http://2012.igem.org/wiki/index.php?title=Special:Contributions/Pepeks&feed=atom&limit=50&target=Pepeks&year=&month=2012.igem.org - User contributions [en]2024-03-28T19:20:20ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:USP-UNESP-Brazil/TeamTeam:USP-UNESP-Brazil/Team2012-09-27T03:49:08Z<p>Pepeks: /* Instructors */</p>
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=iGEM Brazilian Team=<br />
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We are an interdisciplinary group of undergraduate and graduate students from several areas. The undergrads are divided among the Chemistry Institute (USP), the Biosciences Institute (USP), the Pharmaceutical Sciences Faculty (USP and Unesp) and the Polytechnic School (USP). The graduate students are from the Biomedical Sciences Institute (USP), the Chemistry Institute (USP) and the Physics Institute (USP). <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption=USP and UNESP Brazil's Team | image=lasttime2.jpg | size=500px }}<br />
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=Who we are=<br />
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==='''Instructors'''===<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Andrés.JPG | caption= | size=200px }}<br />
*'''Edgar Andrés Ochoa Cruz (Young PhD)''': <br />
I am a scientist interested in technology and inventions. Working for generating awareness of the power available on new technologies, like reverse bioinformatics (Computation in biological organisms) and synthetic biology (Energy and biofuel production applications), and the positive impact that they can generate in the life quality of world population.<br />
<br />
This competition was the opportunity to develop my first project in the synthetic biology field. More ideas for new projects are coming ...(swordfish1313@gmail.com)<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=marieVan.jpg | size=120px }}<br />
*'''Marie Anne Van Sluys (Full professor)''':<br />
Dr. Van Sluys is full professor in the Botany Department of the University of São Paulo. She is the head of the Genomic and Transposable Elements Laboratory (GaTE). The GaTE focus is to study the impact of transposable elements in the structure, function, and diversification of bacterial and plants genomes.<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=carloshotta.jpg | caption= | size=120px }}<br />
*'''Carlos Takeshi Hotta (Assistant Professor)'''<br />
I am an assistant professor in the Biochemistry Department of the University of São Paulo. I am the head of the Plant Molecular Physiology group, that focus on the role of the circadian clock in increasing plant productivity. I also write a science blog [http://scienceblogs.com.br/brontossauros].<br />
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*'''Cleslei Fernando Zanelli'''<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=Cleslei.jpg | size=120px }}<br />
Degree in pharmacy and biochemistry from UNESP (2002) and PhD in Biotechnology from UNESP (2006). I am currently an assistant professor in the Biological Sciences Department of Pharmacy School of Araraquara - UNESP. Our lab is interested in understanding the control of protein synthesis using the budding yeast Saccharomyces cerevisiae as a model for functional genomics analyses. We also use the budding yeast collections of mutants and fusion proteins in high content screenings (HCS) to search for cellular drug targets.<br />
<br />
==='''Advisors:'''===<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Josegregorio.jpg | size=100px}}<br />
*'''José Gregório''':<br />
Degree in Biological Sciences from the Institute of Biosciences Masters in Biotechnology from the University of São Paulo (1994) and Ph.D. in Biological Sciences (Microbiology) from the University of São Paulo (2000). He is currently Professor from the University of São Paulo. He has experience in biochemistry, with emphasis on Biochemistry of Microorganisms, acting on the following topics: polyhydroxyalkanoates, biosurfactant, bacterial metabolism, metabolic engineering. <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Luiziana.jpg | size=100px}}<br />
*'''Luiziana Ferreira da Silva''':<br />
Graduated in Pharmacy and Biochemistry, Faculty of Pharmaceutical Sciences USP (1982), MSc (1990) and Ph.D. (1998) in Biological Sciences (Microbiology) by the Institute of Biomedical Sciences, University of São Paulo. She is currently professor Dr., University of São Paulo. Has experience in the area of microbiology with emphasis on Bacteriology. Her research involving: bacterial metabolism, metabolic flux analysis, biosynthesis of biodegradable plastics and elastomers (polyhydroxyalkanoates - PHA) as well as other compounds of biotechnological interest.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=GlauciaFoto.jpg | size=100px}}<br />
*'''Glaucia Souza'''<br />
Prof. Glaucia Souza is bachelor by the Biosciences Institute at USP (1988), graduate in biochemistry by the Chemistry Institute at USP (1993), post-doctor in molecular genetics by La Jolla Cancer Research Foundation (1994) and post-doctor in molecular genetics by Baylor College of Medicine (1996). Currently she is associated professor and leader of the Signal Transduction Lab at Chemistry Institute (USP).<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Ncaticha.jpg | size=100px}}<br />
*'''Nestor Caticha'''<br />
Nestor Caticha is a professor at the Instituto de Física USP working in Information theory<br />
of artificial and natural systems and Bayesian data analysis. Current research includes <br />
modelling collective properties of societies of information processing agents, theoretical <br />
analysis of decision processes and the development of methods for neuroimaging using<br />
fMRI and EEG. He obtained a B.Sc. from Unicamp and a Ph.D. from Caltech, both<br />
in Physics.<br />
<br />
==='''Graduate:'''===<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Debora.jpg | caption= | size=80px }}<br />
*'''Débora Parrine'''<br />
Débora is a graduate student from Prof. Luiziana's Lab. Her research involves production of biodegradable materials and metabolic engeneering.<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Luiza.JPG | caption= | size=100px }}<br />
*'''Luíza Barros''':<br />
Luíza is a plant scientist. She was the last one to join the team and is always available to talk about how wonderful Molecular Biology can be to solve our lab problems.<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Marcelo.jpg | caption= | size=100px }}<br />
*'''Marcelo Boareto''':<br />
Marcelo is a biological physicist and phD student in Physics. He has two academic goals: to be recognized as a biologist when talking to a biologist and as a physicist when talking to a physicist. Neither of the two has been achieved, yet.<br />
<br />
==='''Undergrads:'''===<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cauã.jpg | caption= | size=80px }}<br />
*'''Cauã Westmann''':<br />
I'm a Biology undergratuate student at USP and an eternal lover of life and all its aspects. I'm always trying to understand and connect everything in an holistic evolutionary aproach.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cleandho.JPG | caption= | size=100px }}<br />
*'''Cleandho Marcos''': <br />
I like to understand how life works and how we are related to it. I also like to help people and get to know them. My hobbies are: plants science, hiking and camping.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Chico.jpg | caption= | size=100px }}<br />
*'''Chico Camargo''': <br />
Chico is a molecular scientist who found his place in the modeling team. He says work and studies are for leisure time - having fun is his obligation. <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Daniel_Ariano.jpg | caption= | size=110px }}<br />
*'''Daniel Ariano''' <br />
Daniel is a Mechanic Engineer who loves every kind of science and technology, what includes synthetic biology. He is the "handyman" of the group.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Fernando_Lindenberg.jpg | caption= | size=110px }}<br />
*'''Fernando Lindenberg'''<br />
I am an undergraduate student at São Paulo State University at Araraquara, School of Pharmaceutical Sciences. I spent the last two years working and learning molecular biology at the Laboratory of Molecular and Cellular Biology.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Joana.jpg | caption= | size=100px }}<br />
*'''Joana Guiro''': <br />
Joana is a third year Biology undergraduate student at USP. She is very hard working and became pretty good in molecular biology basic techniques in the last couple of months. <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Lili.png | caption= | size=100px }}<br />
*'''Lilian Oliveira'''<br />
Lilian is a happy chemistry student at USP. She has the "gold hands" of the group: everything works with her magic touch.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=LucasC.jpg | caption= | size=80px}}<br />
*'''Lucas Cespedes''' <br />
Is an undergraduate of fifth year in biology at USP, besides iGEM, works with industrial microbiology, biodegradable plastics and molecular biology.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Maca.jpg | caption= | size=100px }}<br />
*'''Macarena López''':<br />
Is a chilean biology student at USP. She is at her second year, but has no idea at what field will she specialize. Every living organism appears to be worth and interesting to study for her!<br />
<br />
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<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Otto.png | caption= | size=100px }}<br />
*'''Otto Heringer''' <br />
He is a chemistry undergraduate student with an interdisciplinary feeling. In short: a synthetic biologist wannabe.<br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Pedro.png | caption= | size=100px }}<br />
*'''Pedro Medeiros'''<br />
<br />
I have a degree in Biology and now I'm an undergraduated in Pharmacy and Biochemistry by University of São Paulo. My interests are multidesciplinary, but I have a special attention in informatics, technology, open source development and biological sciences. I want to be a Bio Cyber Punk evil scientist in the future.... soon... soon...<br />
<br />
=What we did=<br />
<br />
The participation of the people related to the Plug&Play project is show in the project's [https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Experiments page].<br />
<br />
<br />
The participation of the people related to the Associative Memory Network project is show in the [https://2012.igem.org/Team:USP-UNESP-Brazil/Notebook Lab Diary page].<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/TeamTeam:USP-UNESP-Brazil/Team2012-09-27T03:48:32Z<p>Pepeks: /* Undergrads: */</p>
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=iGEM Brazilian Team=<br />
<br />
We are an interdisciplinary group of undergraduate and graduate students from several areas. The undergrads are divided among the Chemistry Institute (USP), the Biosciences Institute (USP), the Pharmaceutical Sciences Faculty (USP and Unesp) and the Polytechnic School (USP). The graduate students are from the Biomedical Sciences Institute (USP), the Chemistry Institute (USP) and the Physics Institute (USP). <br />
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=Who we are=<br />
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==='''Instructors'''===<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Andrés.JPG | caption= | size=200px }}<br />
*'''Edgar Andrés Ochoa Cruz (Young PhD)''': <br />
I am a scientist interested in technology and inventions. Working for generating awareness of the power available on new technologies, like reverse bioinformatics (Computation in biological organisms) and synthetic biology (Energy and biofuel production applications), and the positive impact that they can generate in the life quality of world population.<br />
<br />
This competition was the opportunity to develop my first project in the synthetic biology field. More ideas for new projects are coming ...(swordfish1313@gmail.com)<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=marieVan.jpg | size=120px }}<br />
*'''Marie Anne Van Sluys (Full professor)''':<br />
Dr. Van Sluys is full professor in the Botany Department of the University of São Paulo. She is the head of the Genomic and Transposable Elements Laboratory (GaTE). The GaTE focus is to study the impact of transposable elements in the structure, function, and diversification of bacterial and plants genomes.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=carloshotta.jpg | caption= | size=120px }}<br />
*'''Carlos Takeshi Hotta (Assistant Professor)'''<br />
I am an assistant professor in the Biochemistry Department of the University of São Paulo. I am the head of the Plant Molecular Physiology group, that focus on the role of the circadian clock in increasing plant productivity. I also write a science blog [http://scienceblogs.com.br/brontossauros].<br />
<br />
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<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=Cleslei.jpg | size=120px }}<br />
*'''Cleslei Fernando Zanelli'''<br />
Degree in pharmacy and biochemistry from UNESP (2002) and PhD in Biotechnology from UNESP (2006). I am currently an assistant professor in the Biological Sciences Department of Pharmacy School of Araraquara - UNESP. Our lab is interested in understanding the control of protein synthesis using the budding yeast Saccharomyces cerevisiae as a model for functional genomics analyses. We also use the budding yeast collections of mutants and fusion proteins in high content screenings (HCS) to search for cellular drug targets.<br />
<br />
==='''Advisors:'''===<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Josegregorio.jpg | size=100px}}<br />
*'''José Gregório''':<br />
Degree in Biological Sciences from the Institute of Biosciences Masters in Biotechnology from the University of São Paulo (1994) and Ph.D. in Biological Sciences (Microbiology) from the University of São Paulo (2000). He is currently Professor from the University of São Paulo. He has experience in biochemistry, with emphasis on Biochemistry of Microorganisms, acting on the following topics: polyhydroxyalkanoates, biosurfactant, bacterial metabolism, metabolic engineering. <br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Luiziana.jpg | size=100px}}<br />
*'''Luiziana Ferreira da Silva''':<br />
Graduated in Pharmacy and Biochemistry, Faculty of Pharmaceutical Sciences USP (1982), MSc (1990) and Ph.D. (1998) in Biological Sciences (Microbiology) by the Institute of Biomedical Sciences, University of São Paulo. She is currently professor Dr., University of São Paulo. Has experience in the area of microbiology with emphasis on Bacteriology. Her research involving: bacterial metabolism, metabolic flux analysis, biosynthesis of biodegradable plastics and elastomers (polyhydroxyalkanoates - PHA) as well as other compounds of biotechnological interest.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=GlauciaFoto.jpg | size=100px}}<br />
*'''Glaucia Souza'''<br />
Prof. Glaucia Souza is bachelor by the Biosciences Institute at USP (1988), graduate in biochemistry by the Chemistry Institute at USP (1993), post-doctor in molecular genetics by La Jolla Cancer Research Foundation (1994) and post-doctor in molecular genetics by Baylor College of Medicine (1996). Currently she is associated professor and leader of the Signal Transduction Lab at Chemistry Institute (USP).<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Ncaticha.jpg | size=100px}}<br />
*'''Nestor Caticha'''<br />
Nestor Caticha is a professor at the Instituto de Física USP working in Information theory<br />
of artificial and natural systems and Bayesian data analysis. Current research includes <br />
modelling collective properties of societies of information processing agents, theoretical <br />
analysis of decision processes and the development of methods for neuroimaging using<br />
fMRI and EEG. He obtained a B.Sc. from Unicamp and a Ph.D. from Caltech, both<br />
in Physics.<br />
<br />
==='''Graduate:'''===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Debora.jpg | caption= | size=80px }}<br />
*'''Débora Parrine'''<br />
Débora is a graduate student from Prof. Luiziana's Lab. Her research involves production of biodegradable materials and metabolic engeneering.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Luiza.JPG | caption= | size=100px }}<br />
*'''Luíza Barros''':<br />
Luíza is a plant scientist. She was the last one to join the team and is always available to talk about how wonderful Molecular Biology can be to solve our lab problems.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Marcelo.jpg | caption= | size=100px }}<br />
*'''Marcelo Boareto''':<br />
Marcelo is a biological physicist and phD student in Physics. He has two academic goals: to be recognized as a biologist when talking to a biologist and as a physicist when talking to a physicist. Neither of the two has been achieved, yet.<br />
<br />
==='''Undergrads:'''===<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cauã.jpg | caption= | size=80px }}<br />
*'''Cauã Westmann''':<br />
I'm a Biology undergratuate student at USP and an eternal lover of life and all its aspects. I'm always trying to understand and connect everything in an holistic evolutionary aproach.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cleandho.JPG | caption= | size=100px }}<br />
*'''Cleandho Marcos''': <br />
I like to understand how life works and how we are related to it. I also like to help people and get to know them. My hobbies are: plants science, hiking and camping.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Chico.jpg | caption= | size=100px }}<br />
*'''Chico Camargo''': <br />
Chico is a molecular scientist who found his place in the modeling team. He says work and studies are for leisure time - having fun is his obligation. <br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Daniel_Ariano.jpg | caption= | size=110px }}<br />
*'''Daniel Ariano''' <br />
Daniel is a Mechanic Engineer who loves every kind of science and technology, what includes synthetic biology. He is the "handyman" of the group.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Fernando_Lindenberg.jpg | caption= | size=110px }}<br />
*'''Fernando Lindenberg'''<br />
I am an undergraduate student at São Paulo State University at Araraquara, School of Pharmaceutical Sciences. I spent the last two years working and learning molecular biology at the Laboratory of Molecular and Cellular Biology.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Joana.jpg | caption= | size=100px }}<br />
*'''Joana Guiro''': <br />
Joana is a third year Biology undergraduate student at USP. She is very hard working and became pretty good in molecular biology basic techniques in the last couple of months. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Lili.png | caption= | size=100px }}<br />
*'''Lilian Oliveira'''<br />
Lilian is a happy chemistry student at USP. She has the "gold hands" of the group: everything works with her magic touch.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=LucasC.jpg | caption= | size=80px}}<br />
*'''Lucas Cespedes''' <br />
Is an undergraduate of fifth year in biology at USP, besides iGEM, works with industrial microbiology, biodegradable plastics and molecular biology.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Maca.jpg | caption= | size=100px }}<br />
*'''Macarena López''':<br />
Is a chilean biology student at USP. She is at her second year, but has no idea at what field will she specialize. Every living organism appears to be worth and interesting to study for her!<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Otto.png | caption= | size=100px }}<br />
*'''Otto Heringer''' <br />
He is a chemistry undergraduate student with an interdisciplinary feeling. In short: a synthetic biologist wannabe.<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Pedro.png | caption= | size=100px }}<br />
*'''Pedro Medeiros'''<br />
<br />
I have a degree in Biology and now I'm an undergraduated in Pharmacy and Biochemistry by University of São Paulo. My interests are multidesciplinary, but I have a special attention in informatics, technology, open source development and biological sciences. I want to be a Bio Cyber Punk evil scientist in the future.... soon... soon...<br />
<br />
=What we did=<br />
<br />
The participation of the people related to the Plug&Play project is show in the project's [https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Experiments page].<br />
<br />
<br />
The participation of the people related to the Associative Memory Network project is show in the [https://2012.igem.org/Team:USP-UNESP-Brazil/Notebook Lab Diary page].<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:41:39Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
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<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel ([https://static.igem.org/mediawiki/2012/c/ce/QC1_e_QC6_-_2.png QC1 and QC6 Electrophoresis Gel])<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel([https://static.igem.org/mediawiki/2012/c/ce/QC1_e_QC6_-_2.png QC1 and QC6 Electrophoresis Gel])<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel([https://static.igem.org/mediawiki/2012/a/a7/QC4_120813.png QC4 Electrophoresis Gel])<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
([https://static.igem.org/mediawiki/2012/b/b0/QC5_-_120818.png QC5 Electrophoresis Gel])<br />
<br />
<br />
<br />
====QR4====<br />
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.<br />
<br />
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.<br />
<br />
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.<br />
<br />
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.<br />
<br />
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics. <br />
<br />
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.<br />
<br />
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.<br />
<br />
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.<br />
<br />
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).<br />
<br />
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.<br />
<br />
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics. <br />
<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
([https://static.igem.org/mediawiki/2012/3/32/QR4.png QR4 Electrophoresis Gel])<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
([https://static.igem.org/mediawiki/2012/2/29/QR5.png QR5 Electrophoresis Gel])<br />
<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
([https://static.igem.org/mediawiki/2012/7/7f/QR6.jpg QR6 Electrophoresis Gel])<br />
<br />
====QR1====<br />
18/09/2012 - Pedro - Ligation. <br />
<br />
19/09/2012 - Daniel & Luiza - Transformation.<br />
<br />
20/09/2012 - Daniel - Inoculum.<br />
<br />
21/09/2012 - Pedro - Digestion and PCR.<br />
<br />
24/09/2012 - Pedro - Electrophoresis Gel.([https://static.igem.org/mediawiki/2012/9/91/QR1_120924b.png QR1 Electrophoresis Gel])<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:35:57Z<p>Pepeks: /* QC1 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel ([https://static.igem.org/mediawiki/2012/c/ce/QC1_e_QC6_-_2.png QC1 and QC6 Electrophoresis Gel])<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
<br />
<br />
====QR4====<br />
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.<br />
<br />
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.<br />
<br />
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.<br />
<br />
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.<br />
<br />
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics. <br />
<br />
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.<br />
<br />
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.<br />
<br />
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.<br />
<br />
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).<br />
<br />
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.<br />
<br />
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics. <br />
<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Pedro - Ligation. <br />
<br />
19/09/2012 - Daniel & Luiza - Transformation.<br />
<br />
20/09/2012 - Daniel - Inoculum.<br />
<br />
21/09/2012 - Pedro - Digestion and PCR.<br />
<br />
24/09/2012 - Pedro - Electrophoresis Gel.<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:34:54Z<p>Pepeks: /* QC1 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel ([https://2012.igem.org/File:QC1_e_QC6_-_2.png QC1 and QC6 Gel]<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
<br />
<br />
====QR4====<br />
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.<br />
<br />
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.<br />
<br />
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.<br />
<br />
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.<br />
<br />
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics. <br />
<br />
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.<br />
<br />
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.<br />
<br />
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.<br />
<br />
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).<br />
<br />
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.<br />
<br />
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics. <br />
<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Pedro - Ligation. <br />
<br />
19/09/2012 - Daniel & Luiza - Transformation.<br />
<br />
20/09/2012 - Daniel - Inoculum.<br />
<br />
21/09/2012 - Pedro - Digestion and PCR.<br />
<br />
24/09/2012 - Pedro - Electrophoresis Gel.<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:32:28Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
<br />
<br />
====QR4====<br />
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.<br />
<br />
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.<br />
<br />
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.<br />
<br />
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.<br />
<br />
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics. <br />
<br />
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.<br />
<br />
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.<br />
<br />
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.<br />
<br />
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).<br />
<br />
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.<br />
<br />
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics. <br />
<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Pedro - Ligation. <br />
<br />
19/09/2012 - Daniel & Luiza - Transformation.<br />
<br />
20/09/2012 - Daniel - Inoculum.<br />
<br />
21/09/2012 - Pedro - Digestion and PCR.<br />
<br />
24/09/2012 - Pedro - Electrophoresis Gel.<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:31:13Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QR4====<br />
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.<br />
<br />
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.<br />
<br />
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.<br />
<br />
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.<br />
<br />
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics. <br />
<br />
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.<br />
<br />
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.<br />
<br />
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.<br />
<br />
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).<br />
<br />
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.<br />
<br />
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics. <br />
<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Pedro - Ligation. <br />
<br />
19/09/2012 - Daniel & Luiza - Transformation.<br />
<br />
20/09/2012 - Daniel - Inoculum.<br />
<br />
21/09/2012 - Pedro - Digestion and PCR.<br />
<br />
24/09/2012 - Pedro - Electrophoresis Gel.<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:30:44Z<p>Pepeks: /* QR1 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QR4====<br />
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.<br />
<br />
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.<br />
<br />
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.<br />
<br />
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.<br />
<br />
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics. <br />
<br />
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.<br />
<br />
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.<br />
<br />
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.<br />
<br />
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).<br />
<br />
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.<br />
<br />
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics. <br />
<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Pedro - Ligation. <br />
<br />
19/09/2012 - Daniel & Luiza - Transformation.<br />
<br />
20/09/2012 - Daniel - Inoculum.<br />
<br />
21/09/2012 - Pedro - Digestion and PCR.<br />
<br />
24/09/2012 - Pedro - Electrophoresis Gel.<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:28:13Z<p>Pepeks: /* QC6 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Tranformation<br />
<br />
14/06/2012 - Daniel, Inoculum<br />
<br />
15/06/2012 - Otto, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QR4====<br />
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.<br />
<br />
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.<br />
<br />
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Daniel & Luiza - Ligation. <br />
<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:24:54Z<p>Pepeks: /* QC5 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]<br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Pedro, Ligation<br />
<br />
15/08/2012 - Daniel, Transformation<br />
<br />
17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR<br />
<br />
18/08/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
====QR4====<br />
<br />
====QR5====<br />
01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. <br />
A few changes were made to this protocol. <br />
- The enzyme NotI was used instead of DpnI.<br />
- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.<br />
- 2uL T4 DNA ligase was used instead of 1uL<br />
- No water added<br />
<br />
02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.<br />
<br />
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).<br />
<br />
====QR6====<br />
08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
10/08/12 – Fernando: All colonies were red.<br />
<br />
22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly. <br />
<br />
23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.<br />
<br />
26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.<br />
<br />
27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.<br />
The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.<br />
<br />
01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).<br />
<br />
02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.<br />
<br />
03/09/12 – Fernando: All colonies were red<br />
<br />
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.<br />
<br />
====QR1====<br />
18/09/2012 - Daniel & Luiza - Ligation. <br />
<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:21:50Z<p>Pepeks: /* QC4 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
<br />
07/08/2012 - Pedro, Transformation <br />
<br />
10/08/2012 - Daniel, Inoculate<br />
<br />
11/06/2012 - Daniel, Miniprep<br />
<br />
13/06/2012 - Pedro, Digestion<br />
<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
<br />
====QC5==== <br />
14/08/2012 - Daniel, Ligation<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
18/09/2012 - Daniel & Luiza - Ligation. <br />
<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T03:21:34Z<p>Pepeks: /* QC1 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
=Lab Diary=<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
<br />
13/06/2012 - Daniel, Transformation <br />
<br />
14/06/2012 - Daniel, Inoculate<br />
<br />
15/06/2012 - Daniel, Miniprep<br />
<br />
16/06/2012 - Otto, Digestion<br />
<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
07/08/2012 - Pedro, Transformation <br />
10/08/2012 - Daniel, Inoculate<br />
11/06/2012 - Daniel, Miniprep<br />
13/06/2012 - Pedro, Digestion<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
====QC5==== <br />
14/08/2012 - Daniel, Ligation<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
18/09/2012 - Daniel & Luiza - Ligation. <br />
<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/TeamTeam:USP-UNESP-Brazil/Team2012-09-27T03:19:34Z<p>Pepeks: /* Who we are */</p>
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<br />
=iGEM Brazilian Team=<br />
<br />
We are an interdisciplinary group of undergraduate and graduate students from several areas. The undergrads are divided among the Chemistry Institute (USP), the Biosciences Institute (USP), the Pharmaceutical Sciences Faculty (USP and Unesp) and the Polytechnic School (USP). The graduate students are from the Biomedical Sciences Institute (USP), the Chemistry Institute (USP) and the Physics Institute (USP). <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption=USP and UNESP Brazil's Team | image=lasttime2.jpg | size=500px }}<br />
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<br />
=Who we are=<br />
<br />
<br />
<br />
<br />
==='''Instructors'''===<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Andrés.JPG | caption= | size=200px }}<br />
*'''Edgar Andrés Ochoa Cruz (Young PhD)''': <br />
I am a scientist interested in technology and inventions. Working for generating awareness of the power available on current technologies and the positive impact that can be generated through them to improve the life quality of the world population. <br />
<br />
This competition was the opportunity to develop my first project in the synthetic biology field. More ideas for new projects are coming ...(swordfish1313@gmail.com)<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=marieVan.jpg | size=120px }}<br />
*'''Marie Anne Van Sluys (Full professor)''':<br />
Dr. Van Sluys is full professor in the Botany Department of the University of São Paulo. She is the head of the Genomic and Transposable Elements Laboratory (GaTE). The GaTE focus is to study the impact of transposable elements in the structure, function, and diversification of bacterial and plants genomes.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=carloshotta.jpg | caption= | size=120px }}<br />
*'''Carlos Takeshi Hotta (Assistant Professor)'''<br />
I am an assistant professor in the Biochemistry Department of the University of São Paulo. I am the head of the Plant Molecular Physiology group, that focus on the role of the circadian clock in increasing plant productivity. I also write a science blog [http://scienceblogs.com.br/brontossauros].<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=Cleslei.jpg | size=120px }}<br />
*'''Cleslei Fernando Zanelli'''<br />
Degree in pharmacy and biochemistry from UNESP (2002) and PhD in Biotechnology from UNESP (2006). I am currently an assistant professor in the Biological Sciences Department of Pharmacy School of Araraquara - UNESP. Our lab is interested in understanding the control of protein synthesis using the budding yeast Saccharomyces cerevisiae as a model for functional genomics analyses. We also use the budding yeast collections of mutants and fusion proteins in high content screenings (HCS) to search for cellular drug targets.<br />
<br />
==='''Advisors:'''===<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Josegregorio.jpg | size=100px}}<br />
*'''José Gregório''':<br />
Degree in Biological Sciences from the Institute of Biosciences Masters in Biotechnology from the University of São Paulo (1994) and Ph.D. in Biological Sciences (Microbiology) from the University of São Paulo (2000). He is currently Professor from the University of São Paulo. He has experience in biochemistry, with emphasis on Biochemistry of Microorganisms, acting on the following topics: polyhydroxyalkanoates, biosurfactant, bacterial metabolism, metabolic engineering. <br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Luiziana.jpg | size=100px}}<br />
*'''Luiziana Ferreira da Silva''':<br />
Graduated in Pharmacy and Biochemistry, Faculty of Pharmaceutical Sciences USP (1982), MSc (1990) and Ph.D. (1998) in Biological Sciences (Microbiology) by the Institute of Biomedical Sciences, University of São Paulo. She is currently professor Dr., University of São Paulo. Has experience in the area of microbiology with emphasis on Bacteriology. Her research involving: bacterial metabolism, metabolic flux analysis, biosynthesis of biodegradable plastics and elastomers (polyhydroxyalkanoates - PHA) as well as other compounds of biotechnological interest.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=GlauciaFoto.jpg | size=100px}}<br />
*'''Glaucia Souza'''<br />
Prof. Glaucia Souza is bachelor by the Biosciences Institute at USP (1988), graduate in biochemistry by the Chemistry Institute at USP (1993), post-doctor in molecular genetics by La Jolla Cancer Research Foundation (1994) and post-doctor in molecular genetics by Baylor College of Medicine (1996). Currently she is associated professor and leader of the Signal Transduction Lab at Chemistry Institute (USP).<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Ncaticha.jpg | size=100px}}<br />
*'''Nestor Caticha'''<br />
Nestor Caticha is a professor at the Instituto de Física USP working in Information theory<br />
of artificial and natural systems and Bayesian data analysis. Current research includes <br />
modelling collective properties of societies of information processing agents, theoretical <br />
analysis of decision processes and the development of methods for neuroimaging using<br />
fMRI and EEG. He obtained a B.Sc. from Unicamp and a Ph.D. from Caltech, both<br />
in Physics.<br />
<br />
==='''Graduate:'''===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Debora.jpg | caption= | size=80px }}<br />
*'''Débora Parrine'''<br />
Débora is a graduate student from prof.Luiziana's Lab. Her research involves production of biodegradable materials.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Luiza.JPG | caption= | size=100px }}<br />
*'''Luíza Barros''':<br />
Luíza is a plant scientist. She was the last one to join the team and is always available to talk about how wonderful Molecular Biology can be to solve our lab problems.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Marcelo.jpg | caption= | size=100px }}<br />
*'''Marcelo Boareto''':<br />
Marcelo is a biological physicist and phD student in Physics. He has two academic goals: to be recognized as a biologist when talking to a biologist and as a physicist when talking to a physicist. Neither of the two has been achieved, yet.<br />
<br />
<br />
<br />
==='''Undergrads:'''===<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cauã.jpg | caption= | size=80px }}<br />
*'''Cauã Westmann''':<br />
I'm a Biology undergratuate student at USP and an eternal lover of life and all its aspects. I'm always trying to understand and connect everything in an holistic evolutionary aproach.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cleandho.JPG | caption= | size=100px }}<br />
*'''Cleandho Marcos''': <br />
I like to understand how life works and how we are related to it. I also like to help people and get to know them. My hobbies are: plants science, hiking and camping.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Chico.jpg | caption= | size=100px }}<br />
*'''Chico Camargo''': <br />
Chico is a molecular scientist who found his place in the modeling team. He says work and studies are for leisure time - having fun is his obligation. <br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Daniel_Ariano.jpg | caption= | size=110px }}<br />
*'''Daniel Ariano''' <br />
Daniel is a Mechanic Engineer who loves every kind of science and technology, what includes synthetic biology. He is the "handyman" of the group.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Fernando_Lindenberg.jpg | caption= | size=110px }}<br />
*'''Fernando Lindenberg'''<br />
I am an undergraduate student at São Paulo State University at Araraquara, School of Pharmaceutical Sciences. I spent the last two years working and learning molecular biology at the Laboratory of Molecular and Cellular Biology.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Joana.jpg | caption= | size=100px }}<br />
*'''Joana Guiro''': <br />
Joana is a third year Biology undergraduate student at USP. She is very hard working and became pretty good in molecular biology basic techniques in the last couple of months. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Lili.png | caption= | size=100px }}<br />
*'''Lilian Oliveira'''<br />
Lilian is a happy chemistry student at USP. She has the "gold hands" of the group: everything works with her magic touch.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=LucasC.jpg | caption= | size=80px}}<br />
*'''Lucas Cespedes''' <br />
Is an undergraduate of fifth year in biology at USP, besides iGEM, works with industrial microbiology, biodegradable plastics and molecular biology.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Maca.jpg | caption= | size=100px }}<br />
*'''Macarena López''':<br />
I'm a chilean biology student.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Otto.png | caption= | size=100px }}<br />
*'''Otto Heringer''' <br />
He is a chemistry undergraduate student with an interdisciplinary feeling. In short: a synthetic biologist wannabe.<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Pedro.png | caption= | size=100px }}<br />
*'''Pedro Medeiros'''<br />
<br />
I have a degree in biology and now I'm an undergraduated in Pharmacy and Biochemistry by University of São Paulo. I'm still looking for something that could be fun to be writen here....<br />
<br />
=What we did=<br />
<br />
The participation of the people related to the Plug&Play project is show in the project's [https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Experiments page].<br />
<br />
<br />
The participation of the people related to the Associative Memory Network project is show in the [https://2012.igem.org/Team:USP-UNESP-Brazil/Notebook Lab Diary page].<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/TeamTeam:USP-UNESP-Brazil/Team2012-09-27T03:18:26Z<p>Pepeks: /* Who we are */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
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<br />
=iGEM Brazilian Team=<br />
<br />
We are an interdisciplinary group of undergraduate and graduate students from several areas. The undergrads are divided among the Chemistry Institute (USP), the Biosciences Institute (USP), the Pharmaceutical Sciences Faculty (USP and Unesp) and the Polytechnic School (USP). The graduate students are from the Biomedical Sciences Institute (USP), the Chemistry Institute (USP) and the Physics Institute (USP). <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption=USP and UNESP Brazil's Team | image=lasttime2.jpg | size=500px }}<br />
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<br />
<br />
<br />
<br />
=Who we are=<br />
<br />
<br />
<br />
<br />
==='''Instructors'''===<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Andrés.JPG | caption= | size=200px }}<br />
*'''Edgar Andrés Ochoa Cruz (Young PhD)''': <br />
I am a scientist interested in technology and inventions. Working for generating awareness of the power available on current technologies and the positive impact that can be generated through them to improve the life quality of the world population. <br />
<br />
This competition was the opportunity to develop my first project in the synthetic biology field. More ideas for new projects are coming ...(swordfish1313@gmail.com)<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=marieVan.jpg | size=120px }}<br />
*'''Marie Anne Van Sluys (Full professor)''':<br />
Dr. Van Sluys is full professor in the Botany Department of the University of São Paulo. She is the head of the Genomic and Transposable Elements Laboratory (GaTE). The GaTE focus is to study the impact of transposable elements in the structure, function, and diversification of bacterial and plants genomes.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=carloshotta.jpg | caption= | size=120px }}<br />
*'''Carlos Takeshi Hotta (Assistant Professor)'''<br />
I am an assistant professor in the Biochemistry Department of the University of São Paulo. I am the head of the Plant Molecular Physiology group, that focus on the role of the circadian clock in increasing plant productivity. I also write a science blog [http://scienceblogs.com.br/brontossauros].<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=Clesleo.jpg | size=120px }}<br />
*'''Cleslei Fernando Zanelli'''<br />
Degree in pharmacy and biochemistry from UNESP (2002) and PhD in Biotechnology from UNESP (2006). I am currently an assistant professor in the Biological Sciences Department of Pharmacy School of Araraquara - UNESP. Our lab is interested in understanding the control of protein synthesis using the budding yeast Saccharomyces cerevisiae as a model for functional genomics analyses. We also use the budding yeast collections of mutants and fusion proteins in high content screenings (HCS) to search for cellular drug targets.<br />
<br />
==='''Advisors:'''===<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Josegregorio.jpg | size=100px}}<br />
*'''José Gregório''':<br />
Degree in Biological Sciences from the Institute of Biosciences Masters in Biotechnology from the University of São Paulo (1994) and Ph.D. in Biological Sciences (Microbiology) from the University of São Paulo (2000). He is currently Professor from the University of São Paulo. He has experience in biochemistry, with emphasis on Biochemistry of Microorganisms, acting on the following topics: polyhydroxyalkanoates, biosurfactant, bacterial metabolism, metabolic engineering. <br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Luiziana.jpg | size=100px}}<br />
*'''Luiziana Ferreira da Silva''':<br />
Graduated in Pharmacy and Biochemistry, Faculty of Pharmaceutical Sciences USP (1982), MSc (1990) and Ph.D. (1998) in Biological Sciences (Microbiology) by the Institute of Biomedical Sciences, University of São Paulo. She is currently professor Dr., University of São Paulo. Has experience in the area of microbiology with emphasis on Bacteriology. Her research involving: bacterial metabolism, metabolic flux analysis, biosynthesis of biodegradable plastics and elastomers (polyhydroxyalkanoates - PHA) as well as other compounds of biotechnological interest.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=GlauciaFoto.jpg | size=100px}}<br />
*'''Glaucia Souza'''<br />
Prof. Glaucia Souza is bachelor by the Biosciences Institute at USP (1988), graduate in biochemistry by the Chemistry Institute at USP (1993), post-doctor in molecular genetics by La Jolla Cancer Research Foundation (1994) and post-doctor in molecular genetics by Baylor College of Medicine (1996). Currently she is associated professor and leader of the Signal Transduction Lab at Chemistry Institute (USP).<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Ncaticha.jpg | size=100px}}<br />
*'''Nestor Caticha'''<br />
Nestor Caticha is a professor at the Instituto de Física USP working in Information theory<br />
of artificial and natural systems and Bayesian data analysis. Current research includes <br />
modelling collective properties of societies of information processing agents, theoretical <br />
analysis of decision processes and the development of methods for neuroimaging using<br />
fMRI and EEG. He obtained a B.Sc. from Unicamp and a Ph.D. from Caltech, both<br />
in Physics.<br />
<br />
==='''Graduate:'''===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Debora.jpg | caption= | size=80px }}<br />
*'''Débora Parrine'''<br />
Débora is a graduate student from prof.Luiziana's Lab. Her research involves production of biodegradable materials.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Luiza.JPG | caption= | size=100px }}<br />
*'''Luíza Barros''':<br />
Luíza is a plant scientist. She was the last one to join the team and is always available to talk about how wonderful Molecular Biology can be to solve our lab problems.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Marcelo.jpg | caption= | size=100px }}<br />
*'''Marcelo Boareto''':<br />
Marcelo is a biological physicist and phD student in Physics. He has two academic goals: to be recognized as a biologist when talking to a biologist and as a physicist when talking to a physicist. Neither of the two has been achieved, yet.<br />
<br />
<br />
<br />
==='''Undergrads:'''===<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cauã.jpg | caption= | size=80px }}<br />
*'''Cauã Westmann''':<br />
I'm a Biology undergratuate student at USP and an eternal lover of life and all its aspects. I'm always trying to understand and connect everything in an holistic evolutionary aproach.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Cleandho.JPG | caption= | size=100px }}<br />
*'''Cleandho Marcos''': <br />
I like to understand how life works and how we are related to it. I also like to help people and get to know them. My hobbies are: plants science, hiking and camping.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Chico.jpg | caption= | size=100px }}<br />
*'''Chico Camargo''': <br />
Chico is a molecular scientist who found his place in the modeling team. He says work and studies are for leisure time - having fun is his obligation. <br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Daniel_Ariano.jpg | caption= | size=110px }}<br />
*'''Daniel Ariano''' <br />
Daniel is a Mechanic Engineer who loves every kind of science and technology, what includes synthetic biology. He is the "handyman" of the group.<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Fernando_Lindenberg.jpg | caption= | size=110px }}<br />
*'''Fernando Lindenberg'''<br />
I am an undergraduate student at São Paulo State University at Araraquara, School of Pharmaceutical Sciences. I spent the last two years working and learning molecular biology at the Laboratory of Molecular and Cellular Biology.<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=Joana.jpg | caption= | size=100px }}<br />
*'''Joana Guiro''': <br />
Joana is a third year Biology undergraduate student at USP. She is very hard working and became pretty good in molecular biology basic techniques in the last couple of months. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Lili.png | caption= | size=100px }}<br />
*'''Lilian Oliveira'''<br />
Lilian is a happy chemistry student at USP. She has the "gold hands" of the group: everything works with her magic touch.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=LucasC.jpg | caption= | size=80px}}<br />
*'''Lucas Cespedes''' <br />
Is an undergraduate of fifth year in biology at USP, besides iGEM, works with industrial microbiology, biodegradable plastics and molecular biology.<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Maca.jpg | caption= | size=100px }}<br />
*'''Macarena López''':<br />
I'm a chilean biology student.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Otto.png | caption= | size=100px }}<br />
*'''Otto Heringer''' <br />
He is a chemistry undergraduate student with an interdisciplinary feeling. In short: a synthetic biologist wannabe.<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/LImage | image=Pedro.png | caption= | size=100px }}<br />
*'''Pedro Medeiros'''<br />
<br />
I have a degree in biology and now I'm an undergraduated in Pharmacy and Biochemistry by University of São Paulo. I'm still looking for something that could be fun to be writen here....<br />
<br />
=What we did=<br />
<br />
The participation of the people related to the Plug&Play project is show in the project's [https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Experiments page].<br />
<br />
<br />
The participation of the people related to the Associative Memory Network project is show in the [https://2012.igem.org/Team:USP-UNESP-Brazil/Notebook Lab Diary page].<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/File:Cleslei.jpgFile:Cleslei.jpg2012-09-27T03:17:25Z<p>Pepeks: </p>
<hr />
<div></div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-27T02:17:34Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
12/06/2012 - Lilian, Ligation<br />
13/06/2012 - Daniel, Transformation <br />
14/06/2012 - Daniel, Inoculate<br />
15/06/2012 - Daniel, Miniprep<br />
16/06/2012 - Otto, Digestion<br />
17/06/2012 - Daniel, Electrophoresis Gel<br />
====QC4====<br />
06/08/2012 - Pedro, Ligation<br />
07/08/2012 - Pedro, Transformation <br />
10/08/2012 - Daniel, Inoculate<br />
11/06/2012 - Daniel, Miniprep<br />
13/06/2012 - Pedro, Digestion<br />
17/06/2012 - Pedro, Electrophoresis Gel<br />
====QC5==== <br />
14/08/2012 - Daniel, Ligation<br />
====QC6====<br />
12/06/2012 - Lilian, Ligation<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
18/09/2012 - Daniel & Luiza - Ligation. <br />
<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T21:08:25Z<p>Pepeks: /* From 12/06/2012 to 12/08/2012 – Assemblies attempts */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
<br />
====QC4====<br />
<br />
====QC5==== <br />
<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T21:02:08Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
<br />
====QC4====<br />
<br />
====QC5==== <br />
<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T21:00:07Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== <br />
13/08<br />
====QC4====<br />
13/08<br />
<br />
====QC5==== <br />
pag:18<br />
<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
24/09<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:54:20Z<p>Pepeks: /* QC1 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1==== 13/08<br />
<br />
====QC4====<br />
<br />
====QC5==== pag:18<br />
<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:53:02Z<p>Pepeks: /* QC5 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1====<br />
<br />
====QC4====<br />
<br />
====QC5==== pag:18<br />
<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:51:24Z<p>Pepeks: /* First assemblies confirmed */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
<br />
====QC1====<br />
<br />
====QC4====<br />
<br />
====QC5====<br />
<br />
====QR4====<br />
<br />
====QR5====<br />
<br />
====QR1====<br />
<br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:49:10Z<p>Pepeks: /* From 12/06/2012 to 12/08/2012 – Assemblies attempts */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
===First assemblies confirmed===<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:48:35Z<p>Pepeks: /* From 12/06/2012 to 12/08/2012 – Assemblies attempts */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.<br />
<br />
====First assemblies confirmed====<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:48:09Z<p>Pepeks: /* Assembly experiments */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
====From 12/06/2012 to 12/08/2012 – Assemblies attempts====<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice. <br />
<br />
====First assemblies confirmed====<br />
<br />
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows. <br />
<br />
At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:46:52Z<p>Pepeks: /* Assembly experiments */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
From 12/06/2012 to 12/08/2012 – Assemblies attempts<br />
<br />
At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.<br />
<br />
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice. <br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:45:41Z<p>Pepeks: /* Hotta's and Cleslei's Lab experiments */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=== 2- Hotta's and Cleslei's Lab experiments===<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:45:20Z<p>Pepeks: /* Greg's Lab Experiments */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=== 1- Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=Hotta's and Cleslei's Lab experiments=<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:45:03Z<p>Pepeks: /* Cleslei's Lab Experiments */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
===Greg's Lab Experiments===<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=Hotta's and Cleslei's Lab experiments=<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:44:25Z<p>Pepeks: /* Associative Memory Network */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
<br />
=Cleslei's Lab Experiments=<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
=Hotta's and Cleslei's Lab experiments=<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
===Assembly experiments===<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:40:27Z<p>Pepeks: /* Experiments of Biobricks */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
<br />
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
<br />
https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
<br />
<br />
==Associative Memory Network==<br />
<br />
===General experiments===<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
<br />
23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
<br />
===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Clesley's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
<br />
We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
<br />
<br />
<br />
<br />
===Assembly experiments===<br />
<br />
====MR2====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
<br />
11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
<br />
11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
<br />
07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
<br />
09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
<br />
14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
<br />
22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
<br />
04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
<br />
11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
<br />
13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
<br />
19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
<br />
23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
<br />
24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
<br />
02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
<br />
03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
<br />
08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
<br />
14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
<br />
14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/NotebookTeam:USP-UNESP-Brazil/Notebook2012-09-26T20:40:02Z<p>Pepeks: /* Associative Memory Network */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
<br />
<br />
<br />
== <h1 id="Experiments">Experiments</h1> ==<br />
<br />
<br />
==Plasmid Plug&Play==<br />
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The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.<br />
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https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments <br />
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<br />
==Associative Memory Network==<br />
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===General experiments===<br />
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17/07/12 – Amanda and Cleandho:<br />
- Tetracicline test using TOP10 without plasmid: successful, antibiotic is working<br />
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18/07/12 – Aline and Cleandho:<br />
- Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter<br />
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02/08/12 – Cleandho and Lucas:<br />
- Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use.<br />
- Make competent cells for storage and use in -80°C<br />
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03/08/12 – Cleandho and Lucas:<br />
- Competent cells test for viability and competency<br />
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22/08 – Cleandho and Lucas:<br />
- Make competent cell for use and storage at -80°C<br />
- Viability and competency tests of competent TOP 10<br />
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23/08/12 – Débora:<br />
- Digestion of pSB1K3 with PstI and EcoRI<br />
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03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
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05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3<br />
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===Experiments of Biobricks===<br />
At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project. <br />
<br />
The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Clesley's Lab, only Fernando<br />
[[File:BioBricks.png|center|600px|caption|]]<br />
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We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.<br />
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<br />
===Assembly experiments===<br />
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====MR2====<br />
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09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR2 part.<br />
MR2 grew colonies<br />
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10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR2 were inoculated in LB with tetracycline <br />
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11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR2 and quantification by nanodrop<br />
Expected size:<br />
MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR2 – (XbaI and PstI)<br />
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11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
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17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
18/07/12 – Aline and Cleandho:<br />
- Ligation of the parts MR2 using pSB1C3 backbone<br />
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19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR2<br />
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23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR2-2: 119,9 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR2: XbaI + PstI<br />
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24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR2-1 andMR2-2 clones: <br />
MR2-2 is positive and MR2-1 was negative.<br />
<br />
<br />
====MR3====<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation with MR3 part.<br />
MR3 – grew colonies<br />
<br />
10/07/2012 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial clones from MR3 were inoculated in LB with tetracycline <br />
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11/07/2012 – Amanda, Cleandho, Débora and Lucas:<br />
- Plasmid extraction of MR3<br />
Expected size:<br />
MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb<br />
- Digestion with enzymes and incubate 37 ° C for 1 hour<br />
MR3 – ( EcoRI and SpeI)<br />
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11/07/2012 – Aline and Cleandho:<br />
- Electrophoresis to confirmation: no DNA yield<br />
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18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR3 using pSB1C3 backbone<br />
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19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR3.<br />
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23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR3-1: 110,8 ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR3: EcoRI + SpeI<br />
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24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield<br />
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07/08/12 - Cleandho and Lucas:<br />
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3<br />
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09/08/12 – Amanda<br />
- Transformation of MR3 assembly<br />
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14/08/12 – Lucas and Amanda<br />
- Plasmid extraction from two MR3 clones <br />
- Digestion of MR3 clones with (EcoRI + SpeI)<br />
- Electrophoresis to confirm MR3 clones: result failed<br />
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22/08 – Cleandho and Lucas:<br />
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) <br />
- Transformation of MR3 assembly <br />
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04/09/12 – Cleandho:<br />
- Plasmid extraction of six MR3 clones <br />
- Digestion of MR3 clones with EcoRI and SpeI<br />
- Quantification of DNA of MR3 clones:<br />
A 178 ng/uL<br />
B 129 ng/uL<br />
C 119 ng/uL<br />
D 98 ng/uL<br />
E 159 ng/uL<br />
F 110 ng/uL<br />
- Digestion of MR3 using HindIII <br />
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed<br />
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05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 2M, 4G<br />
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
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06/09/12 – Cleandho:<br />
- Transformation of MR3 assembly in three molar proportions.<br />
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11/09/12 – Cleandho and Lucas:<br />
- PCR screening of ten colonies clones of assembly MR3 <br />
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12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon<br />
- Inoculum of clone 4 of MR3 in LBK<br />
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13/09/12 Cleandho and Lucas:<br />
- Plasmid extraction of clone 4 from MR3 assembly<br />
- Digestion of pDNA of clones 4 using EcoRI and SpeI<br />
- Electrophoresis for confirmation of clone 4. Result: failed<br />
<br />
<br />
<br />
====MR6====<br />
<br />
09/07/12 - Amanda, Cleandho, Débora and Lucas:<br />
- Bacterial transformation MR6 part.<br />
MR6 – not grown colonies<br />
<br />
17/07/12 – Amanda and Cleandho:<br />
- Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell<br />
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18/07/12 – Aline and Cleandho:<br />
- Ligation of the part MR6 using pSB1C3 backbone<br />
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19.07.12 – Aline, Cleandho and Débora:<br />
- Transformation of MR6 <br />
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23/07/12 – Cleandho and Débora:<br />
- Quantification of pDNA:<br />
MR6-1: 45,6ng/uL<br />
MR6-5: 46,7ng/uL<br />
- Digestion, using approximately 300ng of DNA<br />
MR6: EcroRI + SpeI<br />
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24/07/12 – Cleandho:<br />
- Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed<br />
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02/08/12 – Cleandho and Lucas:<br />
- Ligation/Assembly 3A of MR6 part<br />
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03/08/12 – Cleandho and Lucas:<br />
- Transformation of MR6<br />
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08/08/12 – Débora:<br />
- Plasmid extraction from MR6 clones<br />
- Digestion of pDNA with EcoRI + PstI from MR6 clones<br />
<br />
09/08/12 – Amanda<br />
- Electrophoresis to confirm MR6 clones. Result: failed<br />
<br />
03/09/12 – Cleandho:<br />
- Preparation of LB culture media<br />
- Inoculum of six MR6 clones <br />
<br />
05/09/12 – Cleandho and Débora:<br />
- Digestion of pSB1K3, pSB1C3, 11J and 16P<br />
- Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3<br />
<br />
06/09/12 – Cleandho:<br />
- Transformation of MR6 assembly in three molar proportions.<br />
<br />
12/09/12 – Cleandho and Lucas:<br />
- Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct<br />
<br />
====MR7====<br />
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14/08/12 – Lucas:<br />
- Ligation of assembly MR7 using pSB1K3 <br />
<br />
24/08/12 – Débora:<br />
- Transformation of MR7 assembly<br />
<br />
28/08/12 – Débora and Lucas:<br />
- Plasmid extraction of MR7 clones <br />
- Digestion of MR7 clones with XbaI and PstI<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Electrophoresis of two clones of MR7 assembly. Result: failed<br />
<br />
<br />
====MR9====<br />
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14/08/12 – Amanda<br />
- Transformation of MR9 assembly<br />
<br />
27/08/12 – Lucas:<br />
- Plasmid extraction for MR9 clones<br />
- Digestion of MR9 clones with EcoRI and SpeI<br />
- Electrophoresis of MR9 clones. Result: failed<br />
<br />
29/08/12 – Débora and Lucas:<br />
- Transformation of MR9 assembly<br />
<br />
<br />
{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/File:BioBricks.pngFile:BioBricks.png2012-09-26T20:39:12Z<p>Pepeks: </p>
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<div></div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/TeamTeam:USP-UNESP-Brazil/Team2012-09-26T04:17:13Z<p>Pepeks: /* Undergrads: */</p>
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=iGEM Brazilian Team=<br />
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The undergraduate students are from the Chemistry Institute (USP), the Biosciences Institute (USP), the Pharmaceutical Sciences Faculty (USP and UNESP) and the Polytechnic School (USP). The graduate students are from the Biomedical Sciences Institute (USP), the Biosciences Institute (USP) and the Physics Institute (USP). <br />
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=Who we are=<br />
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==='''Instructors'''===<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=carloshotta.jpg | caption= | size=120px }}<br />
*'''Carlos Takeshi Hotta (Assistant Professor)'''<br />
I am an assistant professor in the Biochemistry Department of the University of São Paulo. I am the head of the Plant Molecular Physiology group, that focus on the role of the circadian clock in increasing plant productivity. I also write a science blog [http://scienceblogs.com.br/brontossauros].<br />
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*'''Cleslei Fernando Zanelli'''<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Andrés.JPG | caption= | size=200px }}<br />
*'''Edgar Andrés Ochoa Cruz (Young PhD)''': <br />
I am a scientist interested in technology and inventions. Working for generating awareness of the power available on current technologies and the positive impact that can be generated through them to improve the life quality of the world population. <br />
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This competition was the opportunity to develop my first project in the synthetic biology field. More ideas are coming for new projects...(swordfish1313@gmail.com) <br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | caption= | image=marieVan.jpg | size=120px }}<br />
*'''Marie Anne Van Sluys (Full professor)''':<br />
Dr. Van Sluys is full professor in the Botany Department of the University of São Paulo. She is the head of the Genomic and Transposable Elements Laboratory (GaTE), the GaTE focus is to study the impact of transposable elements in the structure, function, and diversification of bacterial genomes and plants. <br />
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==='''Advisors:'''===<br />
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*'''Glaucia Souza''' <br />
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*'''José Gregório''':<br />
Degree in Biological Sciences from the Institute of Biosciences Masters in Biotechnology from the University of São Paulo (1994) and Ph.D. in Biological Sciences (Microbiology) from the University of São Paulo (2000). He is currently Professor from the University of São Paulo. He has experience in biochemistry, with emphasis on Biochemistry of Microorganisms, acting on the following topics: polyhydroxyalkanoates, biosurfactant, bacterial metabolism, metabolic engineering. <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage | caption= | image=Luiziana.jpg | size=100px}}<br />
*'''Luiziana Ferreira da Silva''':<br />
Graduated in Pharmacy and Biochemistry, Faculty of Pharmaceutical Sciences USP (1982), MSc (1990) and Ph.D. (1998) in Biological Sciences (Microbiology) by the Institute of Biomedical Sciences, University of São Paulo. She is currently professor Dr., University of São Paulo. Has experience in the area of microbiology with emphasis on Bacteriology. Her research involving: bacterial metabolism, metabolic flux analysis, biosynthesis of biodegradable plastics and elastomers (polyhydroxyalkanoates - PHA) as well as other compounds of biotechnological interest.<br />
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*'''Nestor Caticha'''<br />
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==='''Graduate:'''===<br />
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*'''Débora Parrine''': Still looking for the camera...<br />
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Luiza.JPG | caption= | size=100px }}<br />
*'''Luíza Barros''': Luíza is a plant scientist. She was the last one to join the team and is always available to talk about how wonderful Molecular Biology can be to solve our lab problems.<br />
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*'''Marcelo Boareto''': Marcelo is a biological physicist and phD student in Physics. He has two academic goals: to be recognized as a biologist when talking to a biologist and as a physicist when talking to a physicist. Neither of the two has been achieved, yet.<br />
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==='''Undergrads:'''===<br />
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*'''Cauã Westmann''': I'm a Biology undergratuate student at USP and an eternal lover of life and all its aspects. I'm always trying to understand and connect evetything in an holistic evolutionary aproach.<br />
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*'''Cleandho Marcos''': <br />
I like to understand how life works and how we are related to it. I also like to help people and get to know them. My hobbies are: plants science, hiking and camping.<br />
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*'''Chico Camargo''': <br />
Chico is a molecular scientist who found his place in the modeling team. He says work and studies are for leisure time - having fun is his obligation. <br />
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*'''Joana Guiro''': <br />
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{{:Team:USP-UNESP-Brazil/Templates/LImage |frameless|border| image=LucasC.jpg | caption= | size=80px}}<br />
*'''Lucas Cespedes''' <br />
Is an undergraduate of fifth year in biology at USP, besides iGEM, works with industrial microbiology, biodegradable plastics and molecular biology.<br />
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*'''Macarena López''':<br />
I'm a biologist.<br />
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*'''Otto Heringer''' <br />
*'''Pedro Medeiros'''<br />
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I have a degree in biology and now I'm an undergraduated in Pharmacy and Biochemistry by University of São Paulo. I'm still looking for something that could be fun to be writen here....<br />
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*'''Daniel Ariano''' <br />
*'''Fernando Lindenberg'''<br />
*'''Liliam Oliveira'''<br />
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=What we did=<br />
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The participation of the people related to the Plug&Play project is show in the Experiments page.<br />
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https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments<br />
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The participation of the people related to the Associative Memory Network project is show in the Lab Diary page.<br />
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https://2012.igem.org/Team:USP-UNESP-Brazil/Notebook<br />
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{{:Team:USP-UNESP-Brazil/Templates/Foot}}</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ModelingTeam:USP-UNESP-Brazil/Associative Memory/Modeling2012-09-23T20:09:14Z<p>Pepeks: </p>
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The modeling of the system will be made of differential equations, describing the intra and extra molecular dynamics using parameters such as genic devices and substances diffusion. Initial trials of the associative behavior were made considering the system as a discrete one. Although it is not definitive due to the continuity of the variables, the program was capable of predict which inputs could generate a proper Hopfield network operation and which couldn't.<br />
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In order to give more precise parameters to the models, measurements of the QS substances diffusibility – an important prediction of the system dynamics. This diffusibility will be estimated using anthocyanins with similar characteristics (polarity and molecular weight) to the real QS substances. Using the spectrometer, will be measured the diffusibility across the wells. In the same experiment, the diffusibility across the membrane will be measured as well. These measurements have great importance to verify the efficiency and viability of the system and of the device.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-23T20:02:02Z<p>Pepeks: /* Assembly diagrams */</p>
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<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
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===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
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3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
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[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
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===HSL production test.===<br />
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Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
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===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
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===Test for Internal feedback and basal production rate===<br />
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In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
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If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
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It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
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===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
<br />
For the Prm promoter system functionality, the follow constructions will be made.<br />
<br />
<br />
[[File:006.JPG|center|600px|caption|]]<br />
"Figure 9"<br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
<br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
<br />
===Assembly diagrams===<br />
<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
<br />
To illustrate all constructions used in all experiments, the Venn diagram ([https://static.igem.org/mediawiki/2012/2/2a/Assembly_Map.png HERE]) was created, gathering all partial constructions in the main construction. [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE] is the assembly fluxogram.<br />
<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/File:Assemblys_in_english_full.jpgFile:Assemblys in english full.jpg2012-09-23T20:01:27Z<p>Pepeks: </p>
<hr />
<div></div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-23T19:58:20Z<p>Pepeks: /* Assembly diagrams */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
===HSL production test.===<br />
<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
<br />
For the Prm promoter system functionality, the follow constructions will be made.<br />
<br />
<br />
[[File:006.JPG|center|600px|caption|]]<br />
"Figure 9"<br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
<br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
<br />
===Assembly diagrams===<br />
<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
<br />
To illustrate all constructions used in all experiments, the Venn diagram ([https://static.igem.org/mediawiki/2012/2/2a/Assembly_Map.png HERE]) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-23T19:57:56Z<p>Pepeks: /* Assembly diagrams */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
===HSL production test.===<br />
<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
<br />
For the Prm promoter system functionality, the follow constructions will be made.<br />
<br />
<br />
[[File:006.JPG|center|600px|caption|]]<br />
"Figure 9"<br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
<br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
<br />
===Assembly diagrams===<br />
<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
<br />
To illustrate all constructions used in all experiments, the Venn diagram ([https://static.igem.org/mediawiki/2012/2/2a/Assembly_Map.png/ HERE]) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/File:Assembly_Map.pngFile:Assembly Map.png2012-09-23T19:53:56Z<p>Pepeks: </p>
<hr />
<div></div>Pepekshttp://2012.igem.org/File:Diragram.pngFile:Diragram.png2012-09-22T21:35:15Z<p>Pepeks: </p>
<hr />
<div></div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:32:05Z<p>Pepeks: /* Test of Repression by cl434 */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
===HSL production test.===<br />
<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
<br />
For the Prm promoter system functionality, the follow constructions will be made.<br />
<br />
<br />
[[File:006.JPG|center|600px|caption|]]<br />
"Figure 9"<br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
<br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
<br />
===Assembly diagrams===<br />
<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:31:25Z<p>Pepeks: /* Experiments of Multi Regulated Promoter */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
===HSL production test.===<br />
<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
<br />
For the Prm promoter system functionality, the follow constructions will be made.<br />
<br />
<br />
[[File:006.JPG|center|600px|caption|]]<br />
"Figure 9"<br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:31:07Z<p>Pepeks: /* Experiments of Multi Regulated Promoter */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
===HSL production test.===<br />
<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
<br />
For the Prm promoter system functionality, the follow constructions will be made.<br />
<br />
<br />
[[File:006.JPG|center|400px|caption|]]<br />
"Figure 9"<br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:27:27Z<p>Pepeks: /* Tests of System "Cin" of quorum sensing */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
===HSL production test.===<br />
<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
FIGURA<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
For the Prm promoter system functionality, the follow constructions will be made. <br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:23:40Z<p>Pepeks: /* Test for Internal feedback and basal production rate */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
HSL production test.<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.JPG|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
FIGURA<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
For the Prm promoter system functionality, the follow constructions will be made. <br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:23:14Z<p>Pepeks: /* Test for Internal feedback and basal production rate */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
HSL production test.<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
<br />
===Tests of Rhl system of Quorum Sensing.===<br />
<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
[[File:0014.jpg|center|600px|caption|]]<br />
"Figure 8"<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
FIGURA<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
For the Prm promoter system functionality, the follow constructions will be made. <br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:19:23Z<p>Pepeks: /* Tests of System "Cin" of quorum sensing */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|600px|caption|]]<br />
"Figure 7"<br />
<br />
HSL production test.<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
Tests of Rhl system of Quorum Sensing.<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
FIGURA<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
For the Prm promoter system functionality, the follow constructions will be made. <br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/ExperimentsTeam:USP-UNESP-Brazil/Associative Memory/Experiments2012-09-22T21:19:10Z<p>Pepeks: /* Tests of System "Cin" of quorum sensing */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
In order to construct the genic design (shown on the introductory part), we Will use bio bricks listed on appendix 1.<br />
At first, before doing assemblies, we intend to confirm all parts that will be used. We will verify the lengths of the inserts in the plasmids using agarose gel and digestion by enzymes that cut in the prefixes and suffixes of BioBricks(EcoRI and PstI).<br />
After this, we intend to make 3 constructions in order to execute 3 experiments (tests) of the genetic device that will be used in future constructions.<br />
<br />
===Tests of System "Cin" of quorum sensing===<br />
This experiment consists in checking the behavior of Biobricks which make part of the Cin system of Quorum Sensing. We will first check the production of QS substance observing the enzymatic activity of cinl (enzyme which produce the QS substance), secondly check the levels of repression and activation of cinR and, lastly, the intensity of positive feedback in the system, comparing the levels of activation and inhibition with the previously checked.<br />
<br />
3 constructions will be made in order to create 3 different lineages of bacteria(figure 7). These lineages will be used in the following tests:<br />
<br />
[[File:0010.JPG|center|400px|caption|]]<br />
"Figure 7"<br />
<br />
HSL production test.<br />
Creating the E.coli lineage 1, we intend to observe the fluorescence in bacterial growth. This experiment doesn’t prove the activity of cinl, but shows signals of its operation since the GFP will be produced in the same ORF of the cinl gene. A graph of fluorescence X IPTG concentration used as input will be made.<br />
<br />
===Test of Response to QS Substance===<br />
For this test we will make the lineage 2 of E.coli. We intend to centrifuge the medium from the bacteria of lineage 1, pouring it on lineage 2 and verify if the members of lineage 2 will display fluorescence. Once we perceive fluoresce, we will have a strong signal that the QS system is working properly.<br />
In the case of any problem involving methodological variables, the GFP from lineage 2 can be replaced by RFP. In the case, we would mix lineage 1 and 2 members and observe the developing of both simultaneously. If a yellow fluorescence glows (Green + Red) – Like on microarrays – we will make sure that the lineage 2 is producing RFP under influence of the lineage 1.<br />
<br />
===Test for Internal feedback and basal production rate===<br />
<br />
In the case of satisfactory results in the previous tests, this one will probably work as well. The main objective is to estimate if the rate of basal expression is not excessive, in other words, if the positive feedback system is low enough. For this, we can compare the rate of basal fluorescence using the graph made on “HSL production test” as parameter.<br />
If the fluorescence doesn’t appear, there are 2 main possibilities: The feedback system isn’t working or it’s too low. In order to verify this issue, the system could be stimulated by insertion of QS substance, measuring the period of glowing and comparing it to the glowing time of bacteria from lineage 2 (which doesn’t have feedback system).<br />
It is possible to verify how much HSL will be necessary to activate the QS of the system. In order to do this, will be necessary to take samples from the growth medium from lineage 1 with several different “IPTG inductions”. It is likely to exists a threshold concentration of the QS substance in which the QS starts and the fluorescence of the system keeps glowing without external influence.<br />
Tests of Rhl system of Quorum Sensing.<br />
Just as previously, we intend to check the operation of the QS system, but this time with the rhl system. We will check: production of rhIL enzyme, levels of activation and repression of rhlR substance and ,lastly, intensity positive feedback system, comparing levels of activation and inhibition with tested previously.<br />
We will do the constructions presented in Figure 8 in addition to tests similar to the system in each Cin.<br />
<br />
<br />
===BHL production test.===<br />
The objective of this test is to confirm if the enzyme responsible by the synthesis of QS substance is being produced. The main evidence of it is the production of GFP under IPTG input. Using this test, a graph relating the quantity of IPTG and fluorescence will be made in order to be a reference for other tests.<br />
Reaction to the QS substance test.<br />
As seen in the test for reaction of Cin system under HSL, a centrifugation of the medium of lineage 2 will be poured on bacteria of lineage 1. If any issue emerge, the same methodology used on the tests of Cin system will be used.<br />
Test of Internal Feedback and Basal production rate Test.<br />
The levels of fluorescence of lineage 3 will be compared with the ones of other lineages and the concentration of BHL required to activate the QS system will be checked. The goals and procedures of this test are equivalent to those in QS Cin system.<br />
<br />
===Experiments of Multi Regulated Promoter=== <br />
FIGURA<br />
The main goal of these experiments is checking if the Biobricks that constitute the system of genic regulation based on multi regulated promoter (Prm) are working properly. This promoter is activated by “cl” factor and inhibited by “cl434” factor. This system will be used to transform all input signals in activation of the system(indicated by cl production) or in inhibition of it(indicated by cl434). The main goal is to make sure if the rates of activating or inhibition of the promoter conform with cl and cl434.<br />
For the Prm promoter system functionality, the follow constructions will be made. <br />
<br />
===Test ofActivationby cl.===<br />
The main goal of this test is to create the “lineage 1” above and induce the production of cl and activation of GFP transcription by use of IPTG. It is expected the appearance of fluorescence, starting in a basal level, in the bacterial growth,<br />
<br />
===Test of Repression by cl434=== <br />
The lineage 2 of E.coli Will be created and induced, by means of IPTG, to produce cl434 and, consequently, inhibit the production of tetR repressor, which represses PtetR promoter, the controller of GFP production. By this way, the transcription of GFP by PtetR will be stimulated. By this way, like in the previous experiment, it is expected to observe the emerging of fluorescence. <br />
There is a possibility that the basal levels of Prm transcription be too high or low to maintain a proper feedback in the QS system. In an ideal situation, the Prm would have transcription rates similar to the QS system promoters (PcinRandPrlhR), emulating the natural feedback of the QS systems- not enough to activate the system. A way to do it would be create mutant Prm promoters with a set of different transcription rates. This would be a good alternative, but probably will not make part in this Project due the deadlines and time required to do it. <br />
Assembly diagrams<br />
Previously, it was intent to use a light receptor (Red Light Suit) as input system. The strategy was to put the light responsive promoter in sequence to the Prm promoter, making the genic regulation of the following ORF. In order to simplify the Project, this part was removed.<br />
To illustrate all constructions used in all experiments, the Venn diagram (HERE(link)) was created, gathering all partial constructions in the main construction. In this LINK, there is the assembly fluxogram.<br />
We intend to assembly all biobricks using the 3A assembly method, except the smaller parts, like RBS and terminal sequences in which will be used the Standard Assembly Method. Depending on the method of assembly, a different scheme of digestion will be used. The different types of digestion are on the assembly fluxogram. Alternatively to the initial plan using the light switch, IPTG will be used as input to induce the initial production of GFP.</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/BackgroundTeam:USP-UNESP-Brazil/Associative Memory/Background2012-09-22T21:13:20Z<p>Pepeks: /* Genetic Construction */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
===Hopfield Associative Memory Networks===<br />
<br />
The main model of the project is the associative memory network made by J.J. Hopfield in the 80’s. On this model, the system tends to converge to a pre-determined equilibrium, restoring the same pattern when exposed to variations of this same pattern.<br />
<br />
The architecture, or geometry of the system, is composed in a way that all neurons are connected .In math terms, a Hopfield Network can be represented as an “Energy” (E) function:<br />
<br />
[[File:equation1.jpg|center|300px|caption|]]<br />
"Equation 1"<br />
<br />
<br />
Where “w” values are chosen such that the stored settings are the minima of the function “E”. The variable “x” is the state of the neuron “i”. <br />
<br />
The state of a given neuron “I”(active or silent) can be mathematically represented as follows: Given that “xi“ is the state of neuron, 1 if is activated or 0 if silent, and a neuron turns active if the sum of all received stimulus (exciting or inhibiting) is more than 0. Mathematically we can represent the state of the neuron xi as:<br />
<br />
[[File:equation2.jpg|center|400px|caption|]]<br />
"Equation 2"<br />
<br />
<br />
In this equation, “wij” is the wheight<br />
<br />
Where "wij" is the weight assigned to the connection from neuron i to neuron j. The summation over j is the sum of all connections made by the neuron i. This dynamics (equation 2) is sufficient for the network to converge the most similar memorized pattern. <br />
<br />
The so called “learning” of a neural network consists on the choice of “w” weights. There are several ways to choose them, what, actually, defines different learning methods<br />
<br />
Set "i" and "j" such as the wheight "wij" is defined as:<br />
<br />
[[File:equation3.png|center|400px|caption|]]<br />
"Equation 3"<br />
<br />
<br />
The Figure 1w shows the selection process of weights of connections between adjacent cells. To add more patterns, we have to sum the network of weights of the new pattern to the old network. (as shown in the Figure 2)<br />
<br />
[[File:009.JPG|center|570px|caption|]]<br />
"Figure 1"<br />
<br />
<br />
The Hopfield model for the construction of an associative memory network using bacteria is a good choice because of its simplicity and strength. The same methodology can be used to the construction of networks with other architectures, such as the “perceptrons”. One step forward is the way how to deal with continuous biological variables, because the standard model uses discrete ones.<br />
<br />
[[File:0018.JPG|center|620px|caption|]]<br />
"Figure 2"<br />
<br />
===Biological Mechanism===<br />
In a neuronal communication system, the cells of the network occupy a specific place of space and the information is addressed through a direct physical contact - the neuron axonal projections. To replicate this in a cell system that moves constantly it would be also necessary some way to specifically address the information flow between the system’s components, otherwise it will not be possible to attach a meaning to a communication without specificity.Furthermore, is possible to verify the neuronal activity[1.4] because of the fixed spatial position of the cells, which conserves the signal observation of a neuron in a specific local.<br />
<br />
It’s much more complicated to observe the dynamics of this cell communicating system using bacteria. Unlike static single cells communicating with each other (see figure 3), we aim to observe the communication between many genetically distinct bacterial populations. And these cells don’t stop moving! In other words: there’s no specific point in space which can be always observed the same phenomenon.<br />
<br />
<br />
[[File:Figura0020.jpg|center|500px|caption|]]<br />
"Figure 3"<br />
<br />
To solve this information addressing problem, it would be necessary that each bacterial population (like neurons) communicate with themselves in a unique way so a receiving signal can be distinguished between populations. We chose the quorum sensing (QS) communication mechanism for this task, using different quorum systems for each “point” (population) in the network. With different QS molecules, it’s possible to build a communication system with unique signals like neurons in a network, where the information addressing specificity is present by the axonal ligation. <br />
<br />
Therefore, the influence of inhibition or activation that one point of the network can have under another in the bacterial system would be given by the particular meaning of each signal for each cell population – this point is where the memory programming of the system occurs which is the determination, for each cell population, the meaning of each communication signal of every point of the network.<br />
<br />
Then, to solve the problems of the visualization of the signals’activation, we planned the construction of a device to make 9 E.coli populations to communicate with each other but still keep their position in space. As an improvement to the example in figure 1, the device enables the communication not only of neighbors but also of all the populations, inhibiting or activating them according to the memory implanted to the network. This way, comparing to the communication between neighbors and of the whole system itself, generates a bigger resolution of the output in response to a similar input, once each position has better information about the pattern of activity of all the other positions. Figure 4 shows a construction of the system that can enable an efficient communication between the populations of different positions of the network, even if it is physically separating them.<br />
<br />
[[File:Physicalsystemforbacterialnetwork.png|center|500px|caption|]]<br />
"Figure 4"<br />
<br />
The device can be easily constructed using a plate of 96 wells with membranes attached to the bottom. The membranes will allow the diffusion of the quorum sensing substances and prevent the flux of bacterial populations between the wells; avoiding, them the mixture of the activation signals (outputs).<br />
<br />
====Genetic Construction====<br />
<br />
Despite the solution we found to the specificity of the communication, another problem appears when we try to genetically build the bacterial populations: there are not enough quorum sensing systems to create 9 bacterial populations with different ones. In the Registry of Parts there are 4 quorum sensing systems well characterized, and there is a strong activation crosstalk between two of them (Las and Rhl), this fact prevent us from using them, therefore, we end up with 3 systems of quorum sensing that can be used.<br />
<br />
Initially,we intended,– only as a concept proof - to create a network associative memory for discrimination activation patterns between the "L" and "T", similar to the one presented in Figure 2. As these memories require that all points of the network have a communication singular, we searched for a case in particular that demanded that memories would require only a small number of quorum sensing substances.<br />
<br />
Asymmetricalmemory, extremely simple, would be the answer: since only two quorum sensing systems are necessary to accomplish the same recognition memory task as an incomplete memory, similarly to the example showed in Figure 2, the "X" and "O" memories. In these patterns there is no intersection between "letter X" and letter"O”, which greatly simplifies the configuration of communication weights between positions (see Figure 5). Because each position of the “X” inhibits all positions of “O” and activates all positions of its own pattern (and vise-versa), there is no need to distinguish between the kind of signal (quorum sense substance) that each position of the “X” emits. The same works for all positions of the “O” pattern. Still, the positions of each pattern can be programmed with the same genetic code, which allow us to use only two systems of quorum sensing to create a network of associative memory that is capable of differentiate between two memories, given a particular input that would be interpreted as an incomplete memory.<br />
<br />
[[File:0019.JPG|center|500px|caption|]]<br />
"Figure 5"<br />
<br />
We decided to use two of the four available quorum sensing systems that have substances different enough for the bacterial communication so there would be little cross-talk between them. The quorum sensing systems chosen were Cin and Rhl[1,16].<br />
<br />
To convert the signals in activation or inhibition, we created a system of transduction of the quorum sensing signal to transcription of an activator or an inhibitor of the transcription of GFP, this will be our system activation reporter. Simultaneous inhibitions and activations of a bacterial population will be converted to a molecular competition of activators and inhibitors by the promoter that controls the production of GFP. It is this molecular competition that promotes the decision between the memories of the communication systems, associating a given input with a more similar memory. As an example, if an input activates more positions of the “X” pattern than the “O”, the competition in the pattern “X” positions will be more favorable to its activation due to the greater number of activators produced by the activated positions, while in the positions of the “O” pattern the opposite occurs, because of its small number of positions activated initially by the given input.<br />
<br />
The promoter multi-regulated by an activator and an inhibitor is called Prm. Its inhibitor is the transcriptional factor cl434 and its activator is the cl factor. The genetic design of the positions of the patterns “X” and “O” can be seen in Figure 6.<br />
<br />
The construction using the signal transduction containing cl434 and cl allows creating different systems of associative memory, limited only by the quantity of quorum sensingsystems available. Figure 6 shows how this generic system would work and elucidates how this system could be applied to different functions involving a genetic control.<br />
<br />
[[File:0022.png|center|600px|caption|]]<br />
"Figure 6"</div>Pepekshttp://2012.igem.org/Team:USP-UNESP-Brazil/Associative_Memory/BackgroundTeam:USP-UNESP-Brazil/Associative Memory/Background2012-09-22T21:13:02Z<p>Pepeks: /* Genetic Construction */</p>
<hr />
<div>{{:Team:USP-UNESP-Brazil/Templates/Header}}<br />
===Hopfield Associative Memory Networks===<br />
<br />
The main model of the project is the associative memory network made by J.J. Hopfield in the 80’s. On this model, the system tends to converge to a pre-determined equilibrium, restoring the same pattern when exposed to variations of this same pattern.<br />
<br />
The architecture, or geometry of the system, is composed in a way that all neurons are connected .In math terms, a Hopfield Network can be represented as an “Energy” (E) function:<br />
<br />
[[File:equation1.jpg|center|300px|caption|]]<br />
"Equation 1"<br />
<br />
<br />
Where “w” values are chosen such that the stored settings are the minima of the function “E”. The variable “x” is the state of the neuron “i”. <br />
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The state of a given neuron “I”(active or silent) can be mathematically represented as follows: Given that “xi“ is the state of neuron, 1 if is activated or 0 if silent, and a neuron turns active if the sum of all received stimulus (exciting or inhibiting) is more than 0. Mathematically we can represent the state of the neuron xi as:<br />
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[[File:equation2.jpg|center|400px|caption|]]<br />
"Equation 2"<br />
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In this equation, “wij” is the wheight<br />
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Where "wij" is the weight assigned to the connection from neuron i to neuron j. The summation over j is the sum of all connections made by the neuron i. This dynamics (equation 2) is sufficient for the network to converge the most similar memorized pattern. <br />
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The so called “learning” of a neural network consists on the choice of “w” weights. There are several ways to choose them, what, actually, defines different learning methods<br />
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Set "i" and "j" such as the wheight "wij" is defined as:<br />
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[[File:equation3.png|center|400px|caption|]]<br />
"Equation 3"<br />
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The Figure 1w shows the selection process of weights of connections between adjacent cells. To add more patterns, we have to sum the network of weights of the new pattern to the old network. (as shown in the Figure 2)<br />
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[[File:009.JPG|center|570px|caption|]]<br />
"Figure 1"<br />
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The Hopfield model for the construction of an associative memory network using bacteria is a good choice because of its simplicity and strength. The same methodology can be used to the construction of networks with other architectures, such as the “perceptrons”. One step forward is the way how to deal with continuous biological variables, because the standard model uses discrete ones.<br />
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[[File:0018.JPG|center|620px|caption|]]<br />
"Figure 2"<br />
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===Biological Mechanism===<br />
In a neuronal communication system, the cells of the network occupy a specific place of space and the information is addressed through a direct physical contact - the neuron axonal projections. To replicate this in a cell system that moves constantly it would be also necessary some way to specifically address the information flow between the system’s components, otherwise it will not be possible to attach a meaning to a communication without specificity.Furthermore, is possible to verify the neuronal activity[1.4] because of the fixed spatial position of the cells, which conserves the signal observation of a neuron in a specific local.<br />
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It’s much more complicated to observe the dynamics of this cell communicating system using bacteria. Unlike static single cells communicating with each other (see figure 3), we aim to observe the communication between many genetically distinct bacterial populations. And these cells don’t stop moving! In other words: there’s no specific point in space which can be always observed the same phenomenon.<br />
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[[File:Figura0020.jpg|center|500px|caption|]]<br />
"Figure 3"<br />
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To solve this information addressing problem, it would be necessary that each bacterial population (like neurons) communicate with themselves in a unique way so a receiving signal can be distinguished between populations. We chose the quorum sensing (QS) communication mechanism for this task, using different quorum systems for each “point” (population) in the network. With different QS molecules, it’s possible to build a communication system with unique signals like neurons in a network, where the information addressing specificity is present by the axonal ligation. <br />
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Therefore, the influence of inhibition or activation that one point of the network can have under another in the bacterial system would be given by the particular meaning of each signal for each cell population – this point is where the memory programming of the system occurs which is the determination, for each cell population, the meaning of each communication signal of every point of the network.<br />
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Then, to solve the problems of the visualization of the signals’activation, we planned the construction of a device to make 9 E.coli populations to communicate with each other but still keep their position in space. As an improvement to the example in figure 1, the device enables the communication not only of neighbors but also of all the populations, inhibiting or activating them according to the memory implanted to the network. This way, comparing to the communication between neighbors and of the whole system itself, generates a bigger resolution of the output in response to a similar input, once each position has better information about the pattern of activity of all the other positions. Figure 4 shows a construction of the system that can enable an efficient communication between the populations of different positions of the network, even if it is physically separating them.<br />
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[[File:Physicalsystemforbacterialnetwork.png|center|500px|caption|]]<br />
"Figure 4"<br />
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The device can be easily constructed using a plate of 96 wells with membranes attached to the bottom. The membranes will allow the diffusion of the quorum sensing substances and prevent the flux of bacterial populations between the wells; avoiding, them the mixture of the activation signals (outputs).<br />
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====Genetic Construction====<br />
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Despite the solution we found to the specificity of the communication, another problem appears when we try to genetically build the bacterial populations: there are not enough quorum sensing systems to create 9 bacterial populations with different ones. In the Registry of Parts there are 4 quorum sensing systems well characterized, and there is a strong activation crosstalk between two of them (Las and Rhl), this fact prevent us from using them, therefore, we end up with 3 systems of quorum sensing that can be used.<br />
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Initially,we intended,– only as a concept proof - to create a network associative memory for discrimination activation patterns between the "L" and "T", similar to the one presented in Figure 2. As these memories require that all points of the network have a communication singular, we searched for a case in particular that demanded that memories would require only a small number of quorum sensing substances.<br />
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Asymmetricalmemory, extremely simple, would be the answer: since only two quorum sensing systems are necessary to accomplish the same recognition memory task as an incomplete memory, similarly to the example showed in Figure 2, the "X" and "O" memories. In these patterns there is no intersection between "letter X" and letter"O”, which greatly simplifies the configuration of communication weights between positions (see Figure 5). Because each position of the “X” inhibits all positions of “O” and activates all positions of its own pattern (and vise-versa), there is no need to distinguish between the kind of signal (quorum sense substance) that each position of the “X” emits. The same works for all positions of the “O” pattern. Still, the positions of each pattern can be programmed with the same genetic code, which allow us to use only two systems of quorum sensing to create a network of associative memory that is capable of differentiate between two memories, given a particular input that would be interpreted as an incomplete memory.<br />
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[[File:0019.JPG|center|500px|caption|]]<br />
"Figure 5"<br />
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We decided to use two of the four available quorum sensing systems that have substances different enough for the bacterial communication so there would be little cross-talk between them. The quorum sensing systems chosen were Cin and Rhl[1,16].<br />
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To convert the signals in activation or inhibition, we created a system of transduction of the quorum sensing signal to transcription of an activator or an inhibitor of the transcription of GFP, this will be our system activation reporter. Simultaneous inhibitions and activations of a bacterial population will be converted to a molecular competition of activators and inhibitors by the promoter that controls the production of GFP. It is this molecular competition that promotes the decision between the memories of the communication systems, associating a given input with a more similar memory. As an example, if an input activates more positions of the “X” pattern than the “O”, the competition in the pattern “X” positions will be more favorable to its activation due to the greater number of activators produced by the activated positions, while in the positions of the “O” pattern the opposite occurs, because of its small number of positions activated initially by the given input.<br />
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The promoter multi-regulated by an activator and an inhibitor is called Prm. Its inhibitor is the transcriptional factor cl434 and its activator is the cl factor. The genetic design of the positions of the patterns “X” and “O” can be seen in Figure 6.<br />
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The construction using the signal transduction containing cl434 and cl allows creating different systems of associative memory, limited only by the quantity of quorum sensingsystems available. Figure 6 shows how this generic system would work and elucidates how this system could be applied to different functions involving a genetic control.<br />
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[[File:0022.png|center|500px|caption|]]<br />
"Figure 6"</div>Pepeks