3 sites are concerned. Cleavage kinetics were determined for four different sites, P4-P1: SWPL, TWPL, RWPL, RRPL with R previously determined as unfavourable at P4 and P3. For P4 an 11-fold difference in activity was determined between favourable S/T and R, while a 3-fold difference was determined for P3 W to R [http://www.jbc.org/content/277/27/24618.abstract (Salter ''et al.'' 2002)]. The most favourable sequence would therefore be SWPL.
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<html>
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<table width="300px" border="0" align=center>
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<tr>
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<td style="background-color:#FFCC66;height:50px;width:50;text-align:center"><b>Uniprot accession</b></td>
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<td style="background-color:#FFCC66;height:50px;width:50;text-align:center"><b>MEROPS ID</b></td>
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<td style="background-color:#FFCC66;height:50px;width:50;text-align:center"><b>Clan</b></td>
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<td style="background-color:#FFCC66;height:50px;width:50;text-align:center"><b>Family</b></td>
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<td style="background-color:#FFCC66;height:50px;width:50;text-align:center"><b>pH optimum</b></td>
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<td style="background-color:#FFCC66;height:50px;width:50;text-align:center"><b>Molecular weight (practical/theoretical)</b></td>
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</tr>
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<tr>
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<td style="background-color:#eeeeee;height:50px;width:50 px;text-align:center;">P12546</td>
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<td style="background-color:#eeeeee;height:50px;width:50 px;text-align:center;">S01.144</td>
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<td style="background-color:#eeeeee;height:50px;width:50 px;text-align:center;">PA(S)</td>
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<td style="background-color:#eeeeee;height:50px;width:50 px;text-align:center;">S1</td>
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<td style="background-color:#eeeeee;height:50px;width:50 px;text-align:center;">4-10.5</td>
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<td style="background-color:#eeeeee;height:50px;width:50 px;text-align:center;">25/29 kDA</td>
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</tr>
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</table>
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</html>
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|-
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|'''The cell wall binding domain anchors our module to the cell surface'''
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'''Why should they work? '''
 
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Several papers, especially those by Kobayashi et al., have demonstrated that protein domains fused the cell wall binding domains of lytC and cwlC are expressed correctly and localized on the surface of the cell whilst maintaining their catalytic ability.
 
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Tsuchiya ''et al'', 1999
 
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Kobayashi ''et al'', 2000
 
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Kobayashi ''et al'', 2002
 
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Yamamoto ''et al'', 2003
 
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Additionally, since we do not intend to knock out the original gene from the B. subtilis strain we will not interfere with it normal expression, avoiding expression at the wrong time or overexpression, and since we aim to remove the catalytic domain from CwlB or CwlC our fusion protein should lose their cell wall turn-over functions and only act as cell wall anchors.
 
 +
The AIP-linker peptide is  anchored to the cell wall by LytC, a protein native to ''B. subtilis'. The process of anchoring the recombinant protein to the cell wall helps to increase the efficiency of detection. It uses electrostatic interactions of several cell wall binding domain repeats to non-covalently bind to the cell wall, and expose our detection apparatus to the elastase. We chose it over other options, such as covalently bound proteins that use the Gram positive sortase system, membrane bound proteins or other non-covalently bound cell wall proteins for several reasons. Firstly, we wanted to minimise the chance of false activation of signal transduction, so we eliminated cell membrane bound proteins, as these would bring the AIP in much greater proximity to the receptor ComD. Whilst the sortase system is well understood for some bacteria such as ''Staphylococcus aureus'', it has not been extensively studies in ''B. subtilis'' and differences in the peptidoglycan structure meant it would have been much more error prone to use this system in our chassis. Thus the most viable option were non-covalently bound surface proteins, and LytC the best candidate within this group as a result of several studies, most notably by Kobayashi et al. (2003), that had previously used it to anchor catalytic domains, as well as short peptides, to the cell wall of ''B. subtilis''.
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'''What is the structure of our construct?'''
 
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We decided to use the CWB of lytC as the basis of our detection module. This decision was based on the fact that more research and recombinant testing of LytC has been done than on CwlC. The gram positive bacteria automatically recognise CWB as such an transport the protein out of the cell where it attaches non-covalently to the cell wall. There is the option to reduce the size of the CWB as it consists of several repeats all but one of which could be removed, however this has not been tested before and thus might not work. Adding the catalytic domain of the protein might help correct localization, but studies have shown that it is not necessary and furthermore ectopic expression of protein at the wrong time might be harmful to the organism. Therefore we have decided to use the cell wall binding domain with the original number of repeats but remove the catalytic domain completely.
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[http://www3.interscience.wiley.com/journal/119078114/abstract?CRETRY=1&SRETRY=0 Tsuchiya et al. 1999]
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Next we added a linker to connect the CWB to the signal peptide ComC. The linker has to fulfil various criteria: It has to contain a specific sequence, recognised by the protease we want to detect, in our case cercarial elastase. Furthermore it has to stretch the ComC far enough away from the CWB to allow access of the protease to the recognition sequence. Additionally the linker should not be too flexible as to allow contact of ComC with its receptor ComC as this might set off false positives. This linker could either be a simple glycine repeat or, like Yamamoto et al. suggested, this sequence: SRGSRA (for lytC). However we opted for two linkers suggested by another paper, one helical and one flexible, as well as a pure glycin linker.
+
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The third component of the detection module is the signal peptide that has to be linear to allow easy expression and avoid the need for post translational modification. The ComCDE quorum sensing system was the best candidate and well be discussed in more detail in the second module. In order to get a particularly strong signal we considered using several repeats of ComC however we ultimately decided not do so for several reasons: The cercarial elastase cleaves at the C-terminus of its recognition sequence. If several ComC molecules were attached to each other these four amino acids would remain attached to the ComC molecule and potentially interfere with recognition of ComC by its receptor ComD.
+
-
Additionally the receptor requires a concentration of only 10ng/ml of ComC for detection which, as our models indicate should be reached easily with just one ComC molecule per surface protein.
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[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSD-426YS50-D&_user=217827&_coverDate=12%2F31%2F2000&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=4764a7bdabc3649925f082f989e835f5 Kobayashi et al. 2000]
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'''How do we test for expression? '''
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[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSD-46G3T2V-4&_user=217827&_coverDate=01%2F31%2F2002&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=c807c9de3e3579777b08fdd77cd32294 Kobayashi et al. 2002]
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In order to be able to detect out protein we want to use His-tags which will only be added to test constructs but not the final module as it would most likely interfere with the module’s function. The his-tags have been added to the end of ComC, probably inhibiting correct detection of ComD by the two component system comD/E. Using a his-tag allow us to test for three different, very important things: We can lyse some of our bacterial culture and try to isolate our his-tagged protein form the lysate. Detection in this step would demonstrate successful expression of our fusion protein. Secondly we can place our bacteria in a solution with high osmolarity. As mentioned this eludes the proteins from the cell wall, which should also work for our fusion proteins. Detection of our protein in this step would demonstrate correct expression as well as localization in the cell wall. Last of all we could place the cells in medium containing the elastase or a protease with the same or very similary specificity. If we are able to pull down the his-tagged peptide then it would demonstrate correct expression, localization and that the cleavage site is accessible to the protease and ComC can be successfully cleaved off.
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[http://jb.asm.org/cgi/reprint/185/22/6666 Yamamoto et al. 2003]
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|
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{| style="width:254px;background:#e7e7e7;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;margin- top:5px;padding: 2px;" cellspacing="5";
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|-
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|[[Image:Picture1.png|250px]]
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|-
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|Crystal structure the protein LytC used to anchor our peptide to the cell wall. (EBI, 2010)
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|}
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</div>
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|-
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|colspan="2"|
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Additionally, since we do not intend to knock out the original gene from the ''B. subtilis'' strain we will not interfere with its normal expression, avoiding expression at the wrong time or over expression. Furthermore since we aim to remove the catalytic domain from LytC or CwlC, our fusion proteins should lose their cell wall turn-over functions and only act as cell wall anchors.
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|-
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|
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'''Summary and Testing'''
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'''Detection of Parasite proteases'''
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Before finalising our system we modelled the requirements for our detection module to analyse its functionality. For more information on this follow this link to our [https://2010.igem.org/Team:Imperial_College_London/Modelling/Protein_Display/Objectives '''Modelling''']. It showed that we could reach the threshold concentration of AIP needed to activate our system, 10ng/ml, quite easily, with the amount of surface protein we expect to express, as well as estimating the time needed to set off our system at a given concentration of elastase in the water.
-
In order to detect schistosoma in water they have to release their elastse. This does not usually occur in the absense of a stimulus however it is easy to trigger this behaviour in vivo. Upon detection of human (or mice) skin lipids and temperatures close to 37°C, the invasive behaviour is triggered. Several proteases are released from pre- and postacetabular glands or the cercaria at the leading edge of the invading parasite. While multiple enzymes are released, only one protease activity was found to be necessary for invasion in S. mansoni, haematobium and douthitti (Kasny ''et al'', 2009): Schistosoma elastase 2a and b (SmCE-1a/b) (Salter et al. 2002). SmCE are trypsin family serine proteases the specificity of which has been investigated by various studies. It appears that the following sites are preferred:
+
The assembly of the protein was done in several steps. We first amplified LytC out of the ''B. subtilis'' genome by PCR, simultaneously using primer extension to add optimised ribosome binding sites to it, and insert the PCR product into pSB1C3. We later ligated the protein with the promoter Pveg. Parallel to this, we ligated the 6 linker sequences ordered from Eurofins|mwg with a double terminator BOO14. We then made use of a natural ACCI site in the LytC gene, to ligate our promoter-LytC sequence with our linker-terminator sequence to create 6 different surface protein constructs, for testing in ''B. subtilis''.
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* P4 - S,T
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|<div ALIGN=CENTER>
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* P3 - S,W,Y
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{| style="width:354px;background:#e7e7e7;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;margin- top:5px;padding: 2px;" cellspacing="5";
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* P2 - P
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|-
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* P1 - L
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|[[Image:Picture2Cell_wall_with_detection_module.png|350px]]
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Subtle differences exist between SmCE-2a and b as far as their P4 and P3 sites are concerned. Cleavage kinetics were determined for four different sites, P4-P1: SWPL, TWPL, RWPL, RRPL with R previously determined as unfavourable at P4 and P3. For P4 an 11-fold difference in activity was determined between favourable S/T and R, while a 3 fold difference was determined for P3 W to R (Salter ''et al'', 2002) (see figure 1). The most favourable sequence would therefore be SWPL.
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|-
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|The detection module is non-covalently bound to the cell wall that overlies the phospholipid bilayer
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|}
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</div>
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|-
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|colspan="2"|
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In order to be able to detect out protein we will use His-tags. However these are only been added to test constructs and not the final module, as the tag would most likely interfere with the module’s function. The his-tags have been added to the C-terminus of the AIP, probably inhibiting correct detection of the AIP by the two component system comD/E. However using a his-tag allows us to test for three different, very important things: We can lyse a bacterial culture to isolate our his-tagged protein from the lysate. Detection in this step would demonstrate successful expression of our fusion protein. Secondly we can place our bacteria in a solution with high osmolarity. As mentioned this eludes the non-covalently bound proteins from the cell wall by interfering with the electrostatic interactions of the cell wall binding domain with the peptidoglycans, which should also work for our fusion proteins. Detection of our protein in this step would demonstrate expression as well as correct localisation in the cell wall. Last of all we could place the cells in medium containing the elastase or a protease with the same or very similarly specificity. If we are able to pull down the his-tagged peptide then it would demonstrate expression, localisation and that the correct cleavage site is accessible to the protease and the AIP can be successfully cleaved off. For a complete list of the BioBrick created for this module see our page on [https://2010.igem.org/Team:Imperial_College_London/Parts '''Parts'''] submitted to the registry.
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Here's a picture of the final construct:
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|-
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|[[Image:IC_Module1.JPG|500px]]
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|}
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</div>

Latest revision as of 01:08, 28 October 2010

Modules Overview | Detection | Signaling | Fast Response
Our design consists of three modules; Detection, Signaling and a Fast Response, each of which can be exchanged with other systems. We used a combination of modelling and human practices to define our specifications. Take a look at the overview page to get a feel for the outline, then head to the full module pages to find out how we did it.
Detection Module
Having discussed with experts if Schistosoma cercaria would be the most useful lifecycle stage of Schistosoma to detect, we developed a surface protein that allows detection of Schistosoma in water. The key motivation for targeting the parasite larvae, called cercaria, is prevention of the disease altogether. The cercaria are often found in water, but their location as well as time of their appearance is unpredictable, putting the population in endemic areas at risk of infection. By providing a fast and reliable test, non-profit organisations would be able to regularly check the water for the presence of cercaria and warn people of the danger. Furthermore we are exploiting an essential aspect of the cercarias’ biochemistry without which they are unable to progress in their life cycle. To learn more about the parasite life cycle follow this link to our section on Schistosoma .
IC Detection2.JPG
Overview of the detection module.

Design

We make use of a cercarial protease, called elastase [http://www.uniprot.org/uniprot/P12546 (UniProt,] [http://merops.sanger.ac.uk/cgi-bin/pepsum?id=S01.144 MEROPS)], which is necessary for cercaria to penetrate human. The surface our B. subtilis is coated with a surface protein which is design to release a sequestered quorum sensing signal upon cleavage by the elastase.

The AIP activates signal transduction

We used the autoinducing peptide (AIP) from the ComCDE system of S. Pneumoniae because it is linear and lacks posttranslational modifications. Using a system foreign to B. subtilis also reduces false activation and the noise our system has to deal with, making it more robust. Because of the nature of the AIP receptor ComD, in order to activate signal transduction the AIP must be in the cleaved form, i.e. not attached to the surface protein anymore. This adds an additional level of robustness to the system.

IC Detection.JPG
The detection module consists of three section: Cell Wall anchor, Linker and AIP

The Linker is a substrate for the cercarial elastase

The AIP is connected to a cell wall anchor via a linker. It is this linker that confers specificity to our surface protein which is to be cleaved by the elastase. The elastase itself recognises a four amino acid sequence, which we included in our linkers. In order to test the optimal design for the linker, we created 6 different linkers which vary in length and flexibility. Furthermore we created a do-it-yourself Software Tool which enables the custom design of the surface protein sequence to include a protease cleavage site of choice. This allows the detection of any protease by incorporating such new surface proteins into our system.


Detection of Parasite proteases

In order to detect Schistosoma in water they have to release their elastase. This does not usually occur in the absence of a stimulus however it is easy to trigger this behaviour in vivo. Upon detection of human (or mouse) skin lipids and temperatures close to 37°C, the invasive behaviour is triggered. Several proteases are released from pre- and postacetabular glands or the cercaria at the leading edge of the invading parasite. While multiple enzymes are released, only one protease activity was found to be necessary for invasion in S. mansoni, and S. haematobium [http://books.google.co.uk/books?id=JL7qs-O3z1MC&pg=PA299&lpg=PA299&dq=advances+in+parasitology+london&source=bl&ots=b8mvkz6zyC&sig=t7Pwy0egEDQ9dW-GIZEK7Pb6xxs&hl=en&ei=n05ATKLnJsqNjAePyOAG&sa=X&oi=book_result&ct=result&resnum=3&ved=0CCEQ6AEwAg#v=onepage&q=advances%20in%20parasitology%20london&f=false (Kasny et al. 2009)]: Schistosoma elastase 2a and b (SmCE-1a/b) [http://www.jbc.org/content/277/27/24618.abstract (Salter et al. 2002)]. SmCE are trypsin family serine proteases the specificity of which has been investigated by various studies. It appears that the following cleavage sites are preferred:

  • P4 - S,T
  • P3 - S,W,Y
  • P2 - P
  • P1 - L

Subtle differences exist between SmCE-2a and b as far as their P4 and P3 sites are concerned. Cleavage kinetics were determined for four different sites, P4-P1: SWPL, TWPL, RWPL, RRPL with R previously determined as unfavourable at P4 and P3. For P4 an 11-fold difference in activity was determined between favourable S/T and R, while a 3-fold difference was determined for P3 W to R [http://www.jbc.org/content/277/27/24618.abstract (Salter et al. 2002)]. The most favourable sequence would therefore be SWPL.

Uniprot accession MEROPS ID Clan Family pH optimum Molecular weight (practical/theoretical)
P12546 S01.144 PA(S) S1 4-10.5 25/29 kDA

The cell wall binding domain anchors our module to the cell surface


The AIP-linker peptide is anchored to the cell wall by LytC, a protein native to B. subtilis'. The process of anchoring the recombinant protein to the cell wall helps to increase the efficiency of detection. It uses electrostatic interactions of several cell wall binding domain repeats to non-covalently bind to the cell wall, and expose our detection apparatus to the elastase. We chose it over other options, such as covalently bound proteins that use the Gram positive sortase system, membrane bound proteins or other non-covalently bound cell wall proteins for several reasons. Firstly, we wanted to minimise the chance of false activation of signal transduction, so we eliminated cell membrane bound proteins, as these would bring the AIP in much greater proximity to the receptor ComD. Whilst the sortase system is well understood for some bacteria such as Staphylococcus aureus, it has not been extensively studies in B. subtilis and differences in the peptidoglycan structure meant it would have been much more error prone to use this system in our chassis. Thus the most viable option were non-covalently bound surface proteins, and LytC the best candidate within this group as a result of several studies, most notably by Kobayashi et al. (2003), that had previously used it to anchor catalytic domains, as well as short peptides, to the cell wall of B. subtilis.


[http://www3.interscience.wiley.com/journal/119078114/abstract?CRETRY=1&SRETRY=0 Tsuchiya et al. 1999]

[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSD-426YS50-D&_user=217827&_coverDate=12%2F31%2F2000&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=4764a7bdabc3649925f082f989e835f5 Kobayashi et al. 2000]

[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSD-46G3T2V-4&_user=217827&_coverDate=01%2F31%2F2002&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=c807c9de3e3579777b08fdd77cd32294 Kobayashi et al. 2002]

[http://jb.asm.org/cgi/reprint/185/22/6666 Yamamoto et al. 2003]

Picture1.png
Crystal structure the protein LytC used to anchor our peptide to the cell wall. (EBI, 2010)

Additionally, since we do not intend to knock out the original gene from the B. subtilis strain we will not interfere with its normal expression, avoiding expression at the wrong time or over expression. Furthermore since we aim to remove the catalytic domain from LytC or CwlC, our fusion proteins should lose their cell wall turn-over functions and only act as cell wall anchors.

Summary and Testing

Before finalising our system we modelled the requirements for our detection module to analyse its functionality. For more information on this follow this link to our Modelling. It showed that we could reach the threshold concentration of AIP needed to activate our system, 10ng/ml, quite easily, with the amount of surface protein we expect to express, as well as estimating the time needed to set off our system at a given concentration of elastase in the water. The assembly of the protein was done in several steps. We first amplified LytC out of the B. subtilis genome by PCR, simultaneously using primer extension to add optimised ribosome binding sites to it, and insert the PCR product into pSB1C3. We later ligated the protein with the promoter Pveg. Parallel to this, we ligated the 6 linker sequences ordered from Eurofins|mwg with a double terminator BOO14. We then made use of a natural ACCI site in the LytC gene, to ligate our promoter-LytC sequence with our linker-terminator sequence to create 6 different surface protein constructs, for testing in B. subtilis.

Picture2Cell wall with detection module.png
The detection module is non-covalently bound to the cell wall that overlies the phospholipid bilayer

In order to be able to detect out protein we will use His-tags. However these are only been added to test constructs and not the final module, as the tag would most likely interfere with the module’s function. The his-tags have been added to the C-terminus of the AIP, probably inhibiting correct detection of the AIP by the two component system comD/E. However using a his-tag allows us to test for three different, very important things: We can lyse a bacterial culture to isolate our his-tagged protein from the lysate. Detection in this step would demonstrate successful expression of our fusion protein. Secondly we can place our bacteria in a solution with high osmolarity. As mentioned this eludes the non-covalently bound proteins from the cell wall by interfering with the electrostatic interactions of the cell wall binding domain with the peptidoglycans, which should also work for our fusion proteins. Detection of our protein in this step would demonstrate expression as well as correct localisation in the cell wall. Last of all we could place the cells in medium containing the elastase or a protease with the same or very similarly specificity. If we are able to pull down the his-tagged peptide then it would demonstrate expression, localisation and that the correct cleavage site is accessible to the protease and the AIP can be successfully cleaved off. For a complete list of the BioBrick created for this module see our page on Parts submitted to the registry.


Here's a picture of the final construct:

IC Module1.JPG