http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Kgizdov&year=&month=2014.igem.org - User contributions [en]2024-03-29T06:32:02ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Aberdeen_ScotlandTeam:Aberdeen Scotland2015-01-21T14:27:19Z<p>Kgizdov: </p>
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<h1>Aberdeen iGEM Team 2014</h1><br />
<h3>Wake up to the Sleeping Sickness</h3><br />
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<img src="https://static.igem.org/mediawiki/2014/a/ae/Igem_logo_exp.png"><br />
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<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<h4>The team was awarded a Gold medal and two special prizes:</h4><br />
<ul><br />
<ul><br />
<li><b>Best Health & Medicine Project</b> - Winner of Health & Medicine Track</li><br />
<li><b>Best Innovation in Measurement</b> - Special Prize for unique assay design</li><br />
</ul><br />
</ul><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Awards_1.jpg"><br />
<h4>*Ana and Konstantin receiving our two special prizes</h4><br />
<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<p>Trypanosoma brucei gambiense, the causative agent of Human African Trypanosomiasis (HAT) (commonly known as African Sleeping Sickness), infects 30,000 people<br />
worldwide, with 60 million at disease risk. Unrecognised, this disease is fatal, but accurate diagnosis, relying on detection of patient serum antibodies against two<br />
different trypanosomal antigens, Variable Surface Glycoprotein LiTat 1.3 and Variable Surface Glycoprotein LiTat 1.5, allows successful treatment.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Parasite.png"><br />
<h3>Our project and achievements</h3><br />
<p>We engineered autotransporters Antigen43 (Ag43) and Ice-Nucleation protein (INP) to successfully express surface epitopes in <i>E.coli</i>, that mimic the two different<br />
trypanosomal antigens. Therefore, we created standardised and user-friendly Ag43 and INP BioBricks ready for peptides of choice to be cloned in. Two <i>E.coli</i> strains<br />
were generated, each expressing a distinct surface epitope and either a quorum sensing sender or receiver module respectively. We delivered a proof-of-principle<br />
demonstration of our project, by showing that the quorum-based system is capable of detecting cell surface Ag43-FLAG ‘Receiver’ <i>E.coli</i> in an anti-FLAG bead pull-<br />
down. These ‘Receivers’ co-cultured with AHL-synthesising ‘Senders’ produced a strong –FLAG-tag dependent, AHL-inducible GFP response. This system if applied in a<br />
real life scenario could effectively diagnose a neglected tropical disease such as Trypanosomiasis, using patient serum-derived antibodies, with minimal facilities.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Diag.png"><br><br><br />
<h3>The user-friendly, portable, inexpensive GFP detection device</h3><br />
<p>We constructed a cheap, Raspberry Pi computer-controlled fluorimeter suitable for developing countries, showing it could detect QS fluorescence output. The<br />
detection device costs around $100 and could replace heavy and expensive microscopes, to make the detection system affordable to remote populations in Africa.</p><br><br />
<img src="https://static.igem.org/mediawiki/2014/7/71/GFP_detector.png"><br><br><br />
<h3>Novel applications</h3><br />
<p>This novel application to diagnose patient exposure to tropical pathogens integrates antigen display and quorum sensing to implement AND logic sensing. It<br />
facilitates cheap, simple diagnosis of neglected tropical diseases such as HAT. Future diagnosis kits can be constructed and modularized using the cheap and self-<br />
replenishing <i>E. coli</i> based detection system we created as a platform for displaying two different mimotopes. Mimotopes are shortened versions of real epitopes<br />
identified for a panel of diseases, all of which are currently stored in a universal database, available to everyone and known as MimoDB [http://immunet.cn/mimodb].<br />
Moreover, it was recently shown that upon heat-treatment of Ag43 autotransporter-foreign peptide complex at 60°C, a chimeric form of the expressed foreign protein<br />
can be isolated and remain fully active. The chimeric protein was then used to induce an immune response and to develop a novel vaccine for cancer therapy (Huang et<br />
al., 2014). Therefore, we suggest that further research can be done on Ag43-trypanosomal mimotope/ other mimotope complexes for a potential first vaccine for HAT/<br />
other diseases.</p><br />
<p><b>Reference:</b> Huang, F., Li, L., Liu, Q., Li, Y., Bai, R., Huang, Y., Zhao, H., Guo, J., Zhou, S., Wang, H. and others, (2014). Bacterial surface display of<br />
endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis. International Journal of Cancer, 134(8),<br />
pp.1981--1990.</p><br />
</div><br />
<br />
<div class="external"><br />
<p>You can find our official iGEM Registry Page <a href="https://igem.org/Team.cgi?year=2014&team_name=Aberdeen_Scotland">here</a>.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_ScotlandTeam:Aberdeen Scotland2015-01-21T14:26:51Z<p>Kgizdov: </p>
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<h1>Aberdeen iGEM Team 2014</h1><br />
<h3>Wake up to the Sleeping Sickness</h3><br />
<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/a/ae/Igem_logo_exp.png"><br />
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<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<h4>The team was awarded a <b>Gold medal</b> and two special prizes:</h4><br />
<ul><br />
<ul><br />
<li><b>Best Health & Medicine Project</b> - Winner of Health & Medicine Track</li><br />
<li><b>Best Innovation in Measurement</b> - Special Prize for unique assay design</li><br />
</ul><br />
</ul><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Awards_1.jpg"><br />
<h4>*Ana and Konstantin receiving our two special prizes</h4><br />
<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<p>Trypanosoma brucei gambiense, the causative agent of Human African Trypanosomiasis (HAT) (commonly known as African Sleeping Sickness), infects 30,000 people<br />
worldwide, with 60 million at disease risk. Unrecognised, this disease is fatal, but accurate diagnosis, relying on detection of patient serum antibodies against two<br />
different trypanosomal antigens, Variable Surface Glycoprotein LiTat 1.3 and Variable Surface Glycoprotein LiTat 1.5, allows successful treatment.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Parasite.png"><br />
<h3>Our project and achievements</h3><br />
<p>We engineered autotransporters Antigen43 (Ag43) and Ice-Nucleation protein (INP) to successfully express surface epitopes in <i>E.coli</i>, that mimic the two different<br />
trypanosomal antigens. Therefore, we created standardised and user-friendly Ag43 and INP BioBricks ready for peptides of choice to be cloned in. Two <i>E.coli</i> strains<br />
were generated, each expressing a distinct surface epitope and either a quorum sensing sender or receiver module respectively. We delivered a proof-of-principle<br />
demonstration of our project, by showing that the quorum-based system is capable of detecting cell surface Ag43-FLAG ‘Receiver’ <i>E.coli</i> in an anti-FLAG bead pull-<br />
down. These ‘Receivers’ co-cultured with AHL-synthesising ‘Senders’ produced a strong –FLAG-tag dependent, AHL-inducible GFP response. This system if applied in a<br />
real life scenario could effectively diagnose a neglected tropical disease such as Trypanosomiasis, using patient serum-derived antibodies, with minimal facilities.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Diag.png"><br><br><br />
<h3>The user-friendly, portable, inexpensive GFP detection device</h3><br />
<p>We constructed a cheap, Raspberry Pi computer-controlled fluorimeter suitable for developing countries, showing it could detect QS fluorescence output. The<br />
detection device costs around $100 and could replace heavy and expensive microscopes, to make the detection system affordable to remote populations in Africa.</p><br><br />
<img src="https://static.igem.org/mediawiki/2014/7/71/GFP_detector.png"><br><br><br />
<h3>Novel applications</h3><br />
<p>This novel application to diagnose patient exposure to tropical pathogens integrates antigen display and quorum sensing to implement AND logic sensing. It<br />
facilitates cheap, simple diagnosis of neglected tropical diseases such as HAT. Future diagnosis kits can be constructed and modularized using the cheap and self-<br />
replenishing <i>E. coli</i> based detection system we created as a platform for displaying two different mimotopes. Mimotopes are shortened versions of real epitopes<br />
identified for a panel of diseases, all of which are currently stored in a universal database, available to everyone and known as MimoDB [http://immunet.cn/mimodb].<br />
Moreover, it was recently shown that upon heat-treatment of Ag43 autotransporter-foreign peptide complex at 60°C, a chimeric form of the expressed foreign protein<br />
can be isolated and remain fully active. The chimeric protein was then used to induce an immune response and to develop a novel vaccine for cancer therapy (Huang et<br />
al., 2014). Therefore, we suggest that further research can be done on Ag43-trypanosomal mimotope/ other mimotope complexes for a potential first vaccine for HAT/<br />
other diseases.</p><br />
<p><b>Reference:</b> Huang, F., Li, L., Liu, Q., Li, Y., Bai, R., Huang, Y., Zhao, H., Guo, J., Zhou, S., Wang, H. and others, (2014). Bacterial surface display of<br />
endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis. International Journal of Cancer, 134(8),<br />
pp.1981--1990.</p><br />
</div><br />
<br />
<div class="external"><br />
<p>You can find our official iGEM Registry Page <a href="https://igem.org/Team.cgi?year=2014&team_name=Aberdeen_Scotland">here</a>.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_ScotlandTeam:Aberdeen Scotland2015-01-21T14:26:11Z<p>Kgizdov: </p>
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<h1>Aberdeen iGEM Team 2014</h1><br />
<h3>Wake up to the Sleeping Sickness</h3><br />
<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/a/ae/Igem_logo_exp.png"><br />
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<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<h4>The team was awarded a Gold medal and two special prizes:</h4><br />
<ul><br />
<ul><br />
<li><b>Best Health & Medicine Project</b> - Winner of Health & Medicine Track</li><br />
<li><b>Best Innovation in Measurement</b> - Special Prize for unique assay design</li><br />
</ul><br />
</ul><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Awards_1.jpg"><br />
<h4>*Ana and Konstantin receiving our two special prizes</h4><br />
<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<p>Trypanosoma brucei gambiense, the causative agent of Human African Trypanosomiasis (HAT) (commonly known as African Sleeping Sickness), infects 30,000 people<br />
worldwide, with 60 million at disease risk. Unrecognised, this disease is fatal, but accurate diagnosis, relying on detection of patient serum antibodies against two<br />
different trypanosomal antigens, Variable Surface Glycoprotein LiTat 1.3 and Variable Surface Glycoprotein LiTat 1.5, allows successful treatment.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Parasite.png"><br />
<h3>Our project and achievements</h3><br />
<p>We engineered autotransporters Antigen43 (Ag43) and Ice-Nucleation protein (INP) to successfully express surface epitopes in <i>E.coli</i>, that mimic the two different<br />
trypanosomal antigens. Therefore, we created standardised and user-friendly Ag43 and INP BioBricks ready for peptides of choice to be cloned in. Two <i>E.coli</i> strains<br />
were generated, each expressing a distinct surface epitope and either a quorum sensing sender or receiver module respectively. We delivered a proof-of-principle<br />
demonstration of our project, by showing that the quorum-based system is capable of detecting cell surface Ag43-FLAG ‘Receiver’ <i>E.coli</i> in an anti-FLAG bead pull-<br />
down. These ‘Receivers’ co-cultured with AHL-synthesising ‘Senders’ produced a strong –FLAG-tag dependent, AHL-inducible GFP response. This system if applied in a<br />
real life scenario could effectively diagnose a neglected tropical disease such as Trypanosomiasis, using patient serum-derived antibodies, with minimal facilities.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Diag.png"><br><br><br />
<h3>The user-friendly, portable, inexpensive GFP detection device</h3><br />
<p>We constructed a cheap, Raspberry Pi computer-controlled fluorimeter suitable for developing countries, showing it could detect QS fluorescence output. The<br />
detection device costs around $100 and could replace heavy and expensive microscopes, to make the detection system affordable to remote populations in Africa.</p><br><br />
<img src="https://static.igem.org/mediawiki/2014/7/71/GFP_detector.png"><br><br><br />
<h3>Novel applications</h3><br />
<p>This novel application to diagnose patient exposure to tropical pathogens integrates antigen display and quorum sensing to implement AND logic sensing. It<br />
facilitates cheap, simple diagnosis of neglected tropical diseases such as HAT. Future diagnosis kits can be constructed and modularized using the cheap and self-<br />
replenishing <i>E. coli</i> based detection system we created as a platform for displaying two different mimotopes. Mimotopes are shortened versions of real epitopes<br />
identified for a panel of diseases, all of which are currently stored in a universal database, available to everyone and known as MimoDB [http://immunet.cn/mimodb].<br />
Moreover, it was recently shown that upon heat-treatment of Ag43 autotransporter-foreign peptide complex at 60°C, a chimeric form of the expressed foreign protein<br />
can be isolated and remain fully active. The chimeric protein was then used to induce an immune response and to develop a novel vaccine for cancer therapy (Huang et<br />
al., 2014). Therefore, we suggest that further research can be done on Ag43-trypanosomal mimotope/ other mimotope complexes for a potential first vaccine for HAT/<br />
other diseases.</p><br />
<p><b>Reference:</b> Huang, F., Li, L., Liu, Q., Li, Y., Bai, R., Huang, Y., Zhao, H., Guo, J., Zhou, S., Wang, H. and others, (2014). Bacterial surface display of<br />
endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis. International Journal of Cancer, 134(8),<br />
pp.1981--1990.</p><br />
</div><br />
<br />
<div class="external"><br />
<p>You can find our official iGEM Registry Page <a href="https://igem.org/Team.cgi?year=2014&team_name=Aberdeen_Scotland">here</a>.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_ScotlandTeam:Aberdeen Scotland2015-01-21T14:22:24Z<p>Kgizdov: </p>
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<h1>Aberdeen iGEM Team 2014</h1><br />
<h3>Wake up to the Sleeping Sickness</h3><br />
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<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/Awards_1.jpg"><br />
<h3>Human African Trypanosomiasis – a Neglected Tropical Disease</h3><br />
<p>Trypanosoma brucei gambiense, the causative agent of Human African Trypanosomiasis (HAT) (commonly known as African Sleeping Sickness), infects 30,000 people<br />
worldwide, with 60 million at disease risk. Unrecognised, this disease is fatal, but accurate diagnosis, relying on detection of patient serum antibodies against two<br />
different trypanosomal antigens, Variable Surface Glycoprotein LiTat 1.3 and Variable Surface Glycoprotein LiTat 1.5, allows successful treatment.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/Parasite.png"><br />
<h3>Our project and achievements</h3><br />
<p>We engineered autotransporters Antigen43 (Ag43) and Ice-Nucleation protein (INP) to successfully express surface epitopes in <i>E.coli</i>, that mimic the two different<br />
trypanosomal antigens. Therefore, we created standardised and user-friendly Ag43 and INP BioBricks ready for peptides of choice to be cloned in. Two <i>E.coli</i> strains<br />
were generated, each expressing a distinct surface epitope and either a quorum sensing sender or receiver module respectively. We delivered a proof-of-principle<br />
demonstration of our project, by showing that the quorum-based system is capable of detecting cell surface Ag43-FLAG ‘Receiver’ <i>E.coli</i> in an anti-FLAG bead pull-<br />
down. These ‘Receivers’ co-cultured with AHL-synthesising ‘Senders’ produced a strong –FLAG-tag dependent, AHL-inducible GFP response. This system if applied in a<br />
real life scenario could effectively diagnose a neglected tropical disease such as Trypanosomiasis, using patient serum-derived antibodies, with minimal facilities.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Diag.png"><br><br><br />
<h3>The user-friendly, portable, inexpensive GFP detection device</h3><br />
<p>We constructed a cheap, Raspberry Pi computer-controlled fluorimeter suitable for developing countries, showing it could detect QS fluorescence output. The<br />
detection device costs around $100 and could replace heavy and expensive microscopes, to make the detection system affordable to remote populations in Africa.</p><br><br />
<img src="https://static.igem.org/mediawiki/2014/7/71/GFP_detector.png"><br><br><br />
<h3>Novel applications</h3><br />
<p>This novel application to diagnose patient exposure to tropical pathogens integrates antigen display and quorum sensing to implement AND logic sensing. It<br />
facilitates cheap, simple diagnosis of neglected tropical diseases such as HAT. Future diagnosis kits can be constructed and modularized using the cheap and self-<br />
replenishing <i>E. coli</i> based detection system we created as a platform for displaying two different mimotopes. Mimotopes are shortened versions of real epitopes<br />
identified for a panel of diseases, all of which are currently stored in a universal database, available to everyone and known as MimoDB [http://immunet.cn/mimodb].<br />
Moreover, it was recently shown that upon heat-treatment of Ag43 autotransporter-foreign peptide complex at 60°C, a chimeric form of the expressed foreign protein<br />
can be isolated and remain fully active. The chimeric protein was then used to induce an immune response and to develop a novel vaccine for cancer therapy (Huang et<br />
al., 2014). Therefore, we suggest that further research can be done on Ag43-trypanosomal mimotope/ other mimotope complexes for a potential first vaccine for HAT/<br />
other diseases.</p><br />
<p><b>Reference:</b> Huang, F., Li, L., Liu, Q., Li, Y., Bai, R., Huang, Y., Zhao, H., Guo, J., Zhou, S., Wang, H. and others, (2014). Bacterial surface display of<br />
endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis. International Journal of Cancer, 134(8),<br />
pp.1981--1990.</p><br />
</div><br />
<br />
<div class="external"><br />
<p>You can find our official iGEM Registry Page <a href="https://igem.org/Team.cgi?year=2014&team_name=Aberdeen_Scotland">here</a>.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/File:Awards_1.jpgFile:Awards 1.jpg2015-01-21T14:20:40Z<p>Kgizdov: </p>
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<div></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Project/DiseaseTeam:Aberdeen Scotland/Project/Disease2014-10-18T02:58:22Z<p>Kgizdov: </p>
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<h1>Trypanosomiasis</h1><br />
<h3>Human African Trypanosomiasis (Sleeping Sickness) Epidemiology</h3><br />
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<p>Over 98% of reported cases of Human African Trypanosomiasis (HAT) are caused by a chronic infection of parasite <i>Trypanosoma brucei gambiense</i>. Its vector is through the bite of an infected Tsetse fly in sub-saharan Africa.</p><br />
<p>WHO estimates as of 2014, 30,000 people are currently infected, with more than 80% of new cases occurring in Democratic Republic of the Congo.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/7/7b/Afr.png" width="60%" height="60%"></center><br />
<p><i>T.b. gambiense</i> sufferers may be infected for months or even years without major signs or symptoms of the disease. When more evident symptoms emerge, the patient is often already in an advanced disease stage where the central nervous system is affected.</p><br />
<p>During epidemic periods prevalence reached 50% in several villages in the Angola, Democratic Republic of Congo, and South Sudan. Sleeping sickness was the first or second greatest cause of mortality in those communities, ahead of even HIV/AIDS.<p><br />
<p>The World Health Organisation included HAT in its roadmap for elimination and control of neglected tropical diseases in 2012, setting a date of 2020 to eliminate the disease as a public health problem.</p><br />
<p>2% of reported cases are due to <i>T.b. rhodesiense</i>, which is an acute infection, however this figure may be skewed by the fact this develops rapidly, invading the central nervous system with symptoms appearing after only a few weeks or months. If it persists, infected have a similar prognosis of almost unanimous mortality.</p><br />
<p>Chagas disease of South America is of a different subgenus, <i>Trypanosoma cruzi</i>, and utilises a different vector, Triatominae (‘assassin bugs’).</p><br />
<h3>Disease Progression</h3><p></p><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/Trypanosoma-evansi-rat-blood-Giemsa-stain.jpg"><br />
Giemsa-stained trypanasoma parasites in blood<br />
<p><i>T. b. gambiense</i> stage 1 exists free in blood serum or lymph, usually pathogens like this will be quickly destroyed by an active immune response, however it exhibits antigenic variation of its LiTat1.3/1.5 coat that changes every 20 minutes with over 100 possible phenotypes encoded in its chromosome; this negates an active immunity from being effectively generated.</p><br />
<p>After a period of approximately 6 months it progresses to stage 2 where the parasite enters the central nervous system, here it is protected from an immune response and may proliferate at will. Clearer symptoms emerge at this point including muscle weakness, sleep disturbance, partial paralysis, parkinsonism, psychosis and other psychiatric symptoms, and coma. If untreated, <i>T. b. gambiense</i> almost always results in death.</p><br />
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<h1>Trypanosomiasis</h1><br />
<h3>Human African Trypanosomiasis (Sleeping Sickness) Epidemiology</h3><br />
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<p>Over 98% of reported cases of Human African Trypanosomiasis (HAT) are caused by a chronic infection of parasite <i>Trypanosoma brucei gambiense</i>. Its vector is through the bite of an infected Tsetse fly in sub-saharan Africa.</p><br />
<p>WHO estimates as of 2014, 30,000 people are currently infected, with more than 80% of new cases occurring in Democratic Republic of the Congo.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/7/7b/Afr.png" width="60%" height="60%"></center><br />
<p><i>T.b. gambiense</i> sufferers may be infected for months or even years without major signs or symptoms of the disease. When more evident symptoms emerge, the patient is often already in an advanced disease stage where the central nervous system is affected.</p><br />
<p>During epidemic periods prevalence reached 50% in several villages in the Angola, Democratic Republic of Congo, and South Sudan. Sleeping sickness was the first or second greatest cause of mortality in those communities, ahead of even HIV/AIDS.<p><br />
<p>The World Health Organisation included HAT in its roadmap for elimination and control of neglected tropical diseases in 2012, setting a date of 2020 to eliminate the disease as a public health problem.</p><br />
<p>2% of reported cases are due to <i>T.b. rhodesiense</i>, which is an acute infection, however this figure may be skewed by the fact this develops rapidly, invading the central nervous system with symptoms appearing after only a few weeks or months. If it persists, infected have a similar prognosis of almost unanimous mortality.</p><br />
<p>Chagas disease of South America is of a different subgenus, <i>Trypanosoma cruzi</i>, and utilises a different vector, Triatominae (‘assassin bugs’).</p><br />
<h3>Disease Progression</h3><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/Trypanosoma-evansi-rat-blood-Giemsa-stain.jpg"><br />
<p><i>T. b. gambiense</i> stage 1 exists free in blood serum or lymph, usually pathogens like this will be quickly destroyed by an active immune response, however it exhibits antigenic variation of its LiTat1.3/1.5 coat that changes every 20 minutes with over 100 possible phenotypes encoded in its chromosome; this negates an active immunity from being effectively generated.</p><br />
<p>After a period of approximately 6 months it progresses to stage 2 where the parasite enters the central nervous system, here it is protected from an immune response and may proliferate at will. Clearer symptoms emerge at this point including muscle weakness, sleep disturbance, partial paralysis, parkinsonism, psychosis and other psychiatric symptoms, and coma. If untreated, <i>T. b. gambiense</i> almost always results in death.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/File:Trypanosoma-evansi-rat-blood-Giemsa-stain.jpgFile:Trypanosoma-evansi-rat-blood-Giemsa-stain.jpg2014-10-18T02:50:31Z<p>Kgizdov: </p>
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<div></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/DeviceTeam:Aberdeen Scotland/Parts/Device2014-10-18T02:31:26Z<p>Kgizdov: </p>
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<p>The initial concept of the fluorescence detector device had a very rough design. Thus it was built from bits and pieces in order to do a preliminary test and<br />
verify its viability.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Rough_set.jpg"><br />
<br />
<p>The preliminary data from the tests showed that the detector was able to differentiate between GFP producing and non-producing bacteria.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Lb_vs_gfp.png"><br />
<br />
<p>Fig.1 Series dilutions to estimate detector sensitivity</p><br />
<br />
<p>From Fig.1 we can see that the detector can easily distinguish the GFP producing culture from below 1/10 dilutions. Thus we continued on and improved the design by soldering some of the circuit and making it more compact. Then we made more test with the Sender and Receiver bacteria.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Send_rec_vs.png"><br />
<p>Fig.2 Detector response to Sender and Receiver cultures</p><br />
<br />
<p>Again the detector is sensitive enough to pick up the GFP in the Receiver.</p><br />
<br />
<p>For the final test, we made clean overnight cultures of the Receiver and Sender and mixed them in a growing culture. This was done to see after how long the detector is going to pick up the GFP production. Hence, if our diagnostic method is going to work.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/79/Resp_time.png"><br />
<p>Fig.3 Detector response to QS GFP-producing Receiver and Sender culture, normalised against LB medium</p><br />
<br />
<p><b>Finally, the simple detector proved capable of detecting the QS culture, thus confirming our design success.</b></p><br />
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<h3>Our part in helping to develop Scottish national science policy in the area of synthetic biology</h3><br />
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<p>The Aberdeen team was fortunate to be able to accept an invitation to an event on 16th October organised by the Chief Scientific Advisor of Scotland, Prof Muffy<br />
Calder. Prof. Calder is a Scottish government-appointed scientist whose role is to advise the Scottish government on science issues as they impact on government<br />
policy.</p><br />
<br />
<p>Prof. Calder co-chairs the <a href="http://www.scottishscience.org.uk/">Scottish Scientific Advisory Council (SSAC)</a>, an government body providing scientific<br />
advice to government, and currently engaged with producing a report on the future development of synthetic biology as a tool for biotechnology in Scotland. We, the<br />
Aberdeen iGEM team were invited to the launch event for the SSAC Report on Synthetic biology: opportunities for Scotland, where we were asked to present a poster on<br />
our iGEM project to evidence the development of synthetic biology in Scotland. We had the opportunity to speak to a number of key figures in this area, including<br />
Prof Calder, and Prof. Nigel Brown, President of the Society for General Microbiology and co-author of the report. We considered that we had contributed in some<br />
small way to the development of synthetic biology policy, and were participants in the process of development of national science policy in this area. We regarded<br />
ourselves as privileged to see how national science policies, including those on synthetic biology are developed in our country, as well as to be able to speak to<br />
Scottish policy makers at the event.</p><br />
<br />
<p>The Scottish SSAC report on Synthetic biology: opportunities for Scotland: A report by the Scottish Science Advisory Council will be published on 17th September<br />
2014.</p><br />
<br />
<div class="float"><a href="http://www.scottishscience.org.uk/"><img src="https://static.igem.org/mediawiki/2014/9/90/SSAClogo.png"></a></div><br />
<br />
<h4>iGEM Aberdeen: public outreach and science communication</h4><br />
<p>The Aberdeen team used a number of outreach opportunities to engage with non-scientists and the general public. Each provided the opportunity to engage with a lay<br />
audience, and find out what the public thinks of the science of synthetic biology. We discussed our science and presented at the following events.</p><br />
<br />
<p><b>SCHMU Radio, Aberdeen:</b> our team was interviewed on the SHMU local radio station for 20 minutes on the Talking Science show, giving us the chance to<br />
communicate our project, and the concept of synthetic biology, to an audience in the Aberdeen city area.</p><br />
<br />
<audio controls><br />
<source src="https://static.igem.org/mediawiki/2014/f/f2/Joe_Radio.mp3" type="audio/mpeg"><br />
Your browser does not support the audio element.<br />
</audio><br />
<br />
<p><b>Explorathon 2014:</b> this annual European event is a vast science public outreach event giving researchers from across Europe the chance to showcase their<br />
science to a general public audience. On <a href="http://www.explorathon.co.uk/aberdeen">Explorathon day</a> (26th September 2014), our contribution was present to<br />
an open audience in a quick-fire Pecha Kucha event (20 slides, 20 seconds each) held in the Belmont cinema in Aberdeen city centre. This open event was attended by a<br />
large number of members of the public, followed by a Q&A session.</p><br />
<br />
<iframe width="560" height="315" src="//www.youtube.com/embed/3qMgndUmz_Q" frameborder="0" allowfullscreen></iframe><br />
<br />
<p><b>iGEM project blog with <a href="http://www.aumag.co.uk/">AU Magazine</a>:</b> our team had a great opportunity to engage with non-scientists through a 3-part<br />
blog we produced in the Aberdeen University science magazine 'Au'. This magazine is written for a non-scientific audience, providing an important outreach<br />
opportunity.</p><br />
<br />
<h5>Our goal for publishing a series of articles was to:</h5><br />
<ul><br />
<li><br />
<ul style="list-style-type:disc"><br />
<li>Address the general public's misconceptions surrounding synthetic biology</li><br />
<li>Popularise SynBio</li><br />
<li>Get people thinking about how synthetic biology can provide solutions to serious global issues we face nowadays</li><br />
<li>To start a discussion about the ethical implication of our actions; Our moral responsibility to use knowledge to do good, as well as looking at the<br />
sustainability of synthetic biology in the future</li><br />
<li>To popularize iGEM</li><br />
<li>To inspire readers of the magazine (fellow students) and show that young people can make a positive contribution and tackle global problems</li><br />
</ul><br />
</li><br />
</ul><br />
<br />
<h4>To date we have published 2 articles:</h4><br />
<ul><br />
<li><br />
<h5><b><a href="https://static.igem.org/mediawiki/2014/9/9e/A_Young_Physicist.pdf">A Young Physicist’s perspective on Synthetic Biology</a></b> by Konstantin Gizdov, in which we<br />
addressed the following:</h5><br />
<ul><br />
<li>- What is iGEM</li><br />
<li>- What is synthetic biology, emphasizing its interdisciplinary aspect and debunking myths surrounding 'the Frankenstein's biology'</li><br />
<li>- Why are physicists involved in biology projects? How the modelling can guide informed design of biological research</li><br />
<li>- The unlimited potential of synbio and how we implement it in everyday life</li><br />
<li>- The risks and ethical implications connected with irresponsible applications of synbio, and how it is tackled by laws and regulations</li><br />
</ul><br />
</li><br />
<li><br />
<h5><a href="https://static.igem.org/mediawiki/2014/a/a4/IGEM-Blogpost_2.pdf"><b>Wake up to the sleeping sickness!</b></a> by Ana-Maria Cujba, our grand plan to engineer an E. coli<br />
System for the diagnosis of neglected tropical diseases:</h5><br />
<ul><br />
<li>- What is Human African Trypanosomiasis (HAT)</li><br />
<li>- The burden of the disease</li><br />
<li>- Difficulties with diagnosing HAT</li><br />
<li>- How to engineer a diagnostic system adapted to function effectively in remote parts of Africa and how synthetic biology enables us to do so</li><br />
<li>- The solution - our E.coli operated diagnosis system and a HAT detector device</li><br />
<li>- The positive impact of our project</li><br />
</ul><br />
</li><br />
</ul><br />
<br />
<p>Our third article about the Giant iGEM Jamboree and an exchange of ideas within an international scientific community will be published in mid-November. We will<br />
emphasize the international aspect of the competition and how all teams work together to perform high quality, safe science.</p><br />
<br />
<h4>Interactions with the Scottish Health Service</h4><br />
<p>In addition to the above outreach, as part of the project planning we also contacted a nearby National Health Service lab (Ms Leslie Bevridge, Grampian health<br />
authority Microbiology pathology lab) to identify possible disease targets for a new diagnosis tool. While we eventually settled on tropical disease diagnosis in<br />
remote areas we were still periodically in contact throughout the project, often regarding our weekly progress.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003Team:Aberdeen Scotland/Parts/ 20032014-10-18T02:24:49Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
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<li class="curr"><a class="curr" href="#">Improved</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_3013">Bba_k523013</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li><br />
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This brick was successfully created but in an ampicillin backbone and as such was not physically submitted to iGEM, details about its characterisation can be found on the registry entry for it here: <a href="http://parts.igem.org/Part:BBa_K1352003">BBa_K1352003</a><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003Team:Aberdeen Scotland/Parts/ 20032014-10-18T02:24:37Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li><br />
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<li class="curr"><a class="curr" href="#">Improved</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_3013">Bba_k523013</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li><br />
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<br />
This brick was successfully created but in an ampicillin backbone and as such was not physically submitted to iGEM, details about its characterisation can be found on the registry entry for it here: <a href="http://parts.igem.org/Part:BBa_K1352003">BBa_K135203</a><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/ModelingTeam:Aberdeen Scotland/Modeling2014-10-18T02:20:33Z<p>Kgizdov: </p>
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<p></p><br />
<p>In order to verify and characterize our diagnostic system we created mathematical models for each individual component. Modeling and simulations were essential<br />
techniques that guided our approach from design to completion. As the main point of interest in our system was inter-cellular communication and its optimization, we<br />
employed ODEs and PDEs as our main mathematical tools.</p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a7/AjkIkdjfmkJ.png"><br />
<h3>Quorum Sensing</h3><br />
<p>We developed a spacial model for analyzing Quorum Sensing between cells and then studied it under our system desired conditions. This gave us insight into how<br />
best structure our assay.</p><br />
<h3>GFP Response</h3><br />
<p>By simulating GFP response we made sure our system will react in a predictable manner and in an appropriate amount of time. We ensured that simulations agreed with<br />
data so that we can rely on reproducibility.</p><br />
<h3>Assay Sensitivity</h3><br />
<p>As with any other test, sensitivity is a main factor. The test needed to be sensitive enough to detect HAT, but also tolerant to noise, so that false-positives<br />
were minimized. We explored the process of antibody binding to make sure we meet those criteria.</p><br />
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</html></div>Kgizdovhttp://2014.igem.org/File:AjkIkdjfmkJ.pngFile:AjkIkdjfmkJ.png2014-10-18T02:20:04Z<p>Kgizdov: </p>
<hr />
<div></div>Kgizdovhttp://2014.igem.org/File:Results1.pngFile:Results1.png2014-10-18T02:16:31Z<p>Kgizdov: uploaded a new version of &quot;File:Results1.png&quot;</p>
<hr />
<div></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002Team:Aberdeen Scotland/Parts/ 90022014-10-18T02:11:58Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
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<br />
<br />
<br />
<h1>Our characterisation of existing BioBrick T9002 </h1><br />
<h2>GFP Producer Controlled by 3OC6HSL Receiver Device</h2><br />
<br><br />
<h3>Aims and Rationale</h3><br />
<br />
BioBrick BBa_T9002 on a pSB1C3 backbone is a composite part encoding a quorum sensing (QS) receiver driving expression of green fluorescent protein. T9002 was previously reported to exhibit increased GFP expression in the presence of AHL (N-acyl homoserine lactone). <br />
<br><br />
<br><br />
We aimed to confirm GFP-expression responsiveness and dependence of AHL, and how the physical proximity of this QS receiver to any QS sender (producer of homoserine lactone) is facilitated by surface-binding affects quorum signalling.<br />
<br><br />
<br><br />
<br />
<h3>Materials and Methods</h3><br />
<br />
T9002 <i>E. coli</i> transformants were grown in conjunction with other <i>E. coli</i> transformed with the AHL ‘Sender’ plasmid BBa_K1090000. BBa_K1090000 codes for AHL-synthesising enzymes, and also constitutively expresses RFP.<br />
<br><br />
<br><br />
<br />
<b><i>Poly-L-lysine Cell Adhesion</i></b><br />
<br><br />
To investigate QS signalling, and how it is affected by binding of sender and receiver bacteria to a solid surface, we immobilised our cells on a surface coated with poly-lysine: <br />
<br />
<br><br />
<br><br />
<br />
1. 50μl 0.01% Poly-Lysine was added to each well of a 96 well glass-bottom Plate (Whatman), and incubated at room temperature for 2 hours<br />
<br />
<br><br />
<br><br />
<br />
2. Excess poly-lysine was removed and the plate washed three times with sterile water and allowed to dry at room temperature.<br />
<br />
<br><br />
<br><br />
<br />
3. <i>E. coli</i> cultures were grown overnight in a 37C shaking water bath, and diluted to an optical density-600nm(OD600) of 0.02. <br />
<br />
<br><br />
<br><br />
<br />
4. K1090000 ‘Sender’ transformants washed by centrifugation at 13000rpm for 1minute and resuspension in phosphate buffered saline (PBS), three times.<br />
<br />
<br><br />
<br><br />
<br />
5. Dual Sender-Receiver wells were diluted and combined in a colorimetric ratio to achieve a total OD600 of 0.02, with a range of 1:1 to 1:10,000,000 Sender:Receiver. 50μl total volume in Liquid Broth (LB) used per well. Incubated at 4C for 1 hour.<br />
<br />
<br><br />
<br><br />
<br />
6. Unbound cells removed by lightly shaking over waste and washed three times with Phosphate-Buffered Saline (PBS). 50μl fresh LB medium was added to each well.<br />
<br />
<br><br />
<br><br />
<br />
7. A FluoSTAR OPTIMA Fluoresence Plate Reader was used to measure Red(Excitation 544nm/Emission 612nm) and Green(Excitation 485nm/Emission 520nm) Fluorescence was recorded every 5 minutes for 7 hours; this incubates the samples at 37 degrees C and shakes (1mm double-orbital) for 30 seconds before each read.<br />
<br />
<br><br><br />
<h3>Results</h3><br />
<br />
<b><i>K1090000 RFP expression</b></i> RFP expression of K1090000 transformed <i>E. coli</i> was compared against untransformed XL1-Blue <i>E. coli</i> negative control and an RFP pSB1C3 plasmid positive control. Analysis of red fluorescence using a fluorimeter plate reader clearly shows that K1090000 constitutively expressed moderate amounts of RFP (Figure 1). <br />
<br><br />
<br><br />
<b><i>T9002 with AHL</b></i><br />
<br><br />
T9002 <i>E. coli</i> was resuspended in conditioned growth medium containing AHL, derived from a filter sterilised K1090000 suspension. The T9002 transformants in this medium as expected displayed a slow, exponential increase in GFP, indicating the T9002 receiver cells were responding to the QS signalling caused by AHL in the medium. However, QS responses by the T9002 receivers were even more marked when T9002 transformants were co-cultured with K109000 QS sender transformants (Figure 2). <br />
<br />
<br><br />
<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/FIG1-T9002.png"><br />
<br><br />
<font size="2">Figure 1 - K1090000 <i>E. coli</i> constitutively expresses RFP. Red (Excitation 544nm/Emission 612nm) fluorescence of K1090000 <i>E. coli</i> (triangles), RFP positive control pSB1C3 (Circles), untransformed XL1-Blue <i>E. coli</i> (dashes).<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<b><i>T9002 with K1090000 </b></i> We observed that optimal conditions for absolute green fluorescent production by T9002 receiver transformants were a ‘sender’ (S) to ‘receiver’ (R) ratio of 1:100; Figure 3. It was inferred that ratios greater or smaller than this resulted in too little AHL or undesirable competition effects, preventing optimal GFP fluorescence. An untransformed XL1-Blue <i>E. coli</i> (X) acted as control.<br />
<br />
<br><br />
<br />
T9002 Receivers in the presence of K1090000 Sender exhibit significantly higher GFP response than T9002 with a control (non-AHL expressing untransformed XL1-Blue <i>E. coli</i>); Figure 4. <br />
<br />
<br><br />
<br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/7/7a/FIG2-T9002.png"><br />
<br><br />
<font size="2">Figure 2 – Production of GFP by T9002, induced either by filtered AHL-containing culture medium, or by actively growing K1090000 sender transformants. AHL derived from culture medium is sufficient for slow rate T9002 Receiver GFP production (-), although T9002 GFP production was more efficient when paired with actively-growing K1090000 senders (circles). Mean GFP fluorescence of <i>E.coli</i> free in 50μl suspensions incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/c/c9/FIG3-T9002.png"><br />
<br><br />
<font size="2">Figure 3 – Greatest absolute fluorescence by T9002 transformants is observed in a Sender (S) 1:100 Receiver (R) cell number initial ratio. T9002 Receiver-produced GFP was compared with that produced when paired with an untransformed XL1-Blue <i>E. coli</i> (X) control or K1090000 Senders (circles), in triplicate. 50μl suspensions were incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/FIG4-T9002.png"><br />
<br><br />
<font size="2">Figure 4 – K1090000 Sender <i>E. coli</i> induction increased GFP expression in T9002 Receivers. Growth started in cell number ratios of aliquots totalling OD600 0.02 densities, suspended in liquid LB. Untransformed XL1-Blue:Receiver 1:100 ratio (Filled Diamonds), Sender:Receiver 1:100 ratio (Open circles). Values are blank corrected.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<i><b>T9002 and K1090000 bound to a Poly-Lysine surface; </i></b> Overall, immobilised Sender/Receiver pairs exhibited a greater extended rate of GFP production response, and higher absolute response after 7 hours than sender-receiver pairs free in suspension; Figure 5.<br />
<br />
<br><br />
<br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/FIG5-T9002.png"><br />
<br><br />
<font size="2">Figure 5: Surface-immobilisation of sender-receiver QS tranformants results in improved GFP expression. Poly-L-lysine wells (triangles) had greater rate of response compared to cells free in suspension (circles). This was performed in a Sender 1:10 Receiver cell number ratio.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<h3>Conclusions</h3><br />
<br />
• BBa_K1090000 ‘Sender’ is an effective AHL expressor that is able to activate BBa_T9002 ‘Receiver’ GFP production. <br />
<br><br />
• BBa_K1090000 has moderate constitutive RFP expression.<br />
<br><br />
• BBa_T9002 is an AHL receiver coupled to a GFP reporter, it has ‘leaky’ GFP expression. <br />
<br><br />
• GFP production significantly increases in the presence of AHL.<br />
<br><br />
• Poly-Lysine surface-immobilisation of T9002 and K1090000 <i>E. coli</i> extends the longterm production rate of GFP expression.<br />
<br><br />
<br><br />
<br><br />
<br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000Team:Aberdeen Scotland/Parts/ 00002014-10-18T02:11:44Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
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<h1>Our characterisation of existing BioBrick K1090000 </h1><br />
<h2>AHL signal sender with RFP reporter under lac promoter control</h2><br />
<br><br />
<h3>Aims and Rationale</h3><br />
<br />
BioBrick BBa_K1090000 on a pSB1C3 backbone is a composite part encoding a quorum sensing (QS) sender driving production expression of AHL (N-acyl homoserine lactone), while also constitutively expressing red fluorescent protein (RFP). <br />
<br><br />
<br><br />
We aimed to confirm the constitutive RFP-expression, but also the ability of this composite part to direct the production of biologically active AHL. In combination with an AHL-responsive QS receiver BioBrick (T9002), we also aimed to investigate how the efficiency of QS signalling was positively or negatively affected by the physical proximity of this QS sender to any QS receiver (responder to homoserine lactone), through coincident surface-binding of the sender and receiver bacteria.<br />
<br><br />
Since the obvious way to audit the effectiveness of the QS Sender K1090000 BioBrick was to use a QS Receiver, we combined our experimental assessment of K1090000 and T9002 in a series of joint experiments. Therefore some of the experiments reported under K1090000 are the same as those reported for T9002 (see adjacent Experience tab, this section of the Wiki).<br />
<br><br><br />
<br />
<h3>Materials and Methods</h3><br />
<br />
K1090000 <i>E. coli</i> transformants were grown in conjunction with other <i>E. coli</i> transformed with the AHL ‘Receiver’ plasmid BBa_T9002. BBa_K1090000 codes for AHL-synthesising enzymes, and also constitutively expresses RFP.<br />
<br><br />
<br><br />
<br />
<b><i>Poly-L-lysine Cell Adhesion</i></b><br />
<br><br />
To investigate QS signalling, and how it is affected by binding of sender and receiver bacteria to a solid surface, we immobilised our cells on a surface coated with poly-lysine: <br />
<br />
<br><br />
<br><br />
<br />
1. 50μl 0.01% Poly-Lysine was added to each well of a 96 well glass-bottom Plate (Whatman), and incubated at room temperature for 2 hours<br />
<br />
<br><br />
<br><br />
<br />
2. Excess poly-lysine was removed and the plate washed three times with sterile water and allowed to dry at room temperature.<br />
<br />
<br><br />
<br><br />
<br />
3. <i>E. coli</i> cultures were grown overnight in a 37C shaking water bath, and diluted to an optical density-600nm(OD600) of 0.02. <br />
<br />
<br><br />
<br><br />
<br />
4. K1090000 ‘Sender’ transformants washed by centrifugation at 13000rpm for 1minute and resuspension in phosphate buffered saline (PBS), three times.<br />
<br />
<br><br />
<br><br />
<br />
5. Dual Sender-Receiver wells were diluted and combined in a colorimetric ratio to achieve a total OD600 of 0.02, with a range of 1:1 to 1:10,000,000 Sender:Receiver. 50μl total volume in Liquid Broth (LB) used per well. Incubated at 4C for 1 hour.<br />
<br />
<br><br />
<br><br />
<br />
6. Unbound cells removed by lightly shaking over waste and washed three times with Phosphate-Buffered Saline (PBS). 50μl fresh LB medium was added to each well.<br />
<br />
<br><br />
<br><br />
<br />
7. A FluoSTAR OPTIMA Fluoresence Plate Reader was used to measure Red(Excitation 544nm/Emission 612nm) and Green(Excitation 485nm/Emission 520nm) Fluorescence was recorded every 5 minutes for 7 hours; this incubates the samples at 37 degrees C and shakes (1mm double-orbital) for 30 seconds before each read.<br />
<br />
<br><br><br />
<h3>Results</h3><br />
<br />
<b><i>K1090000 RFP expression</b></i> RFP expression of K1090000 transformed <i>E. coli</i> was compared against untransformed XL1-Blue <i>E. coli</i> negative control and an RFP pSB1C3 plasmid positive control. Analysis of red fluorescence using a fluorimeter plate reader clearly shows that K1090000 constitutively expressed moderate amounts of RFP (Figure 1).<br />
<br />
<br />
<br />
<br />
<br />
<b><i>K109000 is an efficient producer of QS-active AHL</b></i><br />
<br><br />
K1090000 transformants were grown overnight as a source of AHL. Medium from this culture was taken after the K1090000 cells had been removed by centrifugation and filter sterilisation. <i>E. coli</i> T9002 transformants (QS Receiver producing GFP in response to a QS signal) was resuspended in conditioned growth medium containing AHL, derived from the filter sterilised K1090000 suspension. The T9002 transformants in this medium as expected displayed a slow, exponential increase in GFP, indicating the T9002 receiver cells were responding to the QS signalling caused by AHL in the medium. However, QS responses by the T9002 receivers were even more marked when T9002 transformants were co-cultured with K109000 QS sender transformants (Figure 2). <br />
<br />
<br><br />
<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/FIG1-T9002.png"><br />
<br><br />
<font size="2">Figure 1 - K1090000 <i>E. coli</i> constitutively expresses RFP. Red (Excitation 544nm/Emission 612nm) fluorescence of K1090000 <i>E. coli</i> (triangles), RFP positive control pSB1C3 (Circles), untransformed XL1-Blue <i>E. coli</i> (dashes).<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<b><i>T9002 with K1090000 </b></i> We observed that optimal conditions for absolute green fluorescent production by T9002 receiver transformants were a K1090000 ‘sender’ (S) to ‘receiver’ (R) ratio of 1:100; Figure 3. It was inferred that ratios greater or smaller than this resulted in too little AHL or undesirable competition effects, preventing optimal GFP fluorescence. An untransformed XL1-Blue <i>E. coli</i> (X) acted as control.<br />
<br />
<br><br />
<br />
T9002 Receivers in the presence of K1090000 Sender exhibit significantly higher GFP response than T9002 with a control (non-AHL expressing untransformed XL1-Blue <i>E. coli</i>); Figure 4. This clearly shows the the K1090000 QS Senders were effective and efficient producers of AHL.<br />
<br />
<br><br />
<br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/7/7a/FIG2-T9002.png"><br />
<br><br />
<font size="2">Figure 2 – Production of GFP by T9002 Receiver, driven by either by filtered AHL-containing culture medium derived from K1090000 cultures, or by actively growing K1090000 sender transformants. AHL derived from culture medium is sufficient for slow rate T9002 Receiver GFP production (-), although T9002 GFP production was more efficient when paired with actively-growing K1090000 senders (circles). Mean GFP fluorescence of <i>E.coli</i> free in 50μl suspensions incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/c/c9/FIG3-T9002.png"><br />
<br><br />
<font size="2">Figure 3 – Greatest absolute fluorescence by T9002 transformants is observed in a Sender (S) 1:100 Receiver (R) cell number initial ratio. T9002 Receiver-produced GFP was compared with that produced when paired with an untransformed XL1-Blue <i>E. coli</i> (X) control or K1090000 Senders (circles), in triplicate. 50μl suspensions were incubated at 37C, 1mm double-orbital shaking for 30 seconds every 5 minutes for 355 minutes.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/9/91/FIG4-T9002.png"><br />
<br><br />
<font size="2">Figure 4 – K1090000 Sender <i>E. coli</i> induction increased GFP expression in T9002 Receivers. Growth started in cell number ratios of aliquots totalling OD600 0.02 densities, suspended in liquid LB. Untransformed XL1-Blue:Receiver 1:100 ratio (Filled Diamonds), Sender:Receiver 1:100 ratio (Open circles). Values are blank corrected.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<i><b>T9002 and K1090000 bound to a Poly-Lysine surface; </i></b> Overall, immobilised Sender/Receiver pairs exhibited a greater extended rate of GFP production response, and higher absolute response after 7 hours than sender-receiver pairs free in suspension; Figure 5.<br />
<br />
<br><br />
<br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/FIG5-T9002.png"><br />
<br><br />
<font size="2">Figure 5: Surface-immobilisation of sender-receiver QS tranformants results in improved GFP expression. Poly-L-lysine wells (triangles) had greater rate of response compared to cells free in suspension (circles). This was performed in a Sender 1:10 Receiver cell number ratio.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<h3>Conclusions</h3><br />
<br />
• BBa_K1090000 ‘Sender’ is an effective AHL expressor that is able to activate BBa_T9002 ‘Receiver’ GFP production. <br />
<br><br />
• BBa_K1090000 has moderate constitutive RFP expression.<br />
<br />
<br><br />
• Poly-Lysine surface-immobilisation of T9002 and K1090000 <i>E. coli</i> extends the longterm production rate of GFP expression.<br />
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<li class="curr"><a class="curr" href="#">Created</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li><br />
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<li class="curr"><a class="curr" href="#">Improved</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li><br />
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<div class="t_overview"><br><br />
<h1>Our characterisation of existing BioBrick BBa_K346007</h1><br />
<h2>Antigen 43</h2><br />
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<br />
<br />
<h3>Aim</h3><br />
To verify Assembly Standard 10 compliance<br><br><br />
<br />
<h3>Method</h3><br />
<u>Standard restriction digest and gel electrophoresis</u><br><br />
Escherichia coli bacterial culture containing plasmids BBa_K346007 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 37370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/d/dd/Restriction_digest_table.png"><br />
</center><br />
<br><br />
Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K346007 insert from the pSB1C3 plasmid backbone. Digests were incubated at 37370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.<br><br><br />
<br />
<h3>Results</h3><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/0/06/Restriction_digest_on_BBa_K346007.png"><br><br />
<font size="2">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Gel electrophoresis on standard restriction digests of BBa_K346007<br />
</font><br />
</center><br />
<br><br />
<br />
Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.<br />
<br><br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/d/dd/Restriction_digest_table.jpg"><br><br />
<font size="2">Fig.2&nbsp;&nbsp;&nbsp;&nbsp;Fragment sizes resulted from standard restriction digests on BBa_K346007.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<h3>Results</h3><br />
The pSB1C3-BBa_K346007 construct was shown to contain 6 additional PstI sites, as found in the wild-type gene of Antigen 43. This part is the basic part of BBa_K759001 Biobrick, which we have also shown that contains the same PstI sites (see Experience section of BBa_K759001).<br><br />
Thus we conclude that the BioBrick BBa_K346007 originally deposited with iGEM did not meet assembly standard 10, and the 6 native PstI sites present in Ag43 had not in fact been removed as stated in the original documentation.<br><br />
Based on the results mentioned above, pSB1C3-BBa_K759001 has been chosen as the subject for removal of the PstI sites to ensure that an Ag43 BioBrick is created that meets Assembly Standard 10 requirements (see the RFC10 compatible version of this Biobrick created by 2014 iGEM Aberdeen team:BBa_K1352000)<br><br><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009Team:Aberdeen Scotland/Parts/ 20092014-10-18T02:11:17Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li><br />
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li><br />
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li><br />
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<div class="t_overview"><br />
<h1><br>Our characterisation of existing BioBrick BBa_K542009</h1><br />
<h2>pLacI Regulated AG43</h2><br />
<!-- <h3></h3> --><br />
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<br />
<br />
<br />
<h3>Aim</h3><br />
To verify Assembly Standard 10 compliance<br><br><br />
<br />
<h3>Method</h3><br />
Escherichia coli bacterial culture containing plasmids BBa_K542009 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 37<sup>0</sup>C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/d/dd/Restriction_digest_table.png"></center><br />
<br><br />
<br />
Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K542009 insert from the pSB1C3 plasmid backbone. Digests were incubated at 370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.<br><br><br><br />
<br />
<h3>Results</h3><br />
<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/4/48/Restriction_digest_on_BBa_K542009.png"><br />
<br><br />
<font size="2">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Gel electrophoresis on standard restriction digests of BBa_K542009<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.<br><br><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/7/7e/Fragment_sizes_BBa_K542009.png"><br />
<br><br />
<font size="2">Fig.2&nbsp;&nbsp;&nbsp;&nbsp;Fragment sizes resulted from standard restriction digests on BBa_K542009.<br />
</font><br />
</center><br />
<br><br><br />
<br />
<br />
<br />
<h3>Conclusions</h3><br />
The gel results for the pSB1C3- BBa_K542009 construct suggested that the sequence has a deletion of approximately 1.2 kb, when compared to the theoretical map. Additionally, this Biobrick contains an extra XbaI site. Thus we conclude that the BioBrick BBa_K542009 originally deposited with iGEM did not meet assembly standard 10.<br><br />
Based on the results mentioned above, pSB1C3- BBa_K759001 has been chosen as the subject for removal of the PstI sites to ensure that an Ag43 BioBrick is created that meets Assembly Standard 10 requirements (see the RFC10 compatible version of this Biobrick created by 2014 iGEM Aberdeen team:BBa_K1352000)<br><br><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001Team:Aberdeen Scotland/Parts/ 90012014-10-18T02:11:03Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts">Background</a></li><br />
<li class="curr"><a class="curr" href="#">Created</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/Device">Device Data</a></li><br />
<li class="curr"><a class="curr" href="#">Improved</a></li><br />
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li><br />
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<h1><br>Our characterisation of existing BioBrick BBa_K759001</h1><br />
<h2>Aggregation Module inducible by arabinose in E.coli</h2><br />
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<h3>Aim</h3><br />
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To verify Assembly Standard 10 compliance<br><br><br />
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<h3>Methods</h3><br />
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<u>1. Standard restriction digest and gel electrophoresis</u><br><br />
Escherichia coli bacterial culture containing plasmids BBa_K759001 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:<br><br><br />
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<img src="https://static.igem.org/mediawiki/parts/d/dd/Restriction_digest_table.png"><br />
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Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K759001 insert from the pSB1C3 plasmid backbone. Digests were incubated at 37370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.<br><br><br />
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<br />
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<u>2. Sequencing data</u><br><br />
The plasmid was sequenced by the DNA Sequencing Services at University of Dundee .Sequencing analysis was performed on BBa_K759001 to confirm the suspected additional PstI sites. 6 sequencing primers (see Fig.1) were designed that were located evenly across BBa_K759001 (sequence data was taken from the Registry of Standard Biological Parts) plus both BBa_G00100 (forward primer that amplifies from the prefix) and BBa_G00101 (reverse primer that amplifies from the suffix), as defined by iGEM, were used to sequence Ag43 flanked by the prefix and the suffix.<br />
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<center><br />
<img src="https://static.igem.org/mediawiki/parts/5/51/Sequencing_primers.png"><br />
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<h3> Results</h3><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/parts/c/c1/Restriction_digest_on_BBa_K759001.png"><br />
<br><br />
<font size="2">Fig.1&nbsp;&nbsp;&nbsp;&nbsp;Gel electrophoresis on standard restriction digests of BBa_K759001<br>Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.<br />
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</center><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/parts/2/28/Fragment_sizes_BBa_K759001.png"><br />
<br><br />
<font size="2">Fig.2&nbsp;&nbsp;&nbsp;&nbsp; Fragment sizes resulted from standard restriction digests on BBa_K759001<br />
</center><br />
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Assembly of the sequencing data was performed manually using the CLUSTAL OMEGA multiple alignment program. The resulting sequence (query) was then compared against the original sequence for BBa_K759001 (subject) (sequence of 4493 bp, as taken from the Registry of Standard Biological Parts), using BLAST Pairwise sequence alignment viewer:<br><br><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/parts/9/9b/Assembly_sequence_BBa_K759001.png"><br />
</center><br />
<br><br><br />
Based on the sequencining data, a PstI restriction map of the pSB1C3-BBa_K759001 construct was drawn using SnapGene Viewer:<br><bR><br />
<center><br />
<img src="https://static.igem.org/mediawiki/parts/a/a4/Plasmid_map_BBak759001.png"><br />
</center><br />
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<h3>Conclusions</h3><br />
Both restriction digest and sequencing data show that biobrick BBa_K759001 contains 6 additional PstI sites (CTGCA^G) within the coding region of Antigen 43. Data suggests that the theoretical removal of the 6 additional sites from the coding region of Ag43 within BBa_K759001 is not confirmed by sequencing; therefore the construct is not standardized.<br><br />
Based on the availability of the sequencing data and the results mentioned above, pSB1C3- BBa_K759001 has been chosen as the subject for removal of the PstI sites to ensure that an Ag43 BioBrick is created that meets Assembly Standard 10 requirements (see the RFC10 compatible version of this Biobrick created by 2014 iGEM Aberdeen team: Bba_K1352000)<br><br><br />
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</html></div>Kgizdovhttp://2014.igem.org/Team:Aberdeen_Scotland/Parts/DeviceTeam:Aberdeen Scotland/Parts/Device2014-10-18T02:10:49Z<p>Kgizdov: </p>
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<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts">Background</a></li><br />
<li class="curr"><a class="curr" href="#">Created</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2000">Bba_K1352000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2001">Bba_K1352001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2002">Bba_K1352002</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2003">Bba_K1352003</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2004">Bba_K1352004</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2006">Bba_K1352006</a></li><br />
<li class="curr"><a class="curr" href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/Device">Device Data</a></li><br />
<li class="curr"><a class="curr" href="#">Improved</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9001">Bba_K759001</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_2009">Bba_K542009</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_6007">Bba_K346007</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_0000">Bba_K1090000</a></li><br />
<li><a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts/_9002">Bba_T9002</a></li><br />
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<h1>Detector Test Data</h1><br />
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<p>The initial concept of the fluorescence detector device had a very rough design. Thus it was built from bits and pieces in order to do a preliminary test and<br />
verify its viability.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Rough_set.jpg"><br />
<br />
<p>The preliminary data from the tests showed that the detector was able to differentiate between GFP producing and non-producing bacteria.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/e/e9/Lb_vs_gfp.png"><br />
<br />
<p>Fig.1 Series dilutions to estimate detector sensitivity</p><br />
<br />
<p>From Fig.1 we can see that the detector can easily distinguish the GFP producing culture from below 1/10 dilutions. Thus we continued on and improved the design by soldering some of the circuit and making it more compact. Then we made more test with the Sender and Receiver bacteria.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Send_rec_vs.png"><br />
<p>Fig.2 Detector response to Sender and Receiver cultures</p><br />
<br />
<p>Again the detector is sensitive enough to pick up the GFP in the Receiver.</p><br />
<br />
<p>For the final test, we made clean overnight cultures of the Receiver and Sender and mixed them in a growing culture. This was done to see after how long the detector is going to pick up the GFP production. Hence, if our diagnostic method is going to work.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/79/Resp_time.png"><br />
<p>Fig.3 Detector response to QS GFP-producing Receiver and Sender culture, normalised against LB medium</p><br />
<br />
<p>Finally, the simple detector proved capable of detecting the QS culture, thus confirming our design success.</p><br />
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