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2015-02-20T13:09:01Z
<p>Isro: </p>
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Isro
http://2014.igem.org/Team:Sumbawagen/project/econey
Team:Sumbawagen/project/econey
2015-02-19T05:34:44Z
<p>Isro: </p>
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<h3>ECONEY Project</h3><br />
<h2>Catabolite Repression Phenomenon</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/0/07/Sumbawagen_catabolite_r.png"/><br></center><br />
<br />
<p>There is a unique phenomenon of E.coli activity called “Catabolite Repression”. Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes, even though the inducer of the pathway was present in the environment. </p><br />
<br />
<br />
<p>Glucose is known to repress a large number of inducible enzymes in many differents bacteria. Glucose represses the induction of inducible operons by inhibiting the synthesis of cyclic AMP (cAMP), a nucleotide that is required for the initiation of transcription of a large number of inducible enzyme systems.</p><br />
<br />
<p>The role of cyclic a cAMP is complicated. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNA polymerase to the promoter for the initiation of transcription. In the presence of glucose, cAMP levels are low, and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).<br><br />
<br />
<h2>Plasmid BBA_J04450</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/41/Sumbawagen_econeyred_p.png"/><br></center><br />
<p> We found plasmid BBa_J04450 in iGEM Parts Registry that works based on catabolite repression theory. This plasmid consists of lactose promoter, ribosome binding site, mutant red fluorescent protein gene or mRFP gene and terminator. The LacI promoter works in the presence of lactose, resulting red color in bacteria from mRFP gene expression. Unfortunately, the expression of mRFP gene decreases in the prescence of glucose, resulting yellow color in the bacteria. We use this plasmid to measure glucose concentration.</p><br />
<br />
<h2>Constructing Econey</h2><br />
<p> We faced a problem that in high glucose concentration, the red color from mRFP gene expression goes dimer, while we want the device to express brighter red color in high glucose concentration. Thus, we modified the system by changing sensitivity of catabolite repression in order to make the system has ability to express red color in high glucose concentration. </p><br />
<p> We add 2 new circuits behind plasmid BBa_J04450. The first circuit consists of constitutive promoter, RBS, adenylate cyclase (AC) gene, and a terminator. The second circuit consists of constitutive promoter, RBS, IIA(Glc) gene, and a terminator.</p><br />
<br />
<h2>ECONEY CIRCUITS</h2><br />
<p>Our ECONEY consists of 2 circuits.</p><br />
<strong>Circuit #1</strong><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/29/Sbgen.sir1.png"/><br></center><br />
<p>Circuit #1 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, CyaA gene, and a terminator. CyaA gene is controlled by constitutive promoter and located downstream of ECONEY Basic Circuit. Adenylate cyclase synthesize cAMP. Thus in high concentration of glucose, lac promoter may still active.</p><br><br />
<br />
<strong>Circuit #2</strong><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/56/Sbgen.sir2.png"/><br></center><br />
<p>Circuit #2 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, crr gene, and a terminator. Crr gene is also controlled by constitutive promoter and located downstream of ECONEY Basic Circuit. EIIA or IIAGlc in the PTS pathway helps glucose enter the cell. Thus in LOW concentration of glucose, lac promoter may already be repressed.</p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h>ECONEY Project</h><br />
<h2>Catabolite Repression Phenomenon</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/0/07/Sumbawagen_catabolite_r.png"/><br></center><br />
<br />
<p>There is a unique phenomenon of E.coli activity called “Catabolite Repression”. Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes, even though the inducer of the pathway was present in the environment. </p><br />
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<p>Glucose is known to repress a large number of inducible enzymes in many differents bacteria. Glucose represses the induction of inducible operons by inhibiting the synthesis of cyclic AMP (cAMP), a nucleotide that is required for the initiation of transcription of a large number of inducible enzyme systems.</p><br />
<br />
<p>The role of cyclic a cAMP is complicated. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNA polymerase to the promoter for the initiation of transcription. In the presence of glucose, cAMP levels are low, and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).<br><br />
<br />
<h2>Plasmid BBA_J04450</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/41/Sumbawagen_econeyred_p.png"/><br></center><br />
<p> We found plasmid BBa_J04450 in iGEM Parts Registry that works based on catabolite repression theory. This plasmid consists of lactose promoter, ribosome binding site, mutant red fluorescent protein gene or mRFP gene and terminator. The LacI promoter works in the presence of lactose, resulting red color in bacteria from mRFP gene expression. Unfortunately, the expression of mRFP gene decreases in the prescence of glucose, resulting yellow color in the bacteria. We use this plasmid to measure glucose concentration.</p><br />
<br />
<h2>Constructing Econey</h2><br />
<p> We faced a problem that in high glucose concentration, the red color from mRFP gene expression goes dimer, while we want the device to express brighter red color in high glucose concentration. Thus, we modified the system by changing sensitivity of catabolite repression in order to make the system has ability to express red color in high glucose concentration. </p><br />
<p> We add 2 new circuits behind plasmid BBa_J04450. The first circuit consists of constitutive promoter, RBS, adenylate cyclase (AC) gene, and a terminator. The second circuit consists of constitutive promoter, RBS, IIA(Glc) gene, and a terminator.</p><br />
<br />
<h2>ECONEY CIRCUITS</h2><br />
<p>Our ECONEY consists of 2 circuits.</p><br />
<strong>Circuit #1</strong><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/29/Sbgen.sir1.png"/><br></center><br />
<p>Circuit #1 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, CyaA gene, and a terminator. CyaA gene is controlled by constitutive promoter and located downstream of ECONEY Basic Circuit. Adenylate cyclase synthesize cAMP. Thus in high concentration of glucose, lac promoter may still active.</p><br><br />
<br />
<strong>Circuit #2</strong><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/56/Sbgen.sir2.png"/><br></center><br />
<p>Circuit #2 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, crr gene, and a terminator. Crr gene is also controlled by constitutive promoter and located downstream of ECONEY Basic Circuit. EIIA or IIAGlc in the PTS pathway helps glucose enter the cell. Thus in LOW concentration of glucose, lac promoter may already be repressed.</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/project/econey
Team:Sumbawagen/project/econey
2015-02-19T05:28:02Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h>ECONEY Project</h><br />
<h2>Catabolite Repression Phenomenon</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/0/07/Sumbawagen_catabolite_r.png"/><br></center><br />
<br />
<p>There is a unique phenomenon of E.coli activity called “Catabolite Repression”. Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes, even though the inducer of the pathway was present in the environment. </p><br />
<br />
<br />
<p>Glucose is known to repress a large number of inducible enzymes in many differents bacteria. Glucose represses the induction of inducible operons by inhibiting the synthesis of cyclic AMP (cAMP), a nucleotide that is required for the initiation of transcription of a large number of inducible enzyme systems.</p><br />
<br />
<p>The role of cyclic a cAMP is complicated. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNA polymerase to the promoter for the initiation of transcription. In the presence of glucose, cAMP levels are low, and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).<br><br />
<br />
<h2>Plasmid BBA_J04450</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/41/Sumbawagen_econeyred_p.png"/><br></center><br />
<p> We found plasmid BBa_J04450 in iGEM Parts Registry that works based on catabolite repression theory. This plasmid consists of lactose promoter, ribosome binding site, mutant red fluorescent protein gene or mRFP gene and terminator. The LacI promoter works in the presence of lactose, resulting red color in bacteria from mRFP gene expression. Unfortunately, the expression of mRFP gene decreases in the prescence of glucose, resulting yellow color in the bacteria. We use this plasmid to measure glucose concentration.</p><br />
<br />
<h2>Constructing Econey</h2><br />
<p> We faced a problem that in high glucose concentration, the red color from mRFP gene expression goes dimer, while we want the device to express brighter red color in high glucose concentration. Thus, we modified the system by changing sensitivity of catabolite repression in order to make the system has ability to express red color in high glucose concentration. </p><br />
<p> We add 2 new circuits behind plasmid BBa_J04450. The first circuit consists of constitutive promoter, RBS, adenylate cyclase (AC) gene, and a terminator. The second circuit consists of constitutive promoter, RBS, IIA(Glc) gene, and a terminator.</p><br />
<br />
<h2>ECONEY CIRCUITS</h2><br />
<p>Our ECONEY consists of 2 circuits.</p><br />
<strong>Circuit #1</strong><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/4a/Sumbawagen_circuit12.png"/><br></center><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/29/Sbgen.sir1.png"/><br></center><br />
<p>Circuit #1 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, AC gene, and a terminator. Adenylate Cyclase is an enzyme that can catalyse the shynthezise of cAMP and contain 848 amino acids. Adenylate cyclase will synthesize cAMP that can help lac promoter still active in high glucose concentration. Constitutive promoter is used to guarantee that AC gene will be always expressed. This circuit will allow the system to emit red color in high glucose concentration.</p><br><br />
<br />
<strong>Circuit #2</strong><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/56/Sbgen.sir2.png"/><br></center><br />
<p>Circuit #2 consists of plasmid BBa_J04450 followed by a new circuit that consist of constitutive promoter, RBS, IIAglc gene, and a terminator. IIAglc is one of sub unit of cytoplasmic enzyme that involve in Phosphotransferase system (PTS). Thus with even low glucose concentration outside the cell, more glucose may enter the cells.</p><br />
<br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>SUMBAWAGEN PROJECT OVERVIEW</h2><br />
<h3>Background</h3><br />
<br />
<p>Before taking you to the main description of our project for iGEM 2014, let us give you an introduction of our campus. Biotechnology Faculty, Sumbawa University of Technology is a new university established on May 2013 in Sumbawa Regency of West Nusa Tenggara Province of Indonesia. Located in the island of Sumbawa, the university stands as the only one university that provide Biotechnology education for eastern part of Indonesia. As the impact of the geographical position, Sumbawa receives limited access to the economic and education sector compare with the other neighboring islands especially Java.<br></p><br />
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<p>However, the island is famous for its honey production. Therefore, taking the opportunity of participating iGEM this year, we’d like to do a project in which we can apply synthetic biology which can contribute a positive impact to Sumbawa society not only in education sectors but also in other sectors like economic and politic. That’s how we came up with the idea of ECONEY.<br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/UTS_Campus.jpg"></center><br><br />
<center><strong>Picture 1. Campus view of Sumbawa University of Tecnology, the “Orange Building”</strong><br><br></center><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/20/UTS_Campus_2.jpg"></center><br><br />
<center><strong>Picture 2. Campus view of Sumbawa University of Tecnology, the “Blue Building”</strong><br><br></center><br />
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<p>ECONEY is engineered E. coli that capable to measure different level of glucose concentration in honey. BBa_J04450 has a lac promoter and mRFP gene, thus E. coli harboring this plasmid will give red color when the bacteria were grown with lactose or IPTG. Oppositely, the expression of mRFP will be decreased when glucose was present in medium through a mechanism called catabolite repression. We use this phenomenon to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. Creation of novel circuit which consists of constitutive promoter followed by either Adenylate Cyclase or IIA(Glc) gene, which placed downward lac-mRFP circuit, may affect the sensitivity of catabolite repression. We expect an engineered E. coli, which shows red color expression in different range of glucose concentration. By changing mRFP gene with amilCP, a blue fluorescent protein, we may obtain E. coli expressing either red or blue color when different honey added to the medium due to different glucose concentration.<br></p><br />
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<p>Even though Sumbawagen team consist of sophomore and in shortage condition to the facility and accessibility, that condition in fact push the team to think creatively in solving such limitation. For instance, inspite of using the refrigerated microsentrifuge for the transformation process we use mini centrifuge inside the refrigerator. We also created a homemade shaker for our cultivation project. Those conditions did not made our team lost the spirit to participate in the prestigious competition of iGEM (International Genetically Engineered Machine) 2014. We encourage ourself to overcome the obstacle in front of us, like the quote from our Rector “Where there is a will, there is a way”.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/22/Shaker.png"></center><br><br />
<center><strong>Picture 4. A homemade shaker that we use</strong><br><br></center><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>SUMBAWAGEN PROJECT OVERVIEW</h2><br />
<h3>Background</h3><br />
<br />
<p>Before taking you to the main description of our project for iGEM 2014, let us give you an introduction of our campus. Biotechnology Faculty, Sumbawa University of Technology is a new university established on May 2013 in Sumbawa Regency of West Nusa Tenggara Province of Indonesia. Located in the island of Sumbawa, the university stands as the only one university that provide Biotechnology education for eastern part of Indonesia. As the impact of the geographical position, Sumbawa receives limited access to the economic and education sector compare with the other neighboring islands especially Java.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/3/3f/Jawa-sumbawa.jpg"></center><br><br />
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<p>However, the island is famous for its honey production. Therefore, taking the opportunity of participating iGEM this year, we’d like to do a project in which we can apply synthetic biology which can contribute a positive impact to Sumbawa society not only in education sectors but also in other sectors like economic and politic. That’s how we came up with the idea of ECONEY.<br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/UTS_Campus.jpg"></center><br><br />
<center><strong>Picture 1. Campus view of Sumbawa University of Tecnology, the “Orange Building”</strong><br><br></center><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/20/UTS_Campus_2.jpg"></center><br><br />
<center><strong>Picture 2. Campus view of Sumbawa University of Tecnology, the “Blue Building”</strong><br><br></center><br />
<br />
<p>ECONEY is engineered E. coli that capable to measure different level of glucose concentration in honey. BBa_J04450 has a lac promoter and mRFP gene, thus E. coli harboring this plasmid will give red color when the bacteria were grown with lactose or IPTG. Oppositely, the expression of mRFP will be decreased when glucose was present in medium through a mechanism called catabolite repression. We use this phenomenon to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. Creation of novel circuit which consists of constitutive promoter followed by either Adenylate Cyclase or IIA(Glc) gene, which placed downward lac-mRFP circuit, may affect the sensitivity of catabolite repression. We expect an engineered E. coli, which shows red color expression in different range of glucose concentration. By changing mRFP gene with amilCP, a blue fluorescent protein, we may obtain E. coli expressing either red or blue color when different honey added to the medium due to different glucose concentration.<br></p><br />
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<p>Even though Sumbawagen team consist of sophomore and in shortage condition to the facility and accessibility, that condition in fact push the team to think creatively in solving such limitation. For instance, inspite of using the refrigerated microsentrifuge for the transformation process we use mini centrifuge inside the refrigerator. We also created a homemade shaker for our cultivation project. Those conditions did not made our team lost the spirit to participate in the prestigious competition of iGEM (International Genetically Engineered Machine) 2014. We encourage ourself to overcome the obstacle in front of us, like the quote from our Rector “Where there is a will, there is a way”.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/f/f0/Microcentrifuse.jpg.png"></center><br><br />
<center><strong>Picture 4. Mini centrifuge inside the refrigerator</strong><br><br></center><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/22/Shaker.png"></center><br><br />
<center><strong>Picture 5. A homemade shaker that we use</strong><br><br></center><br />
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<h2>SUMBAWAGEN PROJECT OVERVIEW</h2><br />
<h3>Background</h3><br />
<br />
<p>Before taking you to the main description of our project for iGEM 2014, let us give you an introduction of our campus. Biotechnology Faculty, Sumbawa University of Technology is a new university established on May 2013 in Sumbawa Regency of West Nusa Tenggara Province of Indonesia. Located in the island of Sumbawa, the university stands as the only one university that provide Biotechnology education for eastern part of Indonesia. As the impact of the geographical position, Sumbawa receives limited access to the economic and education sector compare with the other neighboring islands especially Java.<br></p><br />
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<p>However, the island is famous for its honey production. Therefore, taking the opportunity of participating iGEM this year, we’d like to do a project in which we can apply synthetic biology which can contribute a positive impact to Sumbawa society not only in education sectors but also in other sectors like economic and politic. That’s how we came up with the idea of ECONEY.<br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/UTS_Campus.jpg"></center><br><br />
<center><strong>Picture 1. Campus view of Sumbawa University of Tecnology, the “Orange Building”</strong><br><br></center><br />
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<center><strong>Picture 2. Campus view of Sumbawa University of Tecnology, the “Blue Building”</strong><br><br></center><br />
<br />
<p>ECONEY is engineered E. coli that capable to measure different level of glucose concentration in honey. BBa_J04450 has a lac promoter and mRFP gene, thus E. coli harboring this plasmid will give red color when the bacteria were grown with lactose or IPTG. Oppositely, the expression of mRFP will be decreased when glucose was present in medium through a mechanism called catabolite repression. We use this phenomenon to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. Creation of novel circuit which consists of constitutive promoter followed by either Adenylate Cyclase or IIA(Glc) gene, which placed downward lac-mRFP circuit, may affect the sensitivity of catabolite repression. We expect an engineered E. coli, which shows red color expression in different range of glucose concentration. By changing mRFP gene with amilCP, a blue fluorescent protein, we may obtain E. coli expressing either red or blue color when different honey added to the medium due to different glucose concentration.<br></p><br />
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<p>Even though Sumbawagen team consist of sophomore and in shortage condition to the facility and accessibility, that condition in fact push the team to think creatively in solving such limitation. For instance, inspite of using the refrigerated microsentrifuge for the transformation process we use mini centrifuge inside the refrigerator. We also created a homemade shaker for our cultivation project. Those conditions did not made our team lost the spirit to participate in the prestigious competition of iGEM (International Genetically Engineered Machine) 2014. We encourage ourself to overcome the obstacle in front of us, like the quote from our Rector “Where there is a will, there is a way”.</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/overviews/Econey_Project
Team:Sumbawagen/overviews/Econey Project
2015-02-19T04:10:26Z
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<h2>SUMBAWAGEN PROJECT OVERVIEW</h2><br />
<h3>Background</h3><br />
<br />
<p>Before taking you to the main description of our project for iGEM 2014, let us give you an introduction of our campus. Biotechnology Faculty, Sumbawa University of Technology is a new university established on May 2013 in Sumbawa Regency of West Nusa Tenggara Province of Indonesia. Located in the island of Sumbawa, the university stands as the only one university that provide Biotechnology education for eastern part of Indonesia. As the impact of the geographical position, Sumbawa receives limited access to the economic and education sector compare with the other neighboring islands especially Java.<br></p><br />
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<p>However, the island is famous for its honey production. Therefore, taking the opportunity of participating iGEM this year, we’d like to do a project in which we can apply synthetic biology which can contribute a positive impact to Sumbawa society not only in education sectors but also in other sectors like economic and politic. That’s how we came up with the idea of ECONEY.<br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/UTS_Campus.jpg"></center><br><br />
<center><strong>Picture 1. Campus view of Sumbawa University of Tecnology, the “Orange Building”</strong><br><br></center><br />
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<center><strong>Picture 2. Campus view of Sumbawa University of Tecnology, the “Blue Building”</strong><br><br></center><br />
<br />
<p>ECONEY is engineered E. coli that capable to measure different level of glucose concentration in honey. BBa_J04450 has a lac promoter and mRFP gene, thus E. coli harboring this plasmid will give red color when the bacteria were grown with lactose or IPTG. Oppositely, the expression of mRFP will be decreased when glucose was present in medium through a mechanism called catabolite repression. We use this phenomenon to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. Creation of novel circuit which consists of constitutive promoter followed by either Adenylate Cyclase or IIA(Glc) gene, which placed downward lac-mRFP circuit, may affect the sensitivity of catabolite repression. We expect an engineered E. coli, which shows red color expression in different range of glucose concentration. By changing mRFP gene with amilCP, a blue fluorescent protein, we may obtain E. coli expressing either red or blue color when different honey added to the medium due to different glucose concentration.<br></p><br />
<br />
<p>Even though Sumbawagen team consist of sophomore and in shortage condition to the facility and accessibility, that condition in fact push the team to think creatively in solving such limitation. For instance, inspite of using the refrigerated microsentrifuge for the transformation process we use mini centrifuge inside the refrigerator. We also created a homemade shaker for our cultivation project. Those conditions did not made our team lost the spirit to participate in the prestigious competition of iGEM (International Genetically Engineered Machine) 2014. We encourage ourself to overcome the obstacle in front of us, like the quote from our Rector “Where there is a will, there is a way”.</p><br />
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<br />
<br />
<h3>Project Idea</h3><br />
<p>Sumbawagen 2014 project is ECONEY (E. coli for Honey). ECONEY is engineered E.coli that capable to measure different level of glucose consentration in honey. BBa_J04450 has a lac promoter and mRFP gene, thus E. coli harboring this plasmid will give red color when the bacteria were grown with lactose or IPTG. Oppositely, the expression of mRFP will be decreased when glucose was present in medium through a mechanism called catabolite repression. We use this phenomenon to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. Creation of novel circuit which consists of constitutive promoter followed by either Adenylate Cyclase or IIAGlc genes, which placed downward lac-mRFP circuit, may affect the sensitivity of catabolite repression. We expect an engineered E. coli, which shows red color expression in different range of glucose concentration. By changing mRFP gene with amilCP, a blue fluorescent protein, we may obtain E. coli expressing either red or blue color when different honey added to the medium due to different glucose concentration.<br></p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/overviews/Econey_Project
Team:Sumbawagen/overviews/Econey Project
2015-02-19T04:08:02Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>SUMBAWAGEN PROJECT OVERVIEW</h2><br />
<h3>Background</h3><br />
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<p>Biotechnology Faculty, Sumbawa University of Technology (SUT) was a new university, establish at May 2013 in Sumbawa Regency of West Nusa Tenggara Province of Indonesia. An Island located in east part of Indonesia. <br></p><br />
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<p>As the impact of the geographical position according to the capital city of Indonesia which located Java island, Sumbawa has limited accessibility to the economic and education sector. There are only two university in Sumbawa, and the SUT in the one and the only technology university in Sumbawa which has Biotechnology Faculty as their prime major, especially in eastern part of Indonesia.<br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/UTS_Campus.jpg"></center><br><br />
<center><strong>Picture 1. Campus view of Sumbawa University of Tecnology, the “Orange Building”</strong><br><br></center><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/20/UTS_Campus_2.jpg"></center><br><br />
<center><strong>Picture 2. Campus view of Sumbawa University of Tecnology, the “Blue Building”</strong><br><br></center><br />
<br />
<p>Which these such condition Sumbawagen team started their project from the very fresh beginning. The basic reason that motivated the Sumbawagen team is back to their first survey on introducing synthetic biology to Sumbawa society at 2013. Based on the survey results we had conclusion that most of Sumbawa people are already in touch with various of biotechnology product in their daily life. On the other side this fact does not goes in the same direction with the understanding of Sumbawa society to the biotechnology products. This condition lead our team to go deeper into the topic which involves synthetic biology and the development prospect among Sumbawa society.<br></p><br />
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<p>Eventhough Sumbawagen team consist of sophomore and in shortage condition to the facility and accessibility such as, inspite of using the refrigerated microsentrifuge for the transformation process we using minicentrifuge inside the refrigerator. We also created home made shaker for our cultivation project. Those condition did not made our team lost the spirit to participated in the prestigious competition of IGEM (International Genetically Enginereed Machine) 2014. We encourage ourself to overcome the obstacle in front of us, like the quote from our Rector “ When there is a will, there is a way”. </p><br />
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<h3>Project Idea</h3><br />
<p>Sumbawagen 2014 project is ECONEY (E. coli for Honey). ECONEY is engineered E.coli that capable to measure different level of glucose consentration in honey. BBa_J04450 has a lac promoter and mRFP gene, thus E. coli harboring this plasmid will give red color when the bacteria were grown with lactose or IPTG. Oppositely, the expression of mRFP will be decreased when glucose was present in medium through a mechanism called catabolite repression. We use this phenomenon to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. Creation of novel circuit which consists of constitutive promoter followed by either Adenylate Cyclase or IIAGlc genes, which placed downward lac-mRFP circuit, may affect the sensitivity of catabolite repression. We expect an engineered E. coli, which shows red color expression in different range of glucose concentration. By changing mRFP gene with amilCP, a blue fluorescent protein, we may obtain E. coli expressing either red or blue color when different honey added to the medium due to different glucose concentration.<br></p><br />
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<h2>Result – 2. Confirmation of DNA sequence</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/7/73/Sumbawagen_result2_edit.png"></center><br><br />
<center><strong>Figure 1. 2AGL_DT”</strong><br><br></center><br />
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<p>As shown in Figure 1 (2AGL_DT), we confirmed the construction of crr gene encoding IIA(Glc) from E. coli into Biobrick format – flanking by restriction sites of EcoRI and XbaI at the prefix, and SpeI and PstI at the suffix – followed by the sequence of double terminator (BBa_B0015). The other constructs which able to be confirmed by DNA sequencing was the parental plasmid containing crr gene only. While the rest of plasmids did not contain expected sequence.</p><br />
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<p>While the final construct to be expected, namely BBa_J04450 followed by constitutive promoter/RBS/crr/double terminator was actually the sequence of BBa_J04450 only as shown in figure below. The data from forward sequence and reverse sequence (Figure 2; J04450_CP_RB_2AGL_DT REV/FWD) revealed the prefix and suffix sequences clearly.</p><br />
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<center><strong>Figure 2. J04450_CP_RB_2AGL_DT REV/FWD”</strong><br><br></center><br />
<br />
<p>However, we can confirm the sequence of basic part in our project namely BBa_J23119 (constitutive promoter), BBa_B0034 (RBS) and BBa_B0015 (double terminator) alone. Thus for future works, we can confirm to use the plasmid for construction.</p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Result – 2. Confirmation of DNA sequence</h2><br />
<center><img src="https://static.igem.org/mediawiki/2014/7/73/Sumbawagen_result2_edit.png"></center><br><br />
<center><strong>Figure 1. 2AGL_DT”</strong><br><br></center><br />
<br />
<p>As shown in Figure 1 (2AGL_DT), we confirmed the construction of crr gene encoding IIA(Glc) from E. coli into Biobrick format – flanking by restriction sites of EcoRI and XbaI at the prefix, and SpeI and PstI at the suffix – followed by the sequence of double terminator (BBa_B0015). The other constructs which able to be confirmed by DNA sequencing was the parental plasmid containing crr gene only. While the rest of plasmids did not contain expected sequence.</p><br />
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<p>While the final construct to be expected, namely BBa_J04450 followed by constitutive promoter/RBS/crr/double terminator was actually the sequence of BBa_J04450 only as shown in figure below. The data from forward sequence and reverse sequence (Figure 2; J04450_CP_RB_2AGL_DT FWD/REV) revealed the prefix and suffix sequences clearly.</p><br />
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<center><strong>Figure 2. J04450_CP_RB_2AGL_DT FWD/REV”</strong><br><br></center><br />
<br />
<p>However, we can confirm the sequence of basic part in our project namely BBa_J23119 (constitutive promoter), BBa_B0034 (RBS) and BBa_B0015 (double terminator) alone. Thus for future works, we can confirm to use the plasmid for construction.</p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<p>Moyo festival is an annual Sumbawa citizen festival held every year. This year the festival held from September 27th - October 6th, 2014 at Pahlawan Square of Sumbawa Besar and Moyo Island. This festival is eagerly awaited by the people of Sumbawa and even attract domestic and international tourist. People gather to get some fun and to learn about Sumbawa culture. <br></p><br />
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<p>On the third night (September 29 2014) we got the opportunity to introduce synthetic biology and Sumbawagen project to the society because our project is related to Sumbawa honey. We also present some photos of Sumbawagen team activities and introduce some of the tools that we have such as PCR, micropipette, microscope, etc. Visitors were taking turn asking questions in the middle of the crowd that night. We hope that after this event the public will have better understand of synthetic biology and tools related to synthetic biology, to improve knowledge of Sumbawa society.<br></p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Project/Result/Results5
Team:Sumbawagen/Project/Result/Results5
2015-02-19T03:14:06Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Result – 5. Confirmation of protein expression by SDS-PAGE</h2><br />
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<p>The figure here showed data of SDSPAGE. ACFL is sample from E. coli BL21(DE3) supposed to express mRFP and full length Adenylate cyclase. 2AGL is sample from E. coli BL21(DE3) supposed to express mRFP and IIA(Glc). While CTRL is sample from E. coli BL21(DE3) supposed to express mRFP. </p><br />
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<p>Protein band corresponding to mRFP was clearly shown in either total proteins or soluble proteins which mean that mRFP was expressed as soluble protein. Meanwhile protein band corresponding to Adenylate cyclase could not be observed. Protein band corresponding to IIA(Glc) seemed to present.</p><br />
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http://2014.igem.org/Team:Sumbawagen/Project/Result/Results2
Team:Sumbawagen/Project/Result/Results2
2015-02-19T03:13:51Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Result – 2. Confirmation of DNA sequence</h2><br />
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<p>As shown in Figure 1 (2AGL_DT), we confirmed the construction of crr gene encoding IIA(Glc) from E. coli into Biobrick format – flanking by restriction sites of EcoRI and XbaI at the prefix, and SpeI and PstI at the suffix – followed by the sequence of double terminator (BBa_B0015). The other constructs which able to be confirmed by DNA sequencing was the parental plasmid containing crr gene only. While the rest of plasmids did not contain expected sequence.</p><br />
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<p>While the final construct to be expected, namely BBa_J04450 followed by constitutive promoter/RBS/crr/double terminator was actually the sequence of BBa_J04450 only as shown in figure below. The data from forward sequence and reverse sequence (Figure 2; J04450_CP_RB_2AGL_DT FWD/REV) revealed the prefix and suffix sequences clearly.</p><br />
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<p>However, we can confirm the sequence of basic part in our project namely BBa_J23119 (constitutive promoter), BBa_B0034 (RBS) and BBa_B0015 (double terminator) alone. Thus for future works, we can confirm to use the plasmid for construction.</p><br />
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<h2>Result – 2. Confirmation of DNA sequence</h2><br />
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<p>As shown in Figure 1 (2AGL_DT), we confirmed the construction of crr gene encoding IIA(Glc) from E. coli into Biobrick format – flanking by restriction sites of EcoRI and XbaI at the prefix, and SpeI and PstI at the suffix – followed by the sequence of double terminator (BBa_B0015). The other constructs which able to be confirmed by DNA sequencing was the parental plasmid containing crr gene only. While the rest of plasmids did not contain expected sequence.</p><br />
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<p>While the final construct to be expected, namely BBa_J04450 followed by constitutive promoter/RBS/crr/double terminator was actually the sequence of BBa_J04450 only as shown in figure below. The data from forward sequence and reverse sequence (Figure 2; J04450_CP_RB_2AGL_DT FWD/REV) revealed the prefix and suffix sequences clearly.</p><br />
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<p>However, we can confirm the sequence of basic part in our project namely BBa_J23119 (constitutive promoter), BBa_B0034 (RBS) and BBa_B0015 (double terminator) alone. Thus for future works, we can confirm to use the plasmid for construction.</p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2><b>Result – 1. Construction of BBa_J04450 + constitutive promoter/crr or cyaA</b></h2><br />
<br />
<br />
<p>We planned to construct BBa_J04450 as circuit #1 (original circuit) followed downstream by our original circuit #2. Circuit #2 is composed by BBa_J2319 (constitutive promoter) / BBa_B0034 (RBS) / crr (IIA(Glc) or cyaA (Adenylate cyclase) / BBa_B0015 (double terminator).</p><br />
<br />
<p>So the first step was to clone either cyaA or crr from E. coli to Biobrick construct. PCR fragments with correct size were able to be obtained. After direct restriction with EcoRI and SpeI, the fragments were cloned into pSB1C3 backbone. </p><br />
<br />
<p>Next step was to construct, BBa_J2319 (chloramphenicol) / BBa_B0034 (ampicillin) and crr or cyaA (chloramphenicol) / BBa_B0015 (chloramphenicol). For BBa_J2319 / BBa_B0034 construction, prefix insertion was used. Thus ligation product was screened on ampicillin LB agar plate. While for crr or cyaA / BBa_B0015 construction, suffix insertion was used because the large size of crr or cyaA could be isolated by agarose gel excision. After BBa_J2319 / BBa_0034 (ampicillin) and crr or cyaA / BBa_0015 (chloramphenicol) constructs were obtained, prefix insertion was done to get BBa_J2319 / BBa_B0034 / crr or cyaA / BBa_B0015 in pSB1C3 backbone. Final step was prefix insertion of BBa_J00450 the original constructs.</p><br />
<br />
<p>In every steps of works, we only confirmed the insertion by colony selection in appropriate antibiotics containing LB agar plate, and purification of backbone plasmid for insertion by gel electrophoresis to completely separated small insert fragments. Thus in theory there will be no self-ligation. However for either prefix or suffix inserts, the digested samples were not purified, instead used directly for ligation reaction. </p><br />
<br />
<p>Below some representative gel electrophoresis data were shown.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/24/Project_-_1_fig_1.jpg"></center><br><br />
<center>Figure 1. Cloning of adenylate cyclease full sequence (ACFL), adenylate cyclase catalytic domain (ACCD) and IIA(Glc) (2AG) by PCR</center><br><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/45/Sumbawagen.result1.pict2.jpg"></center><br><br />
<center>Figure 2. Confirmation of insertion of ACFL, ACCD and IIA(Glc) into pSB1C3 by colony PCR. Only IIA(Glc) was confirmed to be inserted by increasing band size in this figure.</center><br><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol11
Team:Sumbawagen/Notebook/protocol11
2015-02-19T03:11:52Z
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<h2>Notebook – Protocol – 11. Cultivation</h2><br />
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<p>1. From plate of E. coli BL21/DE3 harboring appropriate plasmid, colony was picked and cultured in 2 ml LB medium containing antibiotic chloramphenicol (Cam) with final concentration of 34 ug/ml. 10 ml size glass tube was used. This was pre-culture.</p><br />
<p>2. The pre-culture was shaken over-night at room temperature.</p> <br />
<p>3. Main culture was prepared for the next day, which contains pre-culture, LB medium, Cam, expression reagent for the expression of reporter gene mRFP (autoinduction medium which includes 20x NPS, 50x 5052 and 1,000x Mg), sugar for catabolite repression induction (either glucose, fructose, glucose+fructose, or honey). Detail receipt with various concentration of sugars was given in Tables below.</p><br />
<p>4. Main culture was shaken over-night at room temperature. </p><br />
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Team:Sumbawagen/Notebook/protocol1
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Notebook – Protocol – 1. Primer design</h2><br />
<br />
<br />
<p>1.DNA sequence of <i>E.coli</i> adenylate cyclase (cyaA; accession code NC_000913 REGION: 3991153..3993699; 2547 bp) and <i>E. coli</i> IIA(Glc) (crr; accession code J02796; 510 bp) were obtained from Genebank <a href ="http://ncbi.nlm.nih.gov/">http://ncbi.nlm.nih.gov/</a></p><br />
<br />
<br />
<p>2.Both DNA sequences were checked for the “in availability” of restriction sites of EcoRI (G^AATT_C), XbaI (T^CTAG_A), SpeI (A^CTAG_T), and PstI (C_TGCA^G) by Bioedit program using menu: Sequence/Nucleic Acid/Restriction Map/Generate Map.</p><br />
<br />
<p>3. Forward primers were designed to contain (from 5’ direction) :<br><br />
<ul style="padding-left:25px;"><br />
<li>additional sequence to accommodate restriction enzyme recognition; TTA</li><br />
<li>sequence from backbone pSB1C3 which has both EcoRI and XbaI sites; <u>GAATTC</u>GCGGCCGCT<u>TCTAGA</u></li><br />
<li>seven codons of the initial part of the genes which include start codon</li></p></ul><br />
<br />
<p>4. Reverse primers were designed to contain (from 5’ direction) :<br><br />
<ul style="padding-left:25px;"><br />
<li>additional sequence to accommodate restriction enzyme recognition; TTA</li><br />
<li>sequence from backbone pSB1C3 which has SpeI site; <u>ACTAGT</u>A</li><br />
<li>double stop codon (in reverse complement sequence); TTATTA </li><br />
<li>six codons of the last part of the genes (in reverse complement sequence)</li><br />
<li>For cyaA, besides the last part of the genes which codes for full length sequence, other reverse primer was also designed to contain the last part of N-terminal catalytic domain (full length sequence, 2547 bp; N-terminal catalytic domain, 1605 bp)</li><br />
</p></ul><br />
<br />
<h2>Resulted primers sequences were as follow</h2><br />
<ul style="padding-left:25px;"><br />
<li>cyaA full length forward primer (primer name “ACEF”)</li><br />
5’-TTA GAA TTC GCG GCC GCT TCT AGA TGT ACC TCT ATA TTG AGA CTC TG-3’<br />
<li>cyaA catalytic domain reverse primer (primer name “ACER1”)</li><br />
5’-TTA ACT AGT ATT ATT ATT CCG AGA GAT CGG GTG AAA T-3’<br />
<li>cyaA full length reverse primer (primer name “ACER2”)</li><br />
5’-TTA ACT AGT ATT ATT ACG AAA AAT ATT GCT GTA ATA G-3’<br />
<li>crr forward primer (primer name “AEF”)</li><br />
5’-TTA GAA TTC GCG GCC GCT TCT AGA TGG GTT TGT TCG ATA AAC TG-3’<br />
<li>crr reverse primer (primer name “AER”)</li><br />
5’-TTA ACT AGT ATT ATT ACT TCT TGA TGC GGA TAA C-3’<br />
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<h2>Notebook – Protocol – 2.PCR (Polymerase Chain Reaction)</h2><br />
<br />
<br />
<p>1. Ordered primers were dissolved in 1x TE buffer to get a 100 uM stock, then diluted to 10 uM stock by 1x TE buffer (usually to final volume of 200 ul). Primers stocks were stored at -20 °C.</p><br />
<br />
<br />
<p>2. There were 3 pairs of primers for the PCR namely <br />
<ul style="padding-left:25px;"><br />
<li>ACEF/ACER1 to clone catalytic domain sequence of adenylate cyclase gene</li><br />
<li>AEF/AER to clone full length sequence of IIA(Glc) gene</strong></li><br />
</p></ul><br />
<br />
<p>3. Reagents compositions for PCR were as follow:<br><br />
<ul style="padding-left:25px;"><br />
<li>2X PCR master mix (Thermoscientific), 25 ul</li><br />
<li>Sterilized distilled water (Akuabides), 15 ul</li><br />
<li>10 uM forward primer, 5 ul</li><br />
<li>10 uM reverse primer, 5 ul</li><br />
<li>Sterilized distilled water (Akuabides), 15 ul</li><br />
<li>10 uM forward primer, 5 ul</li><br />
<li>DNA template, colony from agar plate 0 ul</li><br />
Total volume, 50 ul</p></ul><br />
<br />
<p>4.Reaction conditions for PCR were as follow:<br><br />
<ul style="padding-left:25px;"><br />
<li>95 °C, 2 minutes</li><br />
<li>95 °C, 30 seconds</li><br />
<li>53 °C, 30 seconds</li><br />
<li>72 °C, 2 minutes</li><br />
<li>72 °C, 7 minutes</li><br />
Total cycle, 35 cycles</ul><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol3
Team:Sumbawagen/Notebook/protocol3
2015-02-19T03:10:39Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Notebook – Protocol – 3. Electrophoresis and DNA visualization</h2><br />
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<p>1.50x TAE (Thermoscientific) was diluted to 1x TAE (1 ml 50x TAE + 49 ml distilled water).</p><br />
<p>2. 3 g of Agarose S (Nippongene) was added into 200 ml of 1x TAE to get 1.5 % agarose gel in a glass flask.</p> <br />
<p>3. Agarose was melted by putting the glass flask in boiled water – we have no microwave – and sometimes stirred slowly to avoid bubble formation with stirrer bar on a stirrer.</p><br />
<p>4. Completely melted agarose solution was poured into the casting tray with comb.</p><br />
<p>5. Let the gel became solid by incubating at room temperature for 30 minutes.</p><br />
<p>6. Electrophoresis was done in 1x TAE buffer in an electrophoresis tank (Mupid)</p> <br />
<p>7. The volume of samples used for electrophoresis was 7 ul for 1 kB DNA ladder and 10 ul (PCR product), or 15 ul (Restriction enzyme digestion product) for DNA samples for each well.</p><br />
<p>8. Electrophoresis was done for 20 minutes using 135V setting.</p><br />
<p>9. After electrophoresis, gel was put into 1x TAE solution containing Sybrsafe to stain the DNA for about 20 minutes.</p><br />
<p>10. Then gel was placed on trans illuminator for viewing and documentation using pocket digital camera.</p> <br />
<p>11. The file was viewed with computer using Photoshop.</p><br />
<p>12. For disposal, gel and buffer were dried then burnt together with any tissues or papers in contact.</p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<br />
<h2>Notebook – Protocol – 4. DNA Purification</h2><br />
<p>Spin column based purification was used. The kit is Gel/PCR extraction kit (Nippon Genetics). Protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge. All steps were done at room temperature.</p><br />
<p><u>DNA Purification from Liquid Samples</u></p><br />
<ul style="padding-left:25px;"><br />
<p>1. Into a clean, sterilized 0.5 ul polypropylene tube, add PCR products : buffer GP1 = 1 : 5 (e.g. 40 ul : 200 ul). Vortexing.</p><br />
<p>2. Load the samples onto the spin column equipped with collection tube. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>3. Add 600 ul of GP2. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>4. Centrifuge for 6,000 rpm; 5 minutes to dry the column.</p><br />
<p>5. Replace collecting tube with clean, sterilized 1.5 ml size polypropylene tube.</p><br />
<p>6. Add 30 ul of GP3. Incubate 2 minutes at room temperature. Centrifuge for 6,000 rpm; 5 minutes.</p><br />
<p>7. Obtain the DNA as the flowthrough.</p><br />
</p></ul><br />
<br />
<p><u>DNA Purification from Agarose Gel</u></p><br />
<ul style="padding-left:25px;"><br />
<p>1. Into a clean, sterlized 1.5 ul polypropylene tube, add cutted gel containing DNA of interest : buffer GP1 = < 300 mg : 500 ul. Vortexing. Incubate at 55 oC; 10-15 minutes. Invert the tube.</p><br />
<p>2. Load the samples onto the spin column equipped with collection tube. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>3. Add 600 ul of GP2. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>4. Centrifuge for 6,000 rpm; 5 minutes to dry the column.</p><br />
<p>5. Replace collecting tube with clean, sterilized 1.5 ml size polypropylene tube</p><br />
<p>6. Add 30 ul of GP3. Incubate 2 minutes at room temperature. Centrifuge for 6,000 rpm; 5 minutes.</p><br />
<p>7. Obtain the DNA as the flowthrough.</p><br />
</p></ul><br />
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<h2>Notebook – Protocol – 5. Restriction enzymes digestion</h2><br />
<p>1. Restriction enzymes digestion was done using Fast Digest product (Thermoscientific) using the following composition:</p><br />
<ul style="padding-left:25px;"><br />
<li>DNA sample (1-3 ug), 15 ul</li><br />
<li>Fast Digest restriction enzyme #1, 2 ul</li><br />
<li>Fast Digest restriction enzyme #2, 2 ul</li><br />
<li>10x buffer, 3 ul</li><br />
<li>Sterilized distilled water, 6 ul</li><br />
Total volume, 30 ul</li><br />
</p></ul><br />
<br />
<p>2. Samples were incubated at 37 °C for more than 1 hour (typically 3 hours).</p><br />
<p>3. When necessary, samples were electrophoresed using protocol “3. Electrophoresis”(typically 3 hours).</p><br />
<p>4. Samples were purified using protocol “4. DNA purification”.</p><br />
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<h2>Notebook – Protocol – 6. Plasmid purification</h2><br />
<p>Spin column based purification was used. The kit is Plasmid mini kit (Nippon Genetics). Standard protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge.</p><br />
<br />
<ul><br />
<p>1. Add 2 ml of E. coli overnight culture into 2 ml polypropylene tube. Centrifuge for 6,000 rpm; 5 minutes. Remove the supernatant.</><br />
<p>2. Add 200 ul of mP1; vortexing. Add 200 ul of mP2; invert the tube 10 times. Incubate at 2 minutes at room temperature. Add 300 ul of mP3; invert the tube. </p><br />
<p>3. Immediately centrifuge for 6,000 rpm; 5 minutes.</p><br />
<p>4. Load the supernatant onto spin column. Centrifuge 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>5. Add 400 ul of mP4. Centrifuge 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>6. Add 600 ul of mP5. Centrifuge 6,000 rpm; 1 minute. Discard the flowthrough.</p><br />
<p>7. Centrifuge 6,000 rpm; 5 minute to dry the column.</p><br />
<p>8. Replace collecting tube with clean, sterlized 1,5 ml size polypropylene tube.</p><br />
<p>9. Add 50 ul of mP6. Incubate at room temperature for 2 minutes. Centrifuge for 6,000 rpm; 5 minutes.</p><br />
<p>10. Obtain the plasmid as the flowthrough.</p><br />
<br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol7
Team:Sumbawagen/Notebook/protocol7
2015-02-19T03:08:20Z
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<h2>Notebook – Protocol – 7. Ligation</h2><br />
<p>1. Ligation reaction was as follow:</p><br />
<ul style="padding-left:25px;"><br />
<li>Insert DNA, 2,5 ul*</li><br />
<li>Plasmid DNA, 1.5 ul*</li><br />
<li>Ligation kit (Takara), 5 ul</li><br />
Total volume, 10 ul</li><br><br />
* final quantity insert DNA > plasmid DNA </li><br />
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<p>2. Samples were incubated at 4 oC for overnight (inside a refrigerator).</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol8
Team:Sumbawagen/Notebook/protocol8
2015-02-19T03:07:08Z
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<h2>Notebook – Protocol – 8. Transformation </h2><br />
<br />
<p><strong>A. Purpose</strong></p><br />
<p>Since the amount of DNA that comes with the DNA distribution kit is not enough for assembly, we need to transform this DNA into cells and then make our own stocks with sufficient amount of DNA. The principle of this step is to put the plasmid that contain a small amount DNA of interest into competent cells, let them grow (replicate) and then harvest back the amplified plasmid that contain the gene of interest.</p><br />
<br />
<p><strong>B. Protocol</strong></p><br />
<p><strong>Transformation Method I</strong></p><br />
<p>We used TSS method in this transformation process. The method created and developed by Chung, et al and the protocol was adapted in Dr. Arief Budi Witarto’s experiments.</p><br />
<br />
<p><strong>Preparation</strong></p><br />
<ul style="padding-left:25px;"><br />
<li> Equipments : Centrifuge, horizontal shaker, incubator, micropipette, 1,5 ml microtube, 15 ml tube.</li><br />
<li> Materials : LB culture for culturing the bacteria, E. coli BL21(DE3) competent cells as the cell host, plasmid Bba_J04450 50 pg/ul as the vector, TSS medium, 1 M glucose, TSS medium.</li><br />
<br />
<p>1. Pre-culture</p><br />
<ul style="padding-left:25px;"><br />
<li> Culture the Escherichia coli BL21(DE3) competent cells by adding 100 µl of E. coli. BL21(DE3) glycerol stock into 3 ml of LB liquid medium.</li><br />
<li> Incubate with shaking it at room temperature (28-20 °C) for 3 hours.</li></ul><br />
<br />
<p>2. Plasmid transformation</p><br />
<ul style="padding-left:25px;"><br />
<li> After 3 hours, add 1 ml of the pre-cultured results into 3 microtubes 1,5 ml steril.</li><br />
<li> Pellets the cells by centrifugation at 4°C for 10 minutes.</li><br />
<li> Discard as much as supernatant as possible.</li><br />
<li> Mix the pellets in each tube by adding 1 ml TSS medium into the tubes. Suspend the pellets thoroughly with a micropipette. Suspended pellets then move into another tube that contain pellets, and suspend it again until we get 1 tube contain suspended<i> E. coli.</i></li><br />
<li> Move 100 µl suspended <i> E. coli</i> into 2 cooled microtubes. (One tube using for control and another one for the transformation reaction).</li><br />
<li> Add 2 µl plasmid/part BBa_J04450 50 pg/µl from part registry into 100 µl suspended<i> E. coli.</i></li><br />
<li> Resuspency by pipetting and tapping the tube.</li><br />
<li> Incubate the tubes for 5-6 hours at 4 °C.d<i> E. coli.</i></li><br />
<li> After 5-6 hours, add 1 ml LB liquid medium that has been mixed with 18 µl of glucose 1 M.</li><br />
<li> Incubate overnight with shaking in room temperature (28-30 °C).</li><br />
</ul><br />
<br />
<p>3. Plate out the cultured</p><br />
<ul style="padding-left:25px;"><br />
<li> Pour 100 µl of the transformation results to agar plate containing appropriate antibiotic. We use CAM with 50 µg/µl final concentration in plate. </li><br />
<li> Use spreader to spread evenly then incubate overnight at 37 °C .</li><br />
<li> Centrifuge the remaining transformation results (900 µl) for 3 minutes</li><br />
<li> Move 700 µl supernatant into empty tube.</li><br />
<li> Pipetting the remaining supernatant with pellets (200 µl)</li><br />
<li> Add 100 µl to LB plate containing appropriate antibiotic</i></li><br />
</ul></ul></ul><br />
<div><br />
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<p><strong>Transformation Method II</strong></p><br />
<p>The method used in this transformation process is heat shock method. The principle of this method is by giving high temperature to the cells. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell</p><br />
<br />
<p><strong>Preparation</strong></p><br />
<ul style="padding-left:25px;"><br />
<li> Equipments : Microcentrifuge, horizontal shaker, incubator, micropipette, tubes 15 ml, polipropylene tube (50 ml and 2 ml.</li><br />
<li> Materials needed : LB liquid medium for culturing the bacteria, <i>E. coli</i> DH5-alpha competent cells as the cell host, plasmid Bba_J04450 as the vector. </li><br />
<br />
<p>Steps</p><br />
<ul style="padding-left:25px;"><br />
<li> Add plasmid Bba_J04450 (2 µl for 50 pg/µl, 5 µl for 20 pg/µl, etc) into 3 tube containing <i>E. coli</i> DH5-alpha competent cell (tube 2A, 3A, and 4A) that have made before (see Protocol Making Competent Cell.</li><br />
<li> Mix by pipetting using micropippet (5-10 times pipetting).</li></ul><br />
<li> Incubate in refrigerator for 40 minutes.</li><br />
<li> Prepare the hot water with the temperature of 42 °C, measure the temperature using thermometer.</li><br />
<li> Heatshock the cells by put the bottom of the tube on hot water for 45 seconds.</li><br />
<li> Incubate in refrigerator for 5 minutes. </i></li><br />
<li> Add 1 ml LB liquid medium sterile (without antibiotic) into each tubes (tube 2A, 3A, and 4A). </li><br />
<li> Shake for 2 hours.</li><br />
<li> Plating the transformation by add 100 µl transformation reaction to LB agar plates containing chlorampenicol (final concentration 50 µg/ml).</li><br />
<li> Label the plates with 2a-1x, 2b-1x, and 2c-1x.</i></li><br />
<li> Centrifuge the remaining transformation results for 3 minutes.</li><br />
<li> Discard as much as 700 µl supernatant</li><br />
<li> Suspend the remaining supernatant and pellet by pippeting.</li><br />
<li> Add 100 µl to agar plate containing chloramphenicol (final concentration 50 µg/µl).</li><br />
<li> Label the plates with 2a-1x, 2b-1x, and 2c-1x.</i></li><br />
<li> Incubate at 37 °C overnight.</li><br />
<li> Check the result later.</li><br />
</ul><br />
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http://2014.igem.org/Team:Sumbawagen/Notebook/protocol9
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<h2>Notebook – Protocol – 9. Cell competent preparation</h2><br />
<br />
<br />
<p>1. From LB agar plate containing no antibiotics, colony of either E. coli DH5alpha or BL21(DE3) cell was taken and culture in 2 ml LB medium at room temperature for overnight for pre-culture.</p><br />
<p>2. 900 ul of pre-culture was added into 9 ml of sterilized LB medium in 50 ml polypropylene tube, inside a cleanbench.</p> <br />
<p>3. Tube was shaken for 2 hours at room temperature. Because we have no UV spectrophotometer, OD (optical density) of the main culture could not be measured. However, based on our chief instructor - Dr. Arief experiment, 2 hours shaking at room temperature using the above condition culture was enough to get OD660 nm of about 0.6.</p><br />
<p>4. In a cleanbench, aliquot @1.5 ml of the main culture into cold 4 polypropylene tubes with size of 2 ml. The tubes were pre-chilled by putting inside refrigerator.</p><br />
<p>5. Centrifuge the 4 tubes at 6,000 rpm, 10 minutes at 4 oC by putting the small microcentrifuge inside a refrigerator.</p><br />
<p>6. Inside a cleanbench, the supernatants were discarded. </p> <br />
<p>7. Then @1.5 ml of cold 100 mM MgCl2 was added to each tube. Pellet was suspended.</p><br />
<p>8. Centrifuge the tubes at 6,000 rpm, 10 minutes at 4 °C.</p><br />
<p>9. Then @1.5 ml of cold 100 mM CaCl2 was added to each tube. Pellet was suspended.</p><br />
<p>10. Tubes were incubated on ice for 30 minutes.</p> <br />
<p>11. Centrifuge the tubes at 6,000 rpm, 10 minutes at 4 °C</p><br />
<p>12. Inside a cleanbench, the supernatants were discarded.</p><br />
<p>13. Then add @75 ul of cold 100 mM MgCl2 were added to each tube. Pellet were suspended.</p><br />
<p>14. Cell competent was ready to be used in the same day.</p><br />
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<h2> Notebook – Protocol – 10. DNA Sequencing</h2><br />
<p> DNA sequencing was done by ordering to commercial company.</a></p><br />
<p>1. Premix sample was prepared with the following composition:</p><br />
<ul style="padding-left:25px;"><br />
<li> Plasmid sample, 10 to 20 ul*</li><br />
<li>10 uM primer, 1 ul </li><br />
<li> Sterilized distilled water, 9 to 19 ul</li><br />
Total volume, 21 ul </li><br><br />
Concentration < 900 ng </li><br />
</ul><br />
<br />
<p>2. Samples were sent to the company<br><br />
<p> 3. According to the company, DNA sequencing was done using the following conditions:<br><br />
<ul style="padding-left:25px;"><br />
<li> Sequencing kit, BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)</li><br />
<li> Purification kit, BigDye XTerminator® Purification Kit (Applied Biosystems) </u></li><br />
<li> Sequencer, Applied Biosystems 3730xl DNA Analyzer</li></p></ul><br />
<br />
<al><p>4. DNA sequencing data in .ab1 files were received by email, then analyzed using Bioedit program. For alignment, online CLUSTAL program was used.</p></al><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol12
Team:Sumbawagen/Notebook/protocol12
2015-02-19T03:05:58Z
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<h2>Notebook – Protocol – 12. Honey glucose meter measurement</h2><br />
<ul style="padding-left:25px;"><br />
<p>1. Glucose dissolved in distilled water to a concentrations of 100 mM was used as stock. From that stock, various concentration of glucose were prepared by dilution. The concentrations were 5 / 10 / 15 / 20 / 25 mM.</p><br />
<p>2. Those glucose stocks were measured its mg/dl value by a glucose meter. The values then plotted as y axis, against the concentration as x axis. Linear standard curve was obtained and the formula was used to calculate the concentration of unknown sample.</p><br />
<p>3. Honey in 1.5 ml polypropylene tube was warmed at 60 oC for 10 minutes, then transferred 100 ul to a new tube which contained 900 ul distilled water to obtain 1/10 dilution.</p><br />
<p>4. This sample was used for measurement of glucose concentration by glucose meter, and its glucose concentration was calculated using standard curve obtained before.</p><br />
<p>5. When the mg/dl value still exceeded the linear curve, dilution rate was increased.</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol13
Team:Sumbawagen/Notebook/protocol13
2015-02-19T03:05:36Z
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<h2>Notebook – Protocol – 13. SDS-PAGE</h2><br />
<ul style="padding-left:25px;"><br />
<p>1. 1 ml of E. coli BL21(DE3) overnight culture was added to 1.5 ml propylene tube. Then centrifuge at 6,000 rpm, 5 minutes at room temperature. Then supernatant was discarded.</p><br />
<p>2. Pellet was added with 300 ul of 1x Bug Buster (Novagen). Tube was vortexed.</p><br />
<p>3. Tube was put on boiled water for 5 minutes.</p><br />
<p>4. 50 ul of samples was aliquoted and used as “total protein” sample.</p><br />
<p>5. The rest of the sample in the tube was centrifuged at 6,000 rpm, 10 min, room temperature.</p><br />
<p>6. 50 ul of supernatant was aliquoted and used as “soluble protein’ sample.</p><br />
<p>7. Protein concentration of “total protein” and “soluble protein” were estimated using Lowry method based on the kit from Biorad. We have no UV spectrophotometer for measurement of real concentration at 750 nm. But according to our chief instructor Dr. Arief previous experiment, the concentration of “soluble protein” will be about 1,000 – 2,000 ug/ml, while the concentration of “total protein” will be about 10,000 – 20,000 ug/ml.</p><br />
<p>8. “Soluble protein” sample was used directly, while “total protein” sample was diluted 1/10th to obtain samples with roughly the same concentration for SDS-PAGE.</p><br />
<p>9. 15 ul sample was added with 15 ul dye containing SDS and Coomassie Brilliant Blue (CBB) in 500 ul polypropylene tube, then the tube was put in boiled water for 15 min</p><br />
<p>10. 15 ul of sample – protein quantity about 10 ug – was electrophoresed. Marker used was Blue Star Prestained Protein Marker (Nippon Genetics). The marker has proteins bands of 170 / 125 / 93 / 68 / 53 / 41 / 32 / 23 / 14 / 10 kD.</p><br />
<p>11. SDSPAGE was done with commercial 10-20% SDS gel, and 1x SDS buffer. 10x SDS buffer was prepared using 250 mM Tris, 1,920 mM Glycine, and 1% w/v SDS.</p><br />
<p>12. SDSPAGE was done for 45 minutes with equipment setting of 40 mA.</p><br />
<p>13. Staining of SDSPAGE gel was done using Rapid CBB kit (Kanto Chemicals). Staining was done 1-3 hours.</p><br />
<p>14. Destaining of SDSPAGE gel was done using solution containing 75 ml distilled water, 10 ml acetic acid, 15 ml ethanol. Destaining was done overnight.</p><br />
<p>15. Gel was documented with pocket camera, and file was evaluated with Photoshop program in a computer.</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol14
Team:Sumbawagen/Notebook/protocol14
2015-02-19T03:05:11Z
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<h2>Notebook – Protocol – 14. Measurement</h2><br />
<ul style="padding-left:25px;"><br />
<p>1. Overnight culture of “main culture” was taken from the shaker, then put in rack for measurement.</p><br />
<p>2. First, picture was taken with pocket camera.</p><br />
<p>3. Then color of the medium was measured its RGB value by ColorMeter program from an android-base mobile phone. For each side, 3 pictures were shot.</p><br />
<p>4. For validation, another mobile phone with different resolution of camera was used. Also besides, ColorMeter, we also used different program for RGB measurement namely Color Detector.</p><br />
<p>5. Values of RGB were converted to luminance value to obtain only the brightness of color (http://www.scantips.com/lumin.html; accessed on Sep. 24th, 2014). The formula used is:<br />
Luminance value = (R x 0.30) + (G x 0.59) + (B x 0.11)<br />
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<p>6. Luminance values from 3 RGB data were averaged and calculated its standard deviation.</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol11
Team:Sumbawagen/Notebook/protocol11
2015-02-19T03:03:04Z
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<h2>Notebook – Protocol – 11. Cultivation</h2><br />
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<p>1. From plate of E. coli BL21/DE3 harboring appropriate plasmid, colony was picked and cultured in 2 ml LB medium containing antibiotic chloramphenicol (Cam) with final concentration of 34 ug/ml. 10 ml size glass tube was used. This was pre-culture.</p><br />
<p>2. The pre-culture was shaken over-night at room temperature.</p> <br />
<p>3. Main culture was prepared for the next day, which contains pre-culture, LB medium, Cam, expression reagent for the expression of reporter gene mRFP (autoinduction medium which includes 20x NPS, 50x 5052 and 1,000x Mg), sugar for catabolite repression induction (either glucose, fructose, glucose+fructose, or honey). Detail receipt with various concentration of sugars was given in Tables below.</p><br />
<p>4. Main culture was shaken over-night at room temperature. </p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/protocol11
Team:Sumbawagen/Notebook/protocol11
2015-02-19T03:02:38Z
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<h2>Notebook – Protocol – 11. Cultivation</h2><br />
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<p>1. From plate of E. coli BL21/DE3 harboring appropriate plasmid, colony was picked and cultured in 2 ml LB medium containing antibiotic chloramphenicol (Cam) with final concentration of 34 ug/ml. 10 ml size glass tube was used. This was pre-culture.</p><br />
<p>2. The pre-culture was shaken over-night at room temperature.</p> <br />
<p>3. Main culture was prepared for the next day, which contains pre-culture, LB medium, Cam, expression reagent for the expression of reporter gene mRFP (autoinduction medium which includes 20x NPS, 50x 5052 and 1,000x Mg), sugar for catabolite repression induction (either glucose, fructose, glucose+fructose, or honey). Detail receipt with various concentration of sugars was given in Tables below.</p><br />
<p>4. 4. Main culture was shaken over-night at room temperature. </p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Biobrick</h2><br />
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<strong>BBa_K1508003 (IIA(Glc) + double terminator)</strong><br><br />
This biobrick contains gene coding for IIA(Glc) (BBa_K1508002), followed by double terminator (BBa_B0015). IIA(Glc) is glucose-specific phosphocarrier protein of the phosphoenolpyruvate : glucose phosphotransferase system (PTS). This circuit inserted to the upstream part of plasmid Bba_J04450. We hypothesized that by over-expressing the enzyme, more glucose can be uptook compared to normal condition.<br><br />
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<h2>Coding </h2><br />
<strong>BBa_K1508002 (IIA(Glc) - <i>E.coli</i> glucose-specific phosphotransferase enzyme IIA)</strong><br><br />
Phosphotransferase system (PTS) is a major carbohydrate active -transport system, which is executed by a cascade of protein rally. For glucose uptake, IIA(Glc) is the only enzyme specific to glucose transport. We measure the concentration of glucose in the medium using catabolite repression mechanism toward lacI promoter activity upstream of a reporter gene, mRFP. We expect by over-expressing IIA(Glc) in <i>E. coli</i>, we will obtain different range of glucose standard curve in our measurement system.<br><br><br />
<br />
<strong>BBa_K1508000 (IIA(Glc) - Glucose-specific phosphotransferase enzyme IIA component from <i>E. coli</i>)</strong><br />
<p>IIA(Glc) - previously known as IIA(Glc) - is glucose-specific phosphocarrier protein of the phosphoenolpyruvate: glucose phosphotransferase system (PTS). Therefore, we hypothesized that by over-expressing the enzyme, more glucose can be uptook compared to normal condition. </p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Biobrick</h2><br />
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<strong>BBa_K1508003 (IIA(Glc) + double terminator)</strong><br><br />
This biobrick contains gene coding for IIA(Glc) (BBa_K1508002), followed by double terminator (BBa_B0015). IIA(Glc) is glucose-specific phosphocarrier protein of the phosphoenolpyruvate : glucose phosphotransferase system (PTS). This circuit inserted to the upstream part of plasmid Bba_J04450. We hypothesized that by over-expressing the enzyme, more glucose can be uptook compared to normal condition.<br><br />
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<h2>Coding </h2><br />
<strong>BBa_K1508002 (IIA(Glc) - <i>E.coli</i> glucose-specific phosphotransferase enzyme IIA)</strong><br><br />
Phosphotransferase system (PTS) is a major carbohydrate active -transport system, which is executed by a cascade of protein rally. For glucose uptake, IIA(Glc) is the only enzyme specific to glucose transport. We measure the concentration of glucose in the medium using catabolite repression mechanism toward lacI promoter activity upstream of a reporter gene, mRFP. We expect by over-expressing IIA(Glc) in <i>E. coli</i>, we will obtain different range of glucose standard curve in our measurement system.<br><br><br />
<br />
<strong>BBa_K1508000 (IIA(Glc) - Glucose-specific phosphotransferase enzyme IIA component from <i>E. coli</i>)</strong><br />
<p>IIA(Glc) - previously known as IIA(Glc) - is glucose-specific phosphocarrier protein of the phosphoenolpyruvate: glucose phosphotransferase system (PTS). Therefore, we hypothesized that by over-expressing the enzyme, more glucose can be uptook compared to normal condition. </p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Biobrick</h2><br />
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<h2>Composite</h2><br />
<strong>BBa_K1508003 (IIA(Glc) + double terminator)</strong><br><br />
This biobrick contains gene coding for IIA(Glc) (BBa_K1508002), followed by double terminator (BBa_B0015). IIA(Glc) is glucose-specific phosphocarrier protein of the phosphoenolpyruvate : glucose phosphotransferase system (PTS). This circuit inserted to the upstream part of plasmid Bba_J04450. We hypothesized that by over-expressing the enzyme, more glucose can be uptook compared to normal condition.<br><br />
<br />
<br />
<h2>Coding </h2><br />
<strong>BBa_K1508002 (IIA(Glc) - <i>E.coli</i> glucose-specific phosphotransferase enzyme IIA)</strong><br><br />
Phosphotransferase system (PTS) is a major carbohydrate active -transport system, which is executed by a cascade of protein rally. For glucose uptake, IIA(Glc) is the only enzyme specific to glucose transport. We measure the concentration of glucose in the medium using catabolite repression mechanism toward lacI promoter activity upstream of a reporter gene, mRFP. We expect by over-expressing IIA(Glc) in <i>E. coli</i>, we will obtain different range of glucose standard curve in our measurement system.<br><br />
<br />
<strong>BBa_K1508000 (IIA(Glc) - Glucose-specific phosphotransferase enzyme IIA component from <i>E. coli</i>)</strong><br />
<p>IIA(Glc) - previously known as IIA(Glc) - is glucose-specific phosphocarrier protein of the phosphoenolpyruvate: glucose phosphotransferase system (PTS). Therefore, we hypothesized that by over-expressing the enzyme, more glucose can be uptook compared to normal condition. </p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Project/Result/Results5
Team:Sumbawagen/Project/Result/Results5
2015-02-19T02:49:11Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Result – 5. Confirmation of protein expression by SDS-PAGE</h2><br />
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<p>The figure here showed data of SDSPAGE. ACFL is sample from E. coli BL21(DE3) supposed to express mRFP and full length Adenylate cyclase. 2AGL is sample from E. coli BL21(DE3) supposed to express mRFP and IIA(Glc). While CTRL is sample from E. coli BL21(DE3) supposed to express mRFP. </p><br />
<br />
<p>Protein band corresponding to mRFP was clearly shown in either total proteins or soluble proteins which mean that mRFP was expressed as soluble protein. Meanwhile protein band corresponding to Adenylate cyclase could not be observed. Protein band corresponding to IIA(Glc) seemed to present.</p><br />
<br />
<p>However, after DNA sequencing result, it was confirmed that neither ACFL nor 2AGL coding genes were not able to be constructed. Thus all E. coli samples here were the same, namely only expressing mRFP – harbouring BBa_J04450.</p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Notebook/Results1
Team:Sumbawagen/Notebook/Results1
2015-02-19T02:46:01Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2><b>Notebook – Results – 1. Construction of BBa_J04450 + constitutive promoter/crr or cyaA</b></h2><br />
<br />
<br />
<p>We planned to construct BBa_J04450 as circuit #1 (original circuit) followed downstream by our original circuit #2. Circuit #2 is composed by BBa_J2319 (constitutive promoter) / BBa_B0034 (RBS) / crr (IIA(Glc) or cyaA (Adenylate cyclase) / BBa_B0015 (double terminator).</p><br />
<br />
<p>So the first step was to clone either cyaA or crr from E. coli to Biobrick construct. PCR fragments with correct size were able to be obtained. After direct restriction with EcoRI and SpeI, the fragments were cloned into pSB1C3 backbone. </p><br />
<br />
<p>Next step was to construct, BBa_J2319 (chloramphenicol) / BBa_B0034 (ampicillin) and crr or cyaA (chloramphenicol) / BBa_B0015 (chloramphenicol). For BBa_J2319 / BBa_B0034 construction, prefix insertion was used. Thus ligation product was screened on ampicillin LB agar plate. While for crr or cyaA / BBa_B0015 construction, suffix insertion was used because the large size of crr or cyaA could be isolated by agarose gel excision. After BBa_J2319 / BBa_0034 (ampicillin) and crr or cyaA / BBa_0015 (chloramphenicol) constructs were obtained, prefix insertion was done to get BBa_J2319 / BBa_B0034 / crr or cyaA / BBa_B0015 in pSB1C3 backbone. Final step was prefix insertion of BBa_J00450 the original constructs.</p><br />
<br />
<p>In every steps of works, we only confirmed the insertion by colony selection in appropriate antibiotics containing LB agar plate, and purification of backbone plasmid for insertion by gel electrophoresis to completely separated small insert fragments. Thus in theory there will be no self-ligation. However for either prefix or suffix inserts, the digested samples were not purified, instead used directly for ligation reaction. </p><br />
<br />
<p>Below some representative gel electrophoresis data were shown.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/24/Project_-_1_fig_1.jpg"></center><br><br />
<center>Figure 1. Cloning of adenylate cyclease full sequence (ACFL), adenylate cyclase catalytic domain (ACCD) and IIA(Glc) (2AG) by PCR</center><br><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/45/Sumbawagen.result1.pict2.jpg"></center><br><br />
<center>Figure 2. Confirmation of insertion of ACFL, ACCD and IIA(Glc) into pSB1C3 by colony PCR. Only IIA(Glc) was confirmed to be inserted by increasing band size in this figure.</center><br><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2>Result – 2. Confirmation of DNA sequence</h2><br />
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<p>As shown in Figure 1 (2AGL_DT), we confirmed the construction of crr gene encoding IIA(Glc) from E. coli into Biobrick format – flanking by restriction sites of EcoRI and XbaI at the prefix, and SpeI and PstI at the suffix – followed by the sequence of double terminator (BBa_B0015). The other constructs which able to be confirmed by DNA sequencing was the parental plasmid containing crr gene only. While the rest of plasmids did not contain expected sequence.</p><br />
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<p>While the final construct to be expected, namely BBa_J04450 followed by constitutive promoter/RBS/crr/double terminator was actually the sequence of BBa_J04450 only as shown in figure below. The data from forward sequence and reverse sequence (Figure 2; J04450_CP_RB_2AGL_DT FWD/REV) revealed the prefix and suffix sequences clearly.</p><br />
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<p>However, we can confirm the sequence of basic part in our project namely BBa_J23119 (constitutive promoter), BBa_B0034 (RBS) and BBa_B0015 (double terminator) alone. Thus for future works, we can confirm to use the plasmid for construction.</p><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h2><b>Result – 1. Construction of BBa_J04450 + constitutive promoter/crr or cyaA</b></h2><br />
<br />
<br />
<p>We planned to construct BBa_J04450 as circuit #1 (original circuit) followed downstream by our original circuit #2. Circuit #2 is composed by BBa_J2319 (constitutive promoter) / BBa_B0034 (RBS) / crr (IIA(Glc) or cyaA (Adenylate cyclase) / BBa_B0015 (double terminator).</p><br />
<br />
<p>So the first step was to clone either cyaA or crr from E. coli to Biobrick construct. PCR fragments with correct size were able to be obtained. After direct restriction with EcoRI and SpeI, the fragments were cloned into pSB1C3 backbone. </p><br />
<br />
<p>Next step was to construct, BBa_J2319 (chloramphenicol) / BBa_B0034 (ampicillin) and crr or cyaA (chloramphenicol) / BBa_B0015 (chloramphenicol). For BBa_J2319 / BBa_B0034 construction, prefix insertion was used. Thus ligation product was screened on ampicillin LB agar plate. While for crr or cyaA / BBa_B0015 construction, suffix insertion was used because the large size of crr or cyaA could be isolated by agarose gel excision. After BBa_J2319 / BBa_0034 (ampicillin) and crr or cyaA / BBa_0015 (chloramphenicol) constructs were obtained, prefix insertion was done to get BBa_J2319 / BBa_B0034 / crr or cyaA / BBa_B0015 in pSB1C3 backbone. Final step was prefix insertion of BBa_J00450 the original constructs.</p><br />
<br />
<p>In every steps of works, we only confirmed the insertion by colony selection in appropriate antibiotics containing LB agar plate, and purification of backbone plasmid for insertion by gel electrophoresis to completely separated small insert fragments. Thus in theory there will be no self-ligation. However for either prefix or suffix inserts, the digested samples were not purified, instead used directly for ligation reaction. </p><br />
<br />
<p>Below some representative gel electrophoresis data were shown.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/24/Project_-_1_fig_1.jpg"></center><br><br />
<center>Figure 1. Cloning of adenylate cyclease full sequence (ACFL), adenylate cyclase catalytic domain (ACCD) and IIA(Glc) (2AG) by PCR</center><br><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/45/Sumbawagen.result1.pict2.jpg"></center><br><br />
<center>Figure 2. Confirmation of insertion of ACFL, ACCD and IIA(Glc) into pSB1C3 by colony PCR. Only IIA(Glc) was confirmed to be inserted by increasing band size in this figure.</center><br><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/project/result
Team:Sumbawagen/project/result
2015-02-19T02:42:57Z
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/interlabstudy/results
Team:Sumbawagen/interlabstudy/results
2015-02-19T02:35:49Z
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<h2>SECTION I: Provenance and Release</h2><br />
<ul style="padding-left:15px"><br />
<p>1. The data was taken by member of Sumbawagen team as follow. Cindy Suci Ananda, Muhammad Al Azhar, Adelia Elviantari, and Yulianti. Other person who should be credited is Arief Budi Witarto, Ph.D. (instructor) who supervised the experiments. <br><br />
<br />
<p>2. Date of experiments: September 22nd – 24th, 2014.<br><br />
<br />
<p>3. All persons consented to the condition.</ul> <br><br />
<h2>SECTION II: Protocol</h2><br />
<br />
Introduction: There are no spectrophotometers – neither UV nor fluorescent – in Sumbawa island. Thus our project uses mobile phone camera to obtain data and quantify using customized software. Among fluorescent proteins, mRFP showed strong color visible to naked eye, thus we chose it as the report of measurement in our project. However, this report was made in the request of iGEM 2014 Committee using GFP to show the possibility on using our system for the Measurement Interlab Study. In this study we used different device/part although it has the same GFP gene, aside from which was already been designated by the Committee. We did it because the objective was to show whether GFP is measurable or not in our system.<br><br><br />
<br />
Through this activity, people's understanding of Sumbawa honey is also increasing. We introduce to the public some information about Sumbawa honey such as how to distinguish between genuine and fake honey, a tool used to measure the levels of glucose in honey, until any product that can be produced made from honey. We hope this activity could expand the knowledge and society pride of Sumbawa honey. We also hope that our project will make people aware of the importance of maintaining the natural resource and pride of this region. When the awareness is growing, we believe the existence of honey as the pride of Sumbawa could be maintained.<br><br><br />
<strong>Materials: <i>E. coli</i> BL21(DE3), and <i>E. coli</i> BL21(DE3) / BBa_I13522 </strong><br><br><br />
<br />
Methods: Four glass tubes were prepared to cultivate overnight <i>E. coli</i> in 2 mL LB medium, in room temperature using shaker. The content of each tubes were as follow:<br><br><br />
<ul style="padding-left:55px"><br />
<li>Tube-1. <i>E. coli</i> BL21(DE3) only. No antibiotics. Used as control.</li><br />
<li>Tube-2. <i>E. coli</i> BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final).</li><br />
<li>Tube-3. <i>E. coli</i> BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final); Auto induction reagents (Studier, Protein Expression and Purification<br />
41, 207-234, 2005).</li><br />
<li>Tube-4. <i>E. coli</i> BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final); Glucose 200 mM.</li></ul><br><br><br />
After one night of cultivation, tubes were collected, then photos were taken using pocket digital camera, and color of the medium were measured using free application available on Google Play named ColorMeter, an application developed by vistechprojects.com. The mobile phone used was Samsung GT-P5200 having 3.15 MP camera. The medium were centrifuged (6,000 rpm, 10 min.) in 1,5 ml polypropylene tube to obtain cell pellets, which were used to detect GFP fluorescence above a blue transilluminator (Pearl Biotech).<br><br><br />
<br />
<h2>SECTION III: Results and Discussions</h2><br />
In this study we used BBa_I13522, which already available in our lab, instead of BBa_I20260 which is designated as device no. 1 by Measurement Interlab Study Committee. As shown in Figure 1, both devices have the same RBSs, genes and terminators, except promoters. Because our intention here is to prove whether our system using mobile phone camera is suitable for GFP measurement or not, we use BBa_I13522 for convenient reason.<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/49/SBGI2.jpg"style="width:500px;height:70px;"/></center><br />
<br />
<center><strong>Figure 1. Vector maps of BBa_I13522 (left) and BBa_I20260 (right)</strong><br><br></center><br />
In Figure 2, it is showed that medium from 4 tubes showed similar color, although tube no. 3 has more green. Tube no. 1 and 2 are negative controls because they contain <i>E. coli</i> BL21(DE3) only, and <i>E. coli</i> BL21(DE3) / BBa_I13522 without expression reagent to induce GFP production, respectively. Tube no. 3 contains <i>E. coli</i> BL21(DE3) / BBa_I13522 with GFP was over expressed due to addition of auto induction reagent. On the other hand, glucose was added to tube no. 4 to repress possible leaky expression of GFP through the mechanism of catabolite repression.<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a7/SBGI1.jpg"style="width:300px;height:175px;"/></center><br />
<br />
<center><strong>Figure 2. Color of an overnight LB medium grew with <i>E. coli</i> under different condition.</strong></center><br />
<center>1) <i>E. coli</i> BL21/DE3; 2) <i>E. coli</i> BL21 (DE3)/Bba_I13522; 3) <i>E. coli</i> BL21(DE3)/BBa_I13522 + autoinduction reagent; 4) <i>E. coli</i> BL21(DE3) + glucose 200 mM.<br></center></p><br />
<br><p>ColorMeter program installed in mobile phone, detected RGB values, we then converted the values to luminance value to obtain only the brightness of color (http://www.scantips.com/lumin.html; accessed on Sep. 24th, 2014). The formula used is:</p><br />
<br />
<p>Luminance value = (R x 0.30) + (G x 0.59) + (B x 0.11)</p><br />
<br />
<br>Two measurements of RGB values of the tubes from different angles to correct possible lighting differences, and the calculation of luminance values are given in Table 1 and Figure 3 below.<br></p><br />
<center><strong>Table 1. RGB and Luminance value obtained by ColorMeter program</strong></center><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/5f/Measurment_Interlab_Study_1.jpg"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/c/cf/Grafik_Econey.png"></center><br />
<center><strong>Figure 3. Luminance values of an overnight LB medium grew with <i>E. coli</i> under different condition. </strong></center><br />
<br />
<center>1) <i>E. coli</i> BL21/DE3; 2) <i>E. coli BL21</i> (DE3)/Bba_I13522; 3)<i>E. coli</i> BL21(DE3)/BBa_I13522 + autoinduction reagent; 4)<i>E. coli</i> BL21(DE3) + glucose 200 mM.<br></center><br />
<p>Expression of GFP in theory should make the color of medium brighter thus luminance value will be lower compared to E. coli which did not express GFP. Tube no. 1, and 4 has higher luminance value than tube no. 3, which is as expectation. But tube no. 2 showed low luminance value too. The reason may be because the color differences are too small (luminance value range < 5), thus accuracy of the results was low. In our project using mRFP (data not shown), the difference of luminance value between expressed and non-expressed mRFP were about 50.<br><br><br />
<br />
Finally to validate the expression of GFP, we used the only equipment available in our Lab, which can visualize specifically fluorescent protein including GFP ((http://en.wikipedia.org/wiki/Green_fluorescent_protein; accessed on Sep. 24th, 2014). Two observations were made, in liquid and solid states. As shown in Figure 4 (top), observation of the culture medium showed that tube no. 3 has bright yellow color compared to other tubes. The color should come from GFP expressed by <i>E. coli</i> BL21(DE3)/BBa_I13522 cultured with auto induction reagent. However, the color was not very significant. Thus, cells were pelleted by centrifugation and once again observed. As shown in Figure 4 (bottom), tube no. 3 showed much bright yellow color compared to other tubes. This result concluded that this tube really contains<i>E. coli</i> BL21(DE3)/BBa_I13522 expressing GFP.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/c/ce/Picture4.png"style="width:400px;height:200px;"/></center><br />
<center><strong>Figure 4. Observation of GFP in liquid medium (top) and pellet (bottom) by transilluminator </strong></center><br />
<center>1) <i>E. coli</i> BL21/DE3; 2) <i>E. coli</i> BL21 (DE3)/Bba_I13522; 3) <i>E. coli</i> BL21(DE3)/BBa_I13522 + auto induction reagent; 4) <i>E. coli</i> BL21(DE3) + glucose 200 mM</center><br><br><br />
<br />
<p>In conclusion, our measurement system using mobile phone camera could not detect GFP expression in liquid medium because of small differences observed by naked eyes. Thus, we used mRFP instead for our iGEM project.</P><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<p>Moyo festival is an annual Sumbawa citizen festival held every year. This year the festival held from September 27th - October 6th, 2014 at Pahlawan Square of Sumbawa Besar and Moyo Island. This festival is eagerly awaited by the people of Sumbawa and even a attract domestic and international tourist. People gather to get some fun and to learn about Sumbawa culture. <br></p><br />
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<p>On the third night (September 29 2014) we got the opportunity to introduce synthetic biology and Sumbawagen project to the society because our project is related to Sumbawa honey. We also present some photos of Sumbawagen team activities and introduce some of the tools that we have such as PCR, micropipette, microscope, etc. Visitors were taking turn asking questions in the middle of the crowd that night. We hope that after this event the public will have better understand of synthetic biology and tools related to synthetic biology, to improve knowledge of Sumbawa society.<br></p><br />
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Isro
http://2014.igem.org/Team:Sumbawagen/Human_practice/econey
Team:Sumbawagen/Human practice/econey
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<h1 class="firstHeading">Team:Sumbawagen/Team</h1><br />
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<p>On August 26, 2014 Sumbawagen team had the opportunity to introduce the ECONEY project in @america Hotel Ritz Carlton, Jakarta supported of US government in Indonesia.<br />
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<p>Taking this opportunity, we introduce iGEM and our ECONEY project. In this presentation, Sumbawagen team presents the reason why our team made a device to measure the level of glucose in honey. The representative of US embassy, officials of the State, Rector of Sumbawa University of Technology, and the Dean of our Faculty were present to witness the presentation. In this project presentation, we wear Sumbawa traditional clothes as a special attraction to the audience. After the presentation was completed, our team shares Sumbawa traditional food to the audience which was made from Sumbawa honey to introduce the local wisdom of Sumbawa and as a present of our gratitude to the audience.<br></p><br />
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