http://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&feed=atom&action=historyTeam:TorontoMaRSDiscovery/Project - Revision history2024-03-28T20:45:57ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=166568&oldid=prevShung at 02:50, 22 October 20092009-10-22T02:50:44Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/<del class="diffchange diffchange-inline">Modelling</del>|Modelling]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/<ins class="diffchange diffchange-inline">Modeling</ins>|Modelling]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]</div></td></tr>
</table>Shunghttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=166422&oldid=prevShung at 02:47, 22 October 20092009-10-22T02:47:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/<del class="diffchange diffchange-inline">Modeling</del>|<del class="diffchange diffchange-inline">Modeling</del>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/<ins class="diffchange diffchange-inline">Modelling</ins>|<ins class="diffchange diffchange-inline">Modelling</ins>]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]</div></td></tr>
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</table>Shunghttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=165163&oldid=prevGcromar at 02:20, 22 October 20092009-10-22T02:20:56Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications. For these applications, we will construct recombinant enzymes fused to the targeting sequence. These enzymes will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in dynamic pathway switching.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications. For these applications, we will construct recombinant enzymes fused to the targeting sequence. These enzymes will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system<ins class="diffchange diffchange-inline">. It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to first test an alternative, readily assayable system regardless of commercial relevance, to meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7]</ins>. As a further experiment, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in dynamic pathway switching.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">==Comments==</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">#Size of compartments</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. </del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">#Control of expression</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">#Search for an assayable application</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==References==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==References==</div></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=165045&oldid=prevGcromar: /* Future Work */2009-10-22T02:18:18Z<p><span class="autocomment">Future Work</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications<ins class="diffchange diffchange-inline">. For these applications, </ins>we will construct <ins class="diffchange diffchange-inline">recombinant enzymes </ins>fused to the targeting sequence. These <ins class="diffchange diffchange-inline">enzymes </ins>will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in <ins class="diffchange diffchange-inline">dynamic </ins>pathway switching.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Using pairs of enzymes from pathways modeled in our earlier screens </del>we will construct <del class="diffchange diffchange-inline">recombinants </del>fused to the targeting sequence. These <del class="diffchange diffchange-inline">enzyme pairs </del>will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment<del class="diffchange diffchange-inline">, if possible</del>, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Comments==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Comments==</div></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=164833&oldid=prevGcromar: /* Design */2009-10-22T02:11:50Z<p><span class="autocomment">Design</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#A pair of enzymes that are expected to benefit from the effects of channeling.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#A pair of enzymes that are expected to benefit from the effects of channeling.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our search for a suitable platform to <del class="diffchange diffchange-inline">instigate </del>channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our search for a suitable platform to <ins class="diffchange diffchange-inline">effect </ins>channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. </div></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=164768&oldid=prevGcromar: /* Progress */2009-10-22T02:09:11Z<p><span class="autocomment">Progress</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Our study of the natural biology of microcompartments across bacteria suggests that their occurrence is confined to a protective function, most commonly isolating toxic or reactive intermediates in certain redox reactions. While other microcompartments exist, the lack of a discernible targeting sequence makes them unsuitable for use in our system and encapsulin remains our best option at present. We have short-listed a series of enzyme pairs that we feel are amenable to manipulation by enzyme channeling based on thermodynamic and other properties for use in our future work.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=164364&oldid=prevGcromar at 01:55, 22 October 20092009-10-22T01:55:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see model). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see <ins class="diffchange diffchange-inline">[[Team:TorontoMaRSDiscovery/Modeling|</ins>model<ins class="diffchange diffchange-inline">]]</ins>). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Future Work==</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=163759&oldid=prevGcromar: /* Design */2009-10-22T01:33:30Z<p><span class="autocomment">Design</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<col class='diff-content' />
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]<del class="diffchange diffchange-inline">(<partinfo>BBa_K192001</partinfo>)</del>. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' <ins class="diffchange diffchange-inline">(<partinfo>BBa_K192001</partinfo>) </ins>with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized <ins class="diffchange diffchange-inline">(see model)</ins>. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=163589&oldid=prevGcromar: /* Design */2009-10-22T01:28:48Z<p><span class="autocomment">Design</span></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 01:28, 22 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Using two parts <del class="diffchange diffchange-inline">of our own design </del>and several existing <del class="diffchange diffchange-inline">BioBrick parts</del>[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>). By varying the amounts of inducers aTc and IPTG it is possible to vary the expression of <del class="diffchange diffchange-inline">the microcompartment protein (</del>encapsulin<del class="diffchange diffchange-inline">) </del>and <del class="diffchange diffchange-inline">a </del>targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using <del class="diffchange diffchange-inline">a </del>recombinant, fluorescent marker protein (eCFP) <del class="diffchange diffchange-inline">with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed </del>by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Using two <ins class="diffchange diffchange-inline">new </ins>parts <ins class="diffchange diffchange-inline">(see below) </ins>and several existing <ins class="diffchange diffchange-inline">BioBricks </ins>[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural<ins class="diffchange diffchange-inline">, </ins>microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' <ins class="diffchange diffchange-inline">with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]</ins>(<partinfo>BBa_K192001</partinfo>). By varying the amounts of inducers aTc and IPTG <ins class="diffchange diffchange-inline">in the system </ins>it is possible to vary the expression of encapsulin and <ins class="diffchange diffchange-inline">the </ins>targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach <ins class="diffchange diffchange-inline">to visualize the microcompartments</ins>. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using <ins class="diffchange diffchange-inline">the </ins>recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td></tr>
</table>Gcromarhttp://2009.igem.org/wiki/index.php?title=Team:TorontoMaRSDiscovery/Project&diff=163283&oldid=prevGcromar: /* Design */2009-10-22T01:20:36Z<p><span class="autocomment">Design</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
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<td colspan='2' style="background-color: white; color:black;">Revision as of 01:20, 22 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Components</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Using <del class="diffchange diffchange-inline">standard </del>BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter. By varying the amounts of inducers aTc and IPTG it is possible to vary the expression of the microcompartment protein (encapsulin) and a targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Using <ins class="diffchange diffchange-inline">two parts of our own design and several existing </ins>BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter <ins class="diffchange diffchange-inline">as well as the structural microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>)</ins>. By varying the amounts of inducers aTc and IPTG it is possible to vary the expression of the microcompartment protein (encapsulin) and a targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Progress==</div></td></tr>
</table>Gcromar