http://2012.igem.org/wiki/index.php?title=Special:Contributions/Antresh_kumar&feed=atom&limit=50&target=Antresh_kumar&year=&month=2012.igem.org - User contributions [en]2024-03-28T09:53:04ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:JUIT-India/SafetyTeam:JUIT-India/Safety2012-09-29T04:21:05Z<p>Antresh kumar: </p>
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<h1> Safety Questions </h1><br />
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<h2>Would any of your project ideas raise safety issues in terms of:</h2><br />
<ol><br />
<li><br />
Researcher safety,</li><br />
<ul><br />
<p>In order to work in the lab cartain precautions always need to be undertaken to ensure the safety of the researchers. These include the use of gloves and labcoats to protect from chemicals which are irritant and the use of masks when using powdered media<br />
<br><br />
<p> With regard to our project those were in particular the use for toxic chemicals, including:<br />
<li> <b>Ampicillin</b> is a broad-spectrum bacteriostatic antibiotic which is effective against many Gram-positive and Gram-negative bacteria, including most anaerobic organisms. It can sometimes result in reactions that range in severity from a rash (in the case of patients that may unwittingly have mononucleosis) to potentially lethal allergic reactions such as anaphylaxis</li><br />
<li> <b>Ethidium Bromide</b> is a powerful carcinogen that was used during gel electrophoresis. Gloves were used to protect from ethidium bromide.</li><br />
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<li> Chemicals that have been demonstrated to be toxic or are classed as irritants were not handled by the team at critical concentrations, but rather diluted for us by more experienced members of staff, like our supervisors. This minimised the risk to the all people working in the lab and ensured correct safety practices were taken during the most precarious steps. These substances were stored in locked cupboards and handed to us diluted in aliquots of constant volumes.</li><br />
<li> <i>Pseudomonas</i> strain was used by us and since <i>Pseudomonas aeruginosa</i> is a common bacterium that can cause disease in animals, including humans, therefore proper precautions were undertaken to prevent infection.</li><br />
<li> UV light is used to view EtBr. stained DNA.This can lead to UV exposure especially when DNA bands are excised from the gel. For this reason a blue light box and safety goggles with light filters were used for these steps to prevent damage to the retina as well as our skin.</li><br />
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<p>Generally good laboratory practices were adhered to, including the wearing lab coats and protective gloves when in the laboratory. Furthermore all waste, including live bacteria and toxics, were disposed of by trained members of staff according to sound safety protocols.<br />
</ul></ol><br />
<ul><li> public safety</li><br />
<br />
<ul><li> In order to minimize the risk our project poses to public safety most of our genes were taken from non-pathogenic microbes. Several safety precautions were taken :<br />
</li><br />
<li> We quickly decided to design our project in a way that would avoid harm to humans and the environment and therefore we chose a strain of microbe that was found in the natural conditions of the rice fields.<br />
<li> Genes: As laid out early by our human practices work show, we aimed to minimize the use of genes from pathogenic organisms. We therefore used only a single gene from Pseudomonas. All of our other genes were used from non-pathogenic microbes.</li><br />
<br><br><br />
Overall this system should not be able to cause any harm to the public under any foreseeable circumstances. Our project idea was modified several times during its development in order to fulfill all the safety guide lines we had agreed on in our human practices work shop as well as our panel discussion with experts from many fields, including Synthetic Biology and Ethics.</li></ul><br><br />
<ul><li>environmental safety?<br />
We chose our strains that are easily found in the wild environment of the rice fields so that the impact to the already existing flora and fauna is minimized. Environmental safety was inherent in our design so that no risk should arise of our project. We prepared our plan in order to make Synthetic Biology more environment friendly. <br />
</li></ul></ul><br />
<br><br>Environmental Safety<br />
<br>Our project poses no identifiable threat to environmental safety. The microbes used in our project are not able to survive outside the lab, and all cells were disposed of safely after disinfection with 10% bleach. Bio-hazardous and flammable chemicals were disposed of by following the proper regulations. No gloves were allowed to leave the laboratory, so chemical and biological hazards were restricted to the laboratory.<br />
<br />
<br><br>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,<br />
<br> Did you document these issues in the Registry?<br />
<br> How did you manage to handle the safety issue?<br />
<br> How could other teams learn from your experience?<br />
<br />
<br>No. All bio-bricks made are according to the safety guidelines provided by the Center for Disease Control and Prevention.<br />
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<br><br>Is there a local biosafety group, committee, or review board at your institution?<br />
<br> If yes, what does your local biosafety group think about your project?<br />
<br> If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
<br />
<br><br>Yes, our project safety is governed by the Committee on Bio Safety, Department of Biotech/Bioinformatics, JUIT. We have presented our project proposal to the same committee as mentioned above , and after a review of our materials and procedures, the biosafety office has approved our project and deemed our practices consistent with biosafety regulations.<br />
<br />
<br><br />
<ul><li>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,</li><br />
<ul><li><br />
No, are parts do not raise any safety issues.</li></ul></ul><br />
<br />
<br><br />
<br />
<ul><li> Is there a local biosafety group, committee, or review board at your institution?</li><br />
<ul><li>The biotechnology department in JUIT adequately addresses the biosafety risks associated with modern biotechnology as well as awareness generation on the biosafety aspects and regulations, through an Institutional Biosafety Committee (IBSC) comprising of expert faculty members from institution and DBT appointed nominees. Extension activities, a technical society “RIBOSE” (RESONANCE IN BIOTECHNOLOGISTS AND ENGINEERS) and an e-newsletter spread awareness about the science of Biotechnology. Ecogroup ‘Green-Volunteers’ work towards various environmental issues. One of our advisors, Dr. N.Mehendru, is the biosafety officer for the Department of Pathology. We were able to discuss the safety of our work with him at every stage of the project. We also ensured we complied with the India's biosafety regulations, taking advice from Dr. N.Mahendru.</li><br />
</ul></ul><br><br />
<ul><li> Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</li></ul><br />
<br><br>Antibiotic resistance is a public health problem of increasing magnitude, and finding effective solutions to address this problem is a critical focus of CDC activities.<br />
<br>1. Decreasing the susceptibility of recombinant cells to antibiotics through Antibiotic resistance genes proves to be a high risk to the environment.<br />
<br>2. Our new idea is to use Sac B gene insertion as a tool for selecting transformed mutants. We have designed a new plasmid, that does not contain antibiotic resistant gene. Instead it has Sac B whose expression in the presence of sucrose is lethal to Methylococcus capsulatus. Our results imply that the sacB gene can be used as a positive selection system in Methylococcus capsulatus.<br />
<br />
<br>Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.<br />
The link national biosafety regulations in India [http://dbtbiosafety.nic.in/].<br />
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<!--- The Mission,</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ModelingTeam:JUIT-India/Modeling2012-09-26T19:07:34Z<p>Antresh kumar: </p>
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<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:JUIT-India/Wiki'><span>Wiki Page</span></a></li><br />
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<h1>Experimental Model We are trying to create </h1><br><br />
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ModelingTeam:JUIT-India/Modeling2012-09-26T19:00:02Z<p>Antresh kumar: </p>
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<br />
<h1>Experimental Model We are trying to create </h1><br><br />
<br />
<hr><br />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/58/Final.JPG" width="965" height="600"></div><br />
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<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ExperimentTeam:JUIT-India/Experiment2012-09-26T18:59:22Z<p>Antresh kumar: </p>
<hr />
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<br><br />
<hr><br />
<br />
<h1>Engineered Vector </h1><br><br />
<br />
<hr><br />
<br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/58/Final.JPG" width="965" height="600"></div><br />
<br />
<br />
<br />
<br />
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<br />
</html><br />
=='''Complete Description'''==<br />
<br />
'''Project Description'''<br />
<br />
We are going to use the nif (From M.capsulatus) and nos (from Pseudomonas) genes. The mmo genes are present in to forms in M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO). MMO enzyme catalyzes the conversion of methane to methanol. Once methanol is converted into formaldehyde it enters various biochemical cycles in the cell. Our system involves the utilization of the methanol as an inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for nitrogen fixation. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase. Nif genes then converts the nitrogen into nitrate, which can be easily be taken up by the plant. The main reason for addition of fertilizer is to increase the inorganic nutrients in the soil. By utilizing NosZ and Nif genes we are converting the nitrous oxide into nitrate, thus reducing the need of fertilizers in the soil by increasing the nitrogen available to the plants. This is a small step towards a more environment friendly future.<br />
<br />
Details:<br />
<br />
'''MxaF''':<br />
<br />
Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C1) substrates more reduced than carbon dioxide as their sole source of carbon and energy.Methanotrophs possesses native methanol-inducible promoters, notably promoters which are located upstream of genes that encode methanol dehydrogenase and other proteins required for its activity and enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone. Of these, the promoter PmxaF has been thoroughly scrutinized both biochemically and in expression studies. In its native form in the chromosome, this strong promoter is methanol inducible. However, when this promoter is cloned in expression vectors, it acts essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide. Our system involves the utilization of the methanol as an inducer for MxaF promoter. This would result in the diversion of the flux thus leading to a faster degradation of methane for the cell to survive.<br />
<br />
'''NosZ:'''<br />
<br />
The complete denitrification of nitrate by bacteria to dinitrogen (N,) is generally an anaerobic respiratory process. The last step involves the dissimilatory reduction of nitrous oxide (N,O), the free energy change of which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck, 1990). nosZ is the structural gene for the periplasmic N,O reductase which is required to the conversion of nitrous oxide into free nitrogen. . The nitrous oxide that is emitted in the paddy fields is caused due to the excessive use of fertilizers. The microbes naturally present in the paddy field lead to the emission of nitrous oxide by utilizing these chemical fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for denitrification.<br />
<br />
'''NifA:'''<br />
<br />
The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. Expression of the nif genes is regulated at the transcriptional level by the products of nifA in response to molecular oxygen or ammonia. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase holoenzyme containing the alternative sigma factor, sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA, also known as upstream activation sequence (UAS), that is located more than 100 bp upstream of nif promoters. The NifA protein has three arbitrarily designated domains (20): an amino-terminal domain which is implicated in regulatory function, a catalytic domain that interacts with the sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif which recognizes the UAS on the nif promoters.<br />
<br />
<br />
'''SacB:'''<br />
<br />
Expression from sacB confers sensitivity to sucrose in a wide variety of gram positive and negative bacteria and thus has been used extensively for the last twenty years as a negative selectable marker. The Bacillus subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which is secreted by B. subtilis cells into the culture medium. Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules. In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose<br />
<br />
<br />
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<br><br />
<h1>Our Title Sponsor :</h1><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
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<br />
{|align="justify"<br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/SafetyTeam:JUIT-India/Safety2012-09-26T18:56:33Z<p>Antresh kumar: </p>
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<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
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<br />
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<br><br />
<hr><br />
<br />
<h1>Safety Protocols</h1><br />
<br><br>Experiments were conducted in the Undergraduate lab of the Dept. Of Biotechnology at Jaypee University Of Information Technology. The lab is confirmed to the standards for a bio-safety 1 lab according to the Center for Disease Control and Prevention. Bio-safety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. <br />
<br><br>Standard Microbiological Practices<br />
<br />
<br>a. Statement of Purpose (SOP) have been developed and are kept for easy access which enlists all the safety measures each experiment should be abided for.<br />
<br>b. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.<br />
<br>c. People wash their hands after handling viable materials, after removing gloves, and before leaving the laboratory.<br />
<br>d. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. People who wear contact lenses in laboratories must wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.<br />
<br>e. Mouth pipetting is prohibited .Instead mechanical pipetting devices are to be used.<br />
<br>f. Policies for the safe handling of sharp instruments are clearly instructed.<br />
<br>g. All procedures are performed carefully to minimize the creation of splashes or aerosols.<br />
<br>h. Work surfaces are decontaminated atleast once a day and after any spill of viable material.<br />
<br>i. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method for example Autoclaving. Materials to be decontaminated outside the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. These materials are packaged in accordance with applicable local, state, and federal regulations before removing from the facility.<br />
<br />
<br />
<br><br>Safety Equipment (Primary Barriers)<br />
<br />
<br>a. Special containment devices or equipment such as a biological safety cabinet is generally not required for manipulations of agents assigned to Bio-safety Level 1.<br />
<br>b. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.<br />
<br>c. Gloves are must especially if there is a cut on the hand or if a rash is present. Alternatives to powdered latex gloves should be available.<br />
<br>d. Protective eyewear should be worn while conducting experiments in which splashes of microorganisms or other hazardous materials are anticipated.<br />
<br />
<br><br>Laboratory Facilities (Secondary Barriers)<br />
<br />
<br>a. Laboratory has doors for access control.<br />
<br>b. A sink is provided in the Laboratory for washing hands.<br />
<br>c. The Laboratory is designed such that it can be easily cleaned.<br />
<br>d. Bench tops are impervious to water and are resistant to moderate heat, organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.<br />
<br>e. Laboratory furniture is capable of supporting anticipated loadings and uses. Spaces between benches, cabinets, and equipments are accessible for cleaning.<br />
<br>f. It is made sure that the Laboratory is cut-off from the external environment.<br />
<br />
<br />
Would any of your project ideas raise safety issues in terms of:<br />
<br> researcher safety,<br />
<br> public safety, or<br />
<br> environmental safety?<br />
<br />
<br><br>Researcher Safety<br />
<br>Working in a laboratory environment is not without its risks, so we took a variety of steps to ensure the safety of our team during our lab work. We worked in a laboratory environment certified for Biosafety Level 1 work.<br />
Advanced synthetic biology research does at times require interaction with potentially dangerous substances. Listed below are those substances which posed the most significant hazards to researcher safety. Again, however, it should be noted that our use of these substances was consistent with Harvard's stringent safety standards. These substances included:<br />
<br> Ethidium Bromide (EtBr) was used to stain DNA for gel electrophoresis. Although toxic and a suspected mutagen, the harmful effects of ethidium bromide can be avoided by avoiding direct skin contact and inhalation, which can in turn be avoided by proper observance of safety precautions. In order to minimize primary contact, ethidium bromide is directly contacted only with micropipette tips. Skin contact when handling ethidium bromide-stained gels was avoided by the use of nitrile gloves that were promptly discarded for a fresh pair when switching from the task of pouring gels to other lab work. Secondary contact was avoided by the disposal of ethidium bromide-contaminated gloves and micropipette tips, as well as the designation of a specific bench and set of micropipettes exclusively for use with ethidium bromide.<br />
<br />
<br><br>• Ultraviolet (UV) light was used in visualizing stained DNA in gel electrophoresis. To avoid exposure to UV radiation when reading gels, protective, UV-blocking shields were used at all times. When performing gel extractions, exposure was avoided by wearing protective clothing to cover any exposed skin and safety glasses with UV-protection lenses.<br />
<br><br>• The laboratory strains of E. coli, Methylococcus capsulatus used were non-pathogenic and therefore not a threat to researcher safety. We have conferred various basic antibiotic resistances (including kanamycin resistance, all common features of synthetic biology lab work) to our strains. However, these strains are unlikely to survive in the wild, or in humans specifically—where they would be outcompeted by naturally-occuring bacteria—and thus this resistance does not present a significant problem for researcher safety. In addition, all used materials that contacted the bacteria were decontaminated with 10% bleach, ideally for a 20 minute minimum contact time, before disposal in designated biohazard receptacles.<br />
<br><br>Public Safety<br />
<br><br>• All work with live E.coli , Methylococcus capsulatus cells is carried out in the laminar hood that eliminates the possibility of any GMO(Genetically Modified Organisms) from being released out.<br />
<br>• If toxic, flammable, or chemically reactive substances used in our experiments are released, they may post threat to the general public. These include physical hazards or health hazards. <br />
<br>• It is made sure that appropriate measures for waste disposal are taken. Waste is not washed down the sink or thrown into public dust-bins.<br />
<br />
<br><br>Environmental Safety<br />
<br>Our project poses no identifiable threat to environmental safety. The microbes used in our project are not able to survive outside the lab, and all cells were disposed of safely after disinfection with 10% bleach. Bio-hazardous and flammable chemicals were disposed of by following the proper regulations. No gloves were allowed to leave the laboratory, so chemical and biological hazards were restricted to the laboratory.<br />
<br />
<br><br>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,<br />
<br> Did you document these issues in the Registry?<br />
<br> How did you manage to handle the safety issue?<br />
<br> How could other teams learn from your experience?<br />
<br />
<br>No. All bio-bricks made are according to the safety guidelines provided by the Center for Disease Control and Prevention.<br />
<br />
<br><br>Is there a local biosafety group, committee, or review board at your institution?<br />
<br> If yes, what does your local biosafety group think about your project?<br />
<br> If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
<br />
<br><br>Yes, our project safety is governed by the Committee on Bio Safety, Department of Biotech/Bioinformatics, JUIT. We have presented our project proposal to the same committee as mentioned above , and after a review of our materials and procedures, the biosafety office has approved our project and deemed our practices consistent with biosafety regulations.<br />
<br />
<br />
<br />
<br><br>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
<br />
<br><br>Antibiotic resistance is a public health problem of increasing magnitude, and finding effective solutions to address this problem is a critical focus of CDC activities.<br />
<br>1. Decreasing the susceptibility of recombinant cells to antibiotics through Antibiotic resistance genes proves to be a high risk to the environment.<br />
<br>2. Our new idea is to use Sac B gene insertion as a tool for selecting transformed mutants. We have designed a new plasmid, that does not contain antibiotic resistant gene. Instead it has Sac B whose expression in the presence of sucrose is lethal to Methylococcus capsulatus. Our results imply that the sacB gene can be used as a positive selection system in Methylococcus capsulatus.<br />
<br />
<br>Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.<br />
The link national biosafety regulations in India [http://dbtbiosafety.nic.in/].<br />
<br />
<br />
<br />
</html><br />
<br />
<br />
<br />
<br />
<!--- The Mission,</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ReferencesTeam:JUIT-India/References2012-09-26T18:55:41Z<p>Antresh kumar: </p>
<hr />
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.cssmenu p{<br />
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<div class='cssmenu' z-index:5000><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/55/Idea.jpg" width="965" height="300"></div><br />
<br />
<hr><br />
<h1>BRAINSTOTMING IDEAS </h1><br />
<hr><br />
<h2><br />
<ol><br />
<li>Magnetic Microbes</li><br />
<li>Microbes that can conduct electricity</li><br />
<li>Microbes with high metabolism that can help degrade metal at high altitudes like Siachen Lake</li><br />
<li>Microbes that can help plant survive in air without soil</li><br />
<li>Dyes that can stop greying of hair</li><br />
<br />
<br />
<br />
<br />
</html><br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/WetLabWorkTeam:JUIT-India/WetLabWork2012-09-26T18:55:02Z<p>Antresh kumar: </p>
<hr />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg" width="965" height="300"></div><br />
<br />
<r><br />
<hr><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
Diary: <br />
<br />
== ''' June ''' ==<br />
<br />
• Brainstorming various ideas<br />
<br><br />
• Decide the project to work on for this year<br />
<br />
<br />
==July:==<br />
<br>• Week 1<br />
<br>o Research on the project thoroughly<br />
<br>o Primers Designing<br />
• Week 2<br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of MxaF <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of MxaF<br />
<br>Agarose Gel Electrophoresis<br />
Result: Non-Specific Binding<br><br />
Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
Agarose Gel Electrophoresis<br><br />
Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br />
<br>• Week 3 <br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of NifA <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Results: NO band was observed (due to less duration in the -200c freezer<br />
<br> Wednesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NifA<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br>• Week 4<br />
<br>o Culturing of B.subtilis<br />
<br>o Isolation of genomic DNA from B.subtilis<br />
<br>o PCR reaction for amplification of SacB<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured B.subtilis<br />
<br> Tuesday : Isolated Genomic DNA from B.subtilis<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of SacB<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Non-Specific Binding<br />
<br> Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
==August: ==<br />
<br>• Week 1<br />
<br>o Culturing of P. aeruginosa<br />
<br>o Isolation of genomic DNA from P. aeruginosa<br />
<br>o PCR Reaction for amplification of NosZ<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured P. aeruginosa<br />
<br> Tuesday : Isolated Genomic DNA from P. aeruginosa<br />
Agarose Gel Electrophoresis<br />
Results: NO band was observed <br />
<br> Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method<br />
Agarose Gel Electrophoresis<br />
Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NosZ<br />
<br>Agarose Gel Electrophoresis<br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
Agarose Gel Electrophoresis<br />
Result: No bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
<br />
<br>• Week 2<br />
<br>o PCR reactions for amplification of MxaF & NifA<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture<br />
<br> Monday : PCR reaction performed for MxaF & NifA<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of MxaF into TA Vector<br />
<br> Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: Failed(Contamination)<br />
<br> Thursday : Ligation of MxaF into TA Vector <br />
<br> Friday: Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
<br> Saturday: Ligation of NifA into TA Vector <br />
<br> Sunday: : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
o <br />
<br />
<br>• Week 3<br />
<br>o PCR reactions for amplification of NosZ & SacB<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture\<br />
<br> Monday : PCR reaction performed for NosZ & SacB<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of NosZ into TA Vector<br><br />
Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured <br />
Thursday : Ligation of SacB into TA Vector<br> <br />
Friday: Transformation using MgCl2 & CaCl2 method<br><br />
Result: No colonies were observed<br><br />
Saturday: Ligation of SacB into TA Vector <br><br />
Sunday: : Transformation using MgCl2 & CaCl2 method<br><br />
Result: White Colonies were selected and cultured<br><br />
<br />
• Week 4<br><br />
o Culture transformed Cells<br><br />
o Plasmid Isolation<br><br />
o PCR reactions to confirm the insertion of genes<br><br />
o Agarose Gel Electrophoresis<br><br />
Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells<br><br />
Tuesday : Plasmid Isolation for MxaF & NifA transformed cells<br />
Restriction Digestion of the plasmids<br><br />
Wednesday : PCR reaction performed for amplification of MxaF & NifA<br />
Agarose Gel Electrophoresis. <br><br />
Results: Sharp Bands were observed.<br><br />
Thursday : Plasmid Isolation for NosZ & SacB transformed cells<br />
Restriction Digestion of the plasmids.<br><br />
Agarose Gel Electrophoresis<br><br />
Friday: PCR reaction performed for amplification of MxaF & NifA <br><br />
Saturday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
==September: ==<br />
• Week 1<br><br />
o Restriction Digestion of MxaF containing plasmids<br><br />
o Ligation of MxaF into psb1c3<br><br />
o Restriction Digestion of NosZcontaining plasmids<br><br />
o Ligation of NosZ into psb1c3 containg MxaF<br><br />
Monday : Restriction Digestion of MxaF containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 using EcoR1<br />
Ligation of MxaF into psb1c3<br><br />
Thursday : Restriction Digestion of NosZ containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br> <br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of NosZ into psb1c3 containing MxaF<br><br />
<br />
• <b>Week 2:</b><br><br />
o Restriction Digestion of NifA<br><br />
o Ligation of NifA into psb1c3 containg NifA & SacB<br><br />
o Restriction Digestion of SacB<br><br />
o Ligation of SacB into psb1c3 containg all the other genes<br><br />
Monday : Restriction Digestion of NifA containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1<br />
Ligation of NifA into psb1c3<br><br />
Thursday : Restriction Digestion of SacB containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br><br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of SacB into psb1c3 containing all the other genes.<br><br />
<br />
• <b>Week 3:</b><br><br />
o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes<br><br />
o Growth of transformed cells on selective media.<br><br />
o Plasmid Isolation <br><br />
o PCR reaction to confirm the insertion of genes<br><br />
Monday: Cultures M.Capsulatus Cells<br><br />
Tuesday : Preparation of M.Capsulatus Competent Cells<br><br />
Wednesday: Transformation of competent cells using the prepared plasmids<br><br />
Thursday : Growth of plates on LB plates containg chloramphenicol<br><br />
Friday: Cultured transformed cells in LB agar<br><br />
Saturday: Plasmid Isolation <br><br />
Restriction Digestion<br><br />
Sunday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
<br />
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<h1>Engineered Vector </h1><br><br />
<br />
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=='''Complete Description'''==<br />
<br />
'''Project Description'''<br />
<br />
We are going to use the nif (From M.capsulatus) and nos (from Pseudomonas) genes. The mmo genes are present in to forms in M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO). MMO enzyme catalyzes the conversion of methane to methanol. Once methanol is converted into formaldehyde it enters various biochemical cycles in the cell. Our system involves the utilization of the methanol as an inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for nitrogen fixation. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase. Nif genes then converts the nitrogen into nitrate, which can be easily be taken up by the plant. The main reason for addition of fertilizer is to increase the inorganic nutrients in the soil. By utilizing NosZ and Nif genes we are converting the nitrous oxide into nitrate, thus reducing the need of fertilizers in the soil by increasing the nitrogen available to the plants. This is a small step towards a more environment friendly future.<br />
<br />
Details:<br />
<br />
'''MxaF''':<br />
<br />
Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C1) substrates more reduced than carbon dioxide as their sole source of carbon and energy.Methanotrophs possesses native methanol-inducible promoters, notably promoters which are located upstream of genes that encode methanol dehydrogenase and other proteins required for its activity and enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone. Of these, the promoter PmxaF has been thoroughly scrutinized both biochemically and in expression studies. In its native form in the chromosome, this strong promoter is methanol inducible. However, when this promoter is cloned in expression vectors, it acts essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide. Our system involves the utilization of the methanol as an inducer for MxaF promoter. This would result in the diversion of the flux thus leading to a faster degradation of methane for the cell to survive.<br />
<br />
'''NosZ:'''<br />
<br />
The complete denitrification of nitrate by bacteria to dinitrogen (N,) is generally an anaerobic respiratory process. The last step involves the dissimilatory reduction of nitrous oxide (N,O), the free energy change of which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck, 1990). nosZ is the structural gene for the periplasmic N,O reductase which is required to the conversion of nitrous oxide into free nitrogen. . The nitrous oxide that is emitted in the paddy fields is caused due to the excessive use of fertilizers. The microbes naturally present in the paddy field lead to the emission of nitrous oxide by utilizing these chemical fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for denitrification.<br />
<br />
'''NifA:'''<br />
<br />
The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. Expression of the nif genes is regulated at the transcriptional level by the products of nifA in response to molecular oxygen or ammonia. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase holoenzyme containing the alternative sigma factor, sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA, also known as upstream activation sequence (UAS), that is located more than 100 bp upstream of nif promoters. The NifA protein has three arbitrarily designated domains (20): an amino-terminal domain which is implicated in regulatory function, a catalytic domain that interacts with the sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif which recognizes the UAS on the nif promoters.<br />
<br />
<br />
'''SacB:'''<br />
<br />
Expression from sacB confers sensitivity to sucrose in a wide variety of gram positive and negative bacteria and thus has been used extensively for the last twenty years as a negative selectable marker. The Bacillus subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which is secreted by B. subtilis cells into the culture medium. Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules. In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose<br />
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/File:Vctor.jpgFile:Vctor.jpg2012-09-26T18:52:37Z<p>Antresh kumar: </p>
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<div></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/NotebookTeam:JUIT-India/Notebook2012-09-26T18:51:39Z<p>Antresh kumar: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg" width="965" height="300"></div><br />
<br />
<r><br />
<hr><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
Diary: <br />
<br />
== ''' June ''' ==<br />
<br />
• Brainstorming various ideas<br />
<br><br />
• Decide the project to work on for this year<br />
<br />
<br />
==July:==<br />
<br>• Week 1<br />
<br>o Research on the project thoroughly<br />
<br>o Primers Designing<br />
• Week 2<br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of MxaF <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of MxaF<br />
<br>Agarose Gel Electrophoresis<br />
Result: Non-Specific Binding<br><br />
Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
Agarose Gel Electrophoresis<br><br />
Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br />
<br>• Week 3 <br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of NifA <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Results: NO band was observed (due to less duration in the -200c freezer<br />
<br> Wednesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NifA<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br>• Week 4<br />
<br>o Culturing of B.subtilis<br />
<br>o Isolation of genomic DNA from B.subtilis<br />
<br>o PCR reaction for amplification of SacB<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured B.subtilis<br />
<br> Tuesday : Isolated Genomic DNA from B.subtilis<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of SacB<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Non-Specific Binding<br />
<br> Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
==August: ==<br />
<br>• Week 1<br />
<br>o Culturing of P. aeruginosa<br />
<br>o Isolation of genomic DNA from P. aeruginosa<br />
<br>o PCR Reaction for amplification of NosZ<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured P. aeruginosa<br />
<br> Tuesday : Isolated Genomic DNA from P. aeruginosa<br />
Agarose Gel Electrophoresis<br />
Results: NO band was observed <br />
<br> Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method<br />
Agarose Gel Electrophoresis<br />
Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NosZ<br />
<br>Agarose Gel Electrophoresis<br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
Agarose Gel Electrophoresis<br />
Result: No bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
<br />
<br>• Week 2<br />
<br>o PCR reactions for amplification of MxaF & NifA<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture<br />
<br> Monday : PCR reaction performed for MxaF & NifA<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of MxaF into TA Vector<br />
<br> Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: Failed(Contamination)<br />
<br> Thursday : Ligation of MxaF into TA Vector <br />
<br> Friday: Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
<br> Saturday: Ligation of NifA into TA Vector <br />
<br> Sunday: : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
o <br />
<br />
<br>• Week 3<br />
<br>o PCR reactions for amplification of NosZ & SacB<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture\<br />
<br> Monday : PCR reaction performed for NosZ & SacB<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of NosZ into TA Vector<br><br />
Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured <br />
Thursday : Ligation of SacB into TA Vector<br> <br />
Friday: Transformation using MgCl2 & CaCl2 method<br><br />
Result: No colonies were observed<br><br />
Saturday: Ligation of SacB into TA Vector <br><br />
Sunday: : Transformation using MgCl2 & CaCl2 method<br><br />
Result: White Colonies were selected and cultured<br><br />
<br />
• Week 4<br><br />
o Culture transformed Cells<br><br />
o Plasmid Isolation<br><br />
o PCR reactions to confirm the insertion of genes<br><br />
o Agarose Gel Electrophoresis<br><br />
Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells<br><br />
Tuesday : Plasmid Isolation for MxaF & NifA transformed cells<br />
Restriction Digestion of the plasmids<br><br />
Wednesday : PCR reaction performed for amplification of MxaF & NifA<br />
Agarose Gel Electrophoresis. <br><br />
Results: Sharp Bands were observed.<br><br />
Thursday : Plasmid Isolation for NosZ & SacB transformed cells<br />
Restriction Digestion of the plasmids.<br><br />
Agarose Gel Electrophoresis<br><br />
Friday: PCR reaction performed for amplification of MxaF & NifA <br><br />
Saturday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
==September: ==<br />
• Week 1<br><br />
o Restriction Digestion of MxaF containing plasmids<br><br />
o Ligation of MxaF into psb1c3<br><br />
o Restriction Digestion of NosZcontaining plasmids<br><br />
o Ligation of NosZ into psb1c3 containg MxaF<br><br />
Monday : Restriction Digestion of MxaF containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 using EcoR1<br />
Ligation of MxaF into psb1c3<br><br />
Thursday : Restriction Digestion of NosZ containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br> <br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of NosZ into psb1c3 containing MxaF<br><br />
<br />
• <b>Week 2:</b><br><br />
o Restriction Digestion of NifA<br><br />
o Ligation of NifA into psb1c3 containg NifA & SacB<br><br />
o Restriction Digestion of SacB<br><br />
o Ligation of SacB into psb1c3 containg all the other genes<br><br />
Monday : Restriction Digestion of NifA containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1<br />
Ligation of NifA into psb1c3<br><br />
Thursday : Restriction Digestion of SacB containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br><br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of SacB into psb1c3 containing all the other genes.<br><br />
<br />
• <b>Week 3:</b><br><br />
o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes<br><br />
o Growth of transformed cells on selective media.<br><br />
o Plasmid Isolation <br><br />
o PCR reaction to confirm the insertion of genes<br><br />
Monday: Cultures M.Capsulatus Cells<br><br />
Tuesday : Preparation of M.Capsulatus Competent Cells<br><br />
Wednesday: Transformation of competent cells using the prepared plasmids<br><br />
Thursday : Growth of plates on LB plates containg chloramphenicol<br><br />
Friday: Cultured transformed cells in LB agar<br><br />
Saturday: Plasmid Isolation <br><br />
Restriction Digestion<br><br />
Sunday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
<br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/Human_PracticesTeam:JUIT-India/Human Practices2012-09-26T18:51:12Z<p>Antresh kumar: </p>
<hr />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c9/Survey.jpg" width="965" height="500"></div><br />
<br><br />
<hr><br />
<br />
<h1>SURVEY:</h1><br><br />
<h3>Our team conducted a survey in & around our college. We came up with a lot of interesting answers and ideas. We explained synthetic biology to them in great details and shared their inputs regarding how it can be made safer for the environment and how to make public more open to the idea of synthetic biotechnology. </h3><br />
<hr><br />
<h2>These were the certain questions on which we focused?</h2><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/1/16/Sur.jpg" width="965" height="500"></div><br />
<br />
<br><br />
<h2>Some Unique answers :</h2><br />
<ol><br />
<li>Biotechnologists are people who repair hospital equipment.</li><br />
<li>Biotechnology and Electronics are the similar</li><br />
</ol><br />
<hr><br />
<h2>Next Question</h2><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/9/9c/Sur2.jpg" width="965" height="500"></div><br />
<br><br><br />
<h2>Some interesting ideas put forward by people :</h2><br />
<br>1. Creation of a superhero<br />
<br>2. Growing plants in air<br />
<br>3. Building homes on the moon<br />
<br>4. Jumping bacteria<br />
<br>5. Create fairies<br />
<br>6. Microbes as servants<br />
<br>7. Synthetic Biofuels<br />
<br>8. Flying Cars<br />
<br>9. Green Chemicals form agricultural wastes<br />
<br><br />
<br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/e/ec/Sur3.jpg" width="965" height="500"></div><br />
<h2>How to make Synthetic Biology safer??</h2><br />
<br><br />
Spreading awareness regarding the ethical issues.<br />
<br>1. Use of negative control<br />
<br>2. Prevent horizontal gene transfer<br />
<br>3. Prevent biomagnification<br />
<br>4. Inhibit use of synthetic biology by terrorists.<br />
<br>5. Spreading awareness regarding the ethical issues.<br />
<br />
<br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ModelingTeam:JUIT-India/Modeling2012-09-26T18:50:18Z<p>Antresh kumar: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Advisors'><span>Advisors</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Attributions'><span>Attributions</span></a></li><br />
</ul><br />
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<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>References</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Wiki'><span>Wiki Page</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/0/02/Juit1.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<br />
<h1>Experimental Model We are trying to create </h1><br><br />
<br />
<hr><br />
<br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/58/Final.JPG" width="965" height="600"></div><br />
<br />
<br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/PartsTeam:JUIT-India/Parts2012-09-26T18:49:50Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/e/e8/Parts.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
</html><br />
<h1>We have added 4 parts in the registry :-</h1><br />
<ol><br />
<li>'''BBa_K730001''' :- Mxa F which act as a promoter </li><br />
<li>'''BBa_K730002''' :- NosZ which is a protein Coding part</li><br />
<li>'''BBa_K730003''' :- NifA which is a protein Coding part</li><br />
<li>'''BBa_K730005''' :- Composite Part</li><br />
</ol><br />
<p>For checking full information regarding parts visit Parts Registry page for our team.<br />
<br><br />
PARTS PAGE:-<br />
<br>http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2012&group=JUIT-India&Done=1<br />
<html><br />
<br><br />
<br />
<center><br />
</center><br />
</html><br />
<br />
<br />
=='''JUIT-India 2012 iGEM Team Parts Sandbox'''==<br />
<br />
<br>'''BBa_K730001'''<br />
<br>'''BBa_K730002''' <br />
<br>'''BBa_K730003''' <br />
<br>'''BBa_K730005''' <br />
<hr><br />
<br />
<html><br />
<br><br />
<h1>Our Title Sponsor :</h1><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ProtocolTeam:JUIT-India/Protocol2012-09-26T18:48:42Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
<style> <br />
#contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {<br />
display:none;}<br />
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display: none;}<br />
/* Resizes the menubar to fik the links (default is 400px) */<br />
#menubar {<br />
width: auto;}<br />
body {<br />
margin: 10px 0 0 0;<br />
padding: 5;}<br />
#top-section {<br />
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/*top:1em;<br />
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}<br />
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.cssmenu li:hover li a{<br />
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<br />
}<br />
.cssmenu li ul a{<br />
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font-style:normal;<br />
margin:0px;<br />
padding:0px 10px 0px 15px;<br />
text-align:left;<br />
}<br />
.cssmenu li ul a:hover, .cssmenu li ul li:hover a{<br />
background:#2580a2 url('https://static.igem.org/mediawiki/2012/a/a8/Hover_sub.gif') center left no-repeat;<br />
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text-decoration:none;<br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/1/19/Pro.JPG" width="965" height="300"></div><br />
<br><br />
<hr><br />
</html><br />
<h1>Protocols :-</h1><br />
<h2>Objective:</h2><br />
<hr><br />
To perform restriction digestion of DNA with EcoR I and BamHI enzymes.<br />
<br><br />
Principle:<br />
<br>Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of DNA, found in bacteria. As they cut within the molecule, they are commonly called restriction endonucleases. They specifically cleave the nucleic acids at specific nucleotide sequence called Restriction sites to generate a set of smaller fragments .<br />
Restriction enzymes form part of the restriction-modification system of bacterial cells that provides protection against invasion of the cell by foreign DNA – especially bacteriophage DNA. But the cells own DNA is not cleaved by these Restriction enzymes. This self protection is achieved by the help of the specific DNA methyltransferase enzyme which will methylates the specific DNA sequence for its respective restriction enzymes by transferring methyl groups to adenine or cytosine residues to produce N6-methyladenine or 5-methylcytosine. An interesting feature of restriction endonuclease is that they commonly recognize recognition sequences that are mostly palindromes - they shows the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom strand) sequences. In other words, they are nucleotide sequences or complimentary strands that read the same in opposite direction.<br />
<hr><br />
Materials Required<br />
<ul> <br />
<li> Microcentrifuge tubes</li><br />
<li> Vial stand</li><br />
<li> 10µl pipette</li><br />
<li> Pipette tips</li><br />
<li> Beaker</li><br />
<li> Table top mini centrifuge</li><br />
<li> Incubator</li><br />
<li> Reagents</li><br />
</ul><br />
<hr><br />
Procedure<br />
<ol><br />
<li> Transfer the following solutions in a micro centrifuge tube.</li><br />
<br />
<br />
<li>Incubate the mixture at 37 o C for 1 h to overnight. Keep the tubes in -4o C freezer or in -20o C freezer, after the incubation.</li><br />
</ol><br />
<hr><br />
Precaution<br />
<ul><br />
<li>Make sure that the restriction enzyme does not exceed more than 10% of the total reaction volume, Otherwise the glycerol and the EDTA in the enzyme storage buffer may inhibit digestion process.</li><br />
</ul><br />
<br><br />
Differences Encountered in Real Laboratory<br />
<ul><br />
<li> After performing the experiment, confirm the Digestion of DNA by running a small amount of it in agarose gel with an undigested standard DNA. </li><br />
<li>Some restriction enzymes require BSA. In such cases make sure that, it is added to the reaction mixture. Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction.</li><br />
<li>Before performing the experiment, check whether the restriction enzymes have star activity or not. <br />
</li><br />
<br><br />
<h4>Objectives</h4><br />
<hr><br />
To understand the basic procedures involved in the isolation process of DNA from various sources such as blood, tissue, bacteria etc.<br />
<br />
Requirements for Plasmid Isolation:<br />
<ul><br />
<li>Micro centrifuge.</li><br />
<li> Water bath (37°C).</li><br />
<li> Automatic micropipettes with tips.</li><br />
<li>95-100% isopropanol Ice.</li><br />
</ul><br><br><br />
Buffers and Solutions:<br><br />
1. Alkaline lysis solution I.<br><br />
2.Alkaline lysis solution II.<br><br />
3.Alkaline lysis solution III.<br><br />
4.Antibiotic for plasmid selection.<br><br />
5.Ethanol.<br><br />
6.Phenol: chloroform (1:1, v/v).<br><br />
7.STE.<br><br />
8.TE (pH 8.0) containing 20 μg/ml RNAse A.<br><br />
<br>Media:<br />
<br> <br />
1.Rich medium.<br><br />
Procedure:<br />
<hr> <br />
<br>1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.<br />
<br>2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.<br />
<br>3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.<br />
<br>4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.<br />
<br>5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents well by inverting the tube . Do not vortex! Store the tube in ice.<br />
<br>6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube in ice for 3-5 minutes.<br />
<br>7. Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C in a microfuge. Collect the supernatant to a fresh tube.<br />
<br>8. (Optional) Add equal volume of phenol: chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.<br />
<br>9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.<br />
<br>10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.<br />
<br>11. Discard the supernatant by aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kim wipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.<br />
<br>12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.<br />
<br>13. Remove all of the supernatant by aspiration. Take care with this step, as the pellet sometimes does not adhere tightly to the tube.<br />
<br>14. Remove any beads of ethanol from the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).<br />
<br>15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds and store the DNA at -20°C.<br />
<br><br> <br />
Recipes for Buffers, Solutions and Media:<br />
<br>Alkaline Lysis Solution I :<br />
<br>50 mM glucose.<br />
<br>25 mM Tris-Cl (pH 8.0).<br />
<br>10 mM EDTA (pH 8.0).<br />
<br>Prepare Solution I from standard stocks in batches of approx. 100 ml, sterilize by autoclaving and store at 4°C.<br />
(For plasmid preparation.)<br />
<br>Alkaline Lysis Solution II:<br />
<br>0.2 N NaOH (freshly diluted from a 10 N stock).<br />
<br>1% (w/v) SDS.<br />
<br>Prepare Solution II fresh and use at room temperature.<br />
(For plasmid preparation.)<br />
<br>Alkaline Lysis Solution III:<br />
<br>5 M potassium acetate, 60.0 ml.<br />
<br>Glacial acetic acid, 11.5 ml.<br />
<br>H2O, 28.5 ml.<br />
<br><br>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.<br />
<br>(For plasmid preparation.)<br />
<br><br>EDTA:<br />
<br>To prepare 0.5 M EDTA (pH 8.0): Dissolve 186.1 g of disodium EDTA•2H2O in 800 ml of Distilled 2H2O. Stir well on a magnetic stirrer. EDTA will not dissolve into solution until the pH of the solution is reached to ~ 8.0 . So the pH should adjust to 8.0 with NaOH (~ 20 g of NaOH pellets) and make up the final volume to 1000ml with distilled water. Prepare the aliquots and sterilize by autoclaving.<br />
<br><br>Glycerol:<br />
<br>To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology grade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a pre rinsed 0.22-μm filter. Store in 200-ml aliquots at 4°C.<br />
<br><br>LB Media<br />
<br>Deionized H2O, to 950 ml.<br />
<br>Tryptone, 10 g.<br />
<br>Yeast extract, 5 g.<br />
<br><br>NaCl, 10 g.<br />
<br>To prepare LB (Luria-Bertani) medium, shake the ingredients , mentioned above with Distilled water until the solutes have dissolved. Adjust pH to 7.0 with 5 N NaOH and make up the final volume of the solution to 1 liter with deionized H2O. Then sterilize it for 20 minutes by autoclaving at 15 psi .<br />
<br><br>NaCl:<br />
<br>To prepare 5 M NaCl : Dissolve 292 g of NaCl in 800 ml of sterile H2O and the volume is make up to to 1 liter with deionized H2O. Prepare the aliquots and sterilize it by autoclaving.<br />
<br><br>NaOH:<br />
<br> To 800 ml of H2O, add 400g of NaOH pellets slowly, stirring continuously. After dissolving the pellets,completely, make up the final volume to 1 liter with sterile H2O. Store the solution at room temperature.<br />
<br><br>Potassium Acetate:<br />
<br> 5 M potassium acetate, 60 ml.<br />
<br>Glacial acetic acid, 11.5 ml.<br />
<br>H2O, 28.5 ml.<br />
<br>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at room temperature<br />
<br><br>SDS:<br />
<br>Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of SDS in 900 ml of H2O. Heat to a temperature of 68°C and stir with a magnetic stirrer to help dissolution. Adjust the volume to 1 liter with distilled H2O. Store at room temperature. Autoclaving not necessary.<br />
<br><br>STE:<br />
<br>10 mM Tris-Cl (pH 8.0).<br />
<br>0.1 M NaCl.<br />
<br>1 mM EDTA (pH 8.0).<br />
<br>Sterilize the solution by autoclaving and store at 4°C.<br />
<br><br>TE:<br />
<br>100 mM Tris-Cl (desired pH).<br />
<br>10 mM EDTA (pH 8.0).<br />
<br>(10x Tris EDTA) Sterilize the buffer by autoclaving and store at room temperature.<br />
<br><br>Tris-Cl:<br />
<br>Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH by adding concentrated HCl , to the desired value.The volume of the solution is make up to 1 liter with distilled H2O. Prepare the aliquots and sterilize by autoclaving. <br />
<br>Procedure for Operating the Virtual Lab:<br />
<br>Check whether you have done all the steps listed below:<br />
<br>• Prepare the culture containing the desired plasmid.<br />
<br>• Incubate the culture for 24 hours at 37°C.<br />
<br>• Take the culture from the incubator.<br />
<br>• Transfer 1.5ml of the culture to a microfuge tube.<br />
<br>• Centrifuge the tube for 30seconds at maximum speed (4°C).<br />
<br>• Remove the supernatant.<br />
<br>• Add 100μl alkaline lysis solution I.<br />
<br>• Vortex the sample.<br />
<br>• Add 200μl of alkaline lysis solution II.<br />
<br>• Mix the sample by inverting the tube.<br />
<br>• Store in ice for 1 minute.<br />
<br>• Add alkaline lysis solution III.<br />
<br>• Mix the contents by inverting the tube.<br />
<br>• Store in ice for 3-5 minutes.<br />
<br>• Centrifuge the solution at maximum speed (4°C) for 5 minutes .<br />
<br>• Collect the supernatant to a fresh tube.<br />
<br>• Precipitate the nucleic acid by adding 2 volumes of ethanol.<br />
<br>• Mix by vortexing.<br />
<br>• Stand the tubes for 2 minutes.<br />
<br>• Centrifuge for 5 minutes.<br />
<br>• Collect the precipitated DNA.<br />
<br>• Discard the supernatant by aspiration.<br />
<br>• Stand the tube as inverted to drain the fluid away.<br />
<br>• Add 1ml 70% ethanol.<br />
<br>• Mix by inverting.<br />
<br>• Centrifuge the mixture for 2 minutes.<br />
<br>• Discard the supernatant by aspiration.<br />
<br>• Allow to dry for 3-5 minutes.<br />
<br>• Add TE buffer with RNAse.<br />
<br>• Mix by flickering.<br />
<br>• Detect the plasmid by doing agarose gel electrophoresis.<br><br />
<br><hr><br />
Objective<br />
<br> To separate the DNA fragments based on their Molecular weight.<br />
<br>Materials Required:<br />
<br> Buffers and Solutions:<br />
<br> Agarose solutions.<br />
<br>Ethidium bromide.<br />
<br>Electrophoresis buffer.<br />
<br> Nucleic Acids and Oligonucleotides:<br />
<br> DNA samples.<br />
<br>DNA Ladders.<br />
<br> (Samples of DNAs of known size are typically generated by restriction enzyme digestion of a plasmid or bacteriophage DNA of known sequence).<br />
<br><br>The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:<br />
<br>• An electrophoresis chamber and power supply.<br />
<br>• Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.<br />
<br>• Sample combs, around which molten agarose is poured to form sample wells in the gel.<br />
<br>• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).<br />
<br>• Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.<br />
<br>• Ethidium bromide, a fluorescent dye used for staining nucleic acids.<br />
<br>• Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.<br />
<br> NOTE: Always wear protective eyewear when observing DNA on a Transilluminator to prevent damage to the eyes from UV light.<br />
<br>1. Prepare a 50x stock solution of TAE buffer in 1000m of distilled H2O:<br />
<br><br>For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.<br />
<br>Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml distilled water. EDTA is insoluble and it can be made soluble by adding sodium hydroxide pellets. Check the pH using pH meter. Make the solution 100ml by adding distilled water.<br />
<br>Pipette out 57.1 ml of glacial acetic acid.<br />
<br>Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the volume to 1000ml<br />
<br>2. Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the electrophoresis tank and to cast the gel:<br />
<br>For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA<br />
<br>It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the gel preparation.<br />
<br>3. Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:<br />
<br>For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.<br />
<br><br>Agarose Concentration in Gel (% [w/v]) Range of Separation of Linear DNA Molecules (kb)<br />
<br>0.3 5-60<br />
<br>0.6 1-20<br />
<br>0.7 0.8-10<br />
<br>0.9 0.5-7<br />
<br>1.2 0.4-6<br />
<br>1.5 0-2-3<br />
<br>2.0 0.1-2<br />
<br>4. Loosely plug the neck of the Erlenmeyer flask. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves. The agarose solution can boil over very easily so keep checking it. It is good to stop it after 45 seconds and give it a swirl. It can become superheated and NOT boil until you take it out whereupon it boils out all over you hands. So wear gloves and hold it at arm's length. You can use a Bunsen burner instead of a microwave <br>- just remember to keep watching it.<br />
<br>5. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling. <br />
<br>(For the preparation of ethidium bromide adds 1 g of ethidium bromide to 100 ml of H2O. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room temperature.)<br />
<br>6.While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel.<br />
<br>7.Pour the warm agarose solution into the mold.<br />
<br>(The gel should be between 3 - 5 mm thick. Check that no air bubbles are under or between the teeth of the comb.)<br />
<br>8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer. Mount the gel in the electrophoresis tank.<br />
<br>9.Add just enough electrophoresis buffers to cover the gel to a depth of approx. 1mm.<br />
<br>10. Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.<br />
<br>11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette or an automatic micropipettor or a drawn-out Pasteur pipette or a glass capillary tube. Load size standards into slots on both the right and left sides of the gel.<br />
<br>12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the electrodes are 10cm apart then run the gel at 50V. It is fine to run the gel slower than this but do not run it any faster. Above 5V/cm the agarose may heat up and begin to melt with disastrous effects on your gel's resolution. If the leads have been attached correctly, bubbles should be generated at the anode and cathode.<br />
<br>13. Run the gel until the bromophenol blue and xylenecyanol FF have migrated an appropriate distance through the gel.<br />
<br> (The presence of ethidium bromide allows the gel to be examined by UV illumination at any stage during electrophoresis).<br />
<br>14. The gel tray may be removed and placed directly on a transilluminator. When the UV is switched on we can see orange bands of DNA. <br />
<br><br>Procedure for operating the virtual lab:<br />
<br>Check whether you have done all the steps listed below:<br />
<br>• Prepare TAE buffer.<br />
<br>• Transfer 100ml of the buffer to a conical flask.<br />
<br>• Weigh 2grams of agarose and add to the 100ml buffer solution.<br />
<br>• Keep in oven.<br />
<br>• Take the solution from oven.<br />
<br>• Add ethidium bromide.<br />
<br>• Pour the solution to a gel caster.<br />
<br>• Place the comb.<br />
<br>• Pour the 100ml buffer solution to the electrophoretic chamber.<br />
<br>• Place the gel in the caster in the electrophoretic chamber.<br />
<br>• Connect the electrodes and switch on the current.<br />
<br>• Switch off the power supply.<br />
<br>• Remove the gel from the electrophoretic chamber.<br />
<br>• Place the gel in the UV Transilluminator.<br />
<br>• Switch on the Transilluminator.<br />
<br><br>CAUTION:<br />
<br>• Ethidium bromide is a mutagen and should be handled as a hazardous chemical (so wear gloves while handling)<br />
<br><br>DIFFERENCES ENCOUNTERED IN REAL LABORATORY:<br />
<br>1. Make sure that the Agarose is fully dissolved in the buffer. If it is not dissolved well, again melt it some more time to dissolve completely.<br />
<br>2. Before casting the gel, the tray and comb should wipe with ethanol.<br />
<br>3. Make sure that the gel in the Chamber is immersed in the TAE Buffer.<br />
<br>4. Labelings should be proper.<br />
<br>5. Ensure that the connections should be proper.<br />
<br>6. Before the incubation step, ensure that the water bath is set at the correct temperature that we required or not.<br><br />
<hr><br />
Objective: <br><br />
To familiarize with how cells are made competent which is the primary step for transformation.<br />
<br><br>Materials Required:-<br />
<br>• LB broth<br />
<br>• Culture plates<br />
<br>• Ice cold CaCl2.2H2O (1 M)<br />
<br>• Ice cold MgCl2 CaCl2 solution <br />
<br>• Shaking incubator<br />
<br>• Vortex mixer<br />
<br>• Centrifuge<br />
<br>• Water bath<br />
<br>• Inoculation loop<br />
<br>• Microfuge tubes<br />
<br>• Polypropylene tubes<br />
<br>• Micro pipettes and tips<br />
<br><br>Procedure:-<br />
<br>1. Pick a single bacterial colony from a culture plate which is incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml LB broth a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH 5 alpha contains approx. 109 bacteria/ml.<br />
<br>2. Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes. Cool the cultures to 0°C by storing them on ice for 10 minutes.<br />
<br>3. Recover the cells by centrifugation at 2700g at 4°C for 10 minutes .<br />
<br>4. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for<br> 1 minute to allow the last traces of media to drain away.<br />
<br>5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.<br />
<br>6. Recover the cells by centrifugation at 2700g at 4°C for 10 minutes .<br />
<br>7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.<br />
<br>8. Resuspend the pellet in 2 ml of ice-cold 0.1 M CaCl2 (or TFB) by gentle vortexing for each 50ml of original culture. Standard TFB may be used instead of CaCl2 for most strains of E. coli.<br />
<br>9. At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.<br />
<hr><br />
Objectives <br />
<br>1) To develop an understanding about the transformation.<br />
<br>2) Transform bacteria cells with a foreign DNA.<br />
<br>Materials Required<br />
<br>• LB broth<br />
<br>• LA- Amp plate<br />
<br>• Micropipette and tips<br />
<br>• Incubator<br />
<br>• Water bath<br />
<br>• Microfuge tube<br />
<br>• Calcium chloride treated competent cells<br />
<br>• L-rod<br />
<br>Reagents Required<br />
<br>• X Gal: Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. The X-gal tube should wrap with aluminium foil in order to prevent the damage caused by light and store at -20°C.<br />
<br>• IPTG: Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG with 8 ml of distilled H2O. Prepare the aliquots and store them at -20°C.<br />
<br>Procedure<br />
<br>1. To transform the CaCl2- treated cells directly, transfer 200 µl of each suspension of competent cells to a sterile, chilled polypropylene tube using a chilled micropipette tip.<br />
<br>2. Add DNA (<50ng in a volume of 10 µl) to each tube. Mix the contents of the tubes by gentle swirling. Keep the tubes in ice for 30 minutes. <br />
<br>3. Transfer the tubes to rack placed in a preheated 42 ºC circulating water bath. Keep the tubes in rack for 90 seconds.<br />
<br>4. Transfer the tubes to an ice bath immediately. Allow the cells to chill for 1-2 minutes.<br />
<br>5. Add 800 µl of LB medium to each tube. Incubate the cultures for 45 minutes in a water bath at 37 ºC .<br />
<br>6. After incubation, add 40 µl of X –Gal and 7 µl of IPTG to the LA-Amp plate.<br />
<br>7. Add appropriate volume of transformed competent cells into the plate.<br />
<br>8. Spread all the contents uniformly using an L-rod. Keep the plates at room temperature until the liquid has been absorbed.<br />
<br>9. Invert the plates and keep for incubation at 37 ºC. Transformed colonies will appear in 12-16 hours of incubation. <br />
<br>Differences Encountered in Real Laboratory<br />
<br>1. After taking the competent cells from the freezer, it should thaw at room temperature.<br />
<br>2. There should not be much time lag between adding and spreading of EDTA ,Beta-gal and Transformed cells. If so, the components will not spread uniformly in the plate.<br />
<br>3. Make sure that the samples are uniformly spread in to the plate(should care the spreading).<br />
<br>4. Make sure that the plates with transformed cells should be in inverted position while incubation.<br />
Objective<br />
<hr> <br />
To extract specific bands of DNA from agarose gels in which they are separated through electrophoresis.<br />
<br>Materials Required<br />
<br>• Elution buffer<br />
<br>• Scalpel blade<br />
<br>• UV transilluminator<br />
<br>• Agarose<br />
<br>• Micro pipettes<br />
<br>• Micro pipette tips<br />
<br>• Dry bath incubator<br />
<br>• Microfuge tubes<br />
<br>• Centrifuge<br />
<br>• N-Butanol<br />
<br>• Cryo box<br />
<br>• Cyclomixer<br />
<br>• 70% Ethanol<br />
<br>• 95% Ethanol<br />
<br>• TE buffer<br />
<br>• -20oC freezer<br />
<br>• -70oC freezer<br />
<br>Procedure<br />
<br>1. Visualize the low melting point agarose gel with DNA bands under a UV transilluminator and locate the desired DNA band to cut.<br />
<br>2. Carefully cut around the desired DNA band using a scalpel blade.<br />
<br>3. Transfer the gel piece into a microfuge tube.<br />
<br>4. Add elution buffer into the microfuge tube until the level of buffer is just above the level of gel slice.<br />
<br>5. Heat the gel slice at 65oC until it melts.<br />
<br>6. Freeze the melted gel with DNA by placing in a -70oC freezer for10minuts.<br />
<br>7. After freezing, centrifuge for 10minutes and transfer the supernatant into a new microfuge tube.<br />
<br>8. Again add half amount of elution buffer that you added in the previous step into the pellet.<br />
<br>9. Heat at 65oC until the agarose melts.<br />
<br>10. Freeze the melted gel with DNA by placing in a -70oC freezer for10minuts.<br />
<br>11. Centrifuge the tube again for 10 minutes and transfer (pool) the supernatant into the previous tube with supernatant.<br />
<br>12. Discard the tube with pellet.<br />
<br>13. Add an equal volume of n-Butanol to the supernatant and mix the contents well.<br />
<br>14. Vortex the tube for 15 minutes in order to remove the Ethidium bromide. <br />
<br>15. Discard the upper phase of butanol and repeat the process by adding n-butanol again for one or more times.<br />
<br>16. Add 2 times volume of 95% ethanol and mix thoroughly.<br />
<br>17. Keep for precipitation in -70oC freezer for 30minutes to overnight.<br />
<br>18. After precipitation, centrifuge for 15 minutes.<br />
<br>19. Discard the supernatant into a waste beaker and add 200µl of 70% ethanol to the pellet.<br />
<br>20. Centrifuge for 5minutes and discard the supernatant again.<br />
<br>21. Allow the pellets to dry well.<br />
<br>22. Suspend the pellets in 20µl of TE buffer. (If you want to confirm the recovered DNA, run (1µl) it on a gel.<br />
<br>23. The recovered DNA can be now used for further process of cloning otherwise can stored in -20oC freezer. <br />
<br>Objective:-<br />
<hr><br />
<br>To perform ligation reaction using T4 DNA ligase.<br />
<br>Materials Required:-<br />
<br>• Vials and Vial stand<br />
<br>• Micro pipette<br />
<br>• pipette tips<br />
<br>• Table top mini centrifuge<br />
<br>• Beaker<br />
<br>• Ice box<br />
<br>Reagents:-<br />
<br>Procedure:-<br />
<br>1. Take one clean fresh microfuge tube(Sample) from the rack.<br />
<br>2. In this microfuge tube(Vial), add 5µL water, 1µL Vector, 2.5µL insert and 1µL T4 DNA ligase buffer.<br />
<br>3. 0.5 µL of T4 DNA Ligase enzyme was added to the sample tube ( total reaction volume is 10 µl).<br />
<br>4. The vial is kept in the micro centrifuge and just spin for a few seconds.<br />
<br>5. Incubate the vial at room temperature (22˚C ) for 2 hours.<br />
<br>6. After 2 hours, the ligated mixture is taken for doing transformation.<br />
<br>1.REAL LAB VERSUS VIRTUAL LAB:<br />
<br />
<br>1. T4 DNA Ligase Buffer contains ATP, so repeated freeze thaw cycles can degrade ATP, thereby decreasing the efficiency of Ligation. <br />
<br>2. It is better to vortex or spin the T4 DNA ligase enzyme before pipetting to ensure that it is mixed well.<br />
<br>Objectives<br />
<hr>To amplify a given region of DNA(region of interest).<br />
<br>Procedure:<br />
<br>The protocol describes how to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase. It is the foundation for all subsequent variations of the polymerase chain reaction.<br />
<br>Materials<br />
<br>Buffers and Solutions<br />
<br>10x Amplification buffer<br />
<br>Chloroform<br />
<br>dNTP solution (20 mM) containing all four dNTPs (pH 8.0)<br />
<br>Enzymes and Buffers<br />
<br>Thermostable DNA polymerase<br />
<br>Nucleic Acids and Oligonucleotides<br />
<br>Forward primer (20 μM) in H2O<br />
<br>Reverse primer (20 μM) in H2O<br />
<br>Template DNA.<br />
<br>Dissolve template DNA in 10 mM Tris-Cl (pH 7.6) containing a low concentration of EDTA (<0.1 mM) at the following concentrations: mammalian genomic DNA, 100 μg/ml; yeast genomic DNA, 1 μg/ml; bacterial genomic DNA, 0.1 μg/ml; and plasmid DNA, 1-5 ng/ml.<br />
<br>Method<br />
<br>1. In a sterile 0.5-ml microfuge tube, mix in the following order:<br />
<br>REAGENTS AMOUNT(μl)<br />
<br>Deionized water 37.5 μl<br />
<br>Taq assay buffer(10x) 5 μl<br />
<br>Template DNA 1μl<br />
<br>dNTPs mix 2 μl<br />
<br>Forward primer 2 μl<br />
<br>Reverse primer 2 μl<br />
<br>Taq DNA polymerase 5 μl<br />
<br>The table below provides standard reaction conditions for PCR. Mg2+ (1.5 mM) ;KCl(50 mM) ;dNTPs (200 μM) ;Primers(1 μM );DNA polymerase (1-5 units); Template DNA(1 pg to 1 μg ).<br />
<br> The amount of template DNA required varies according to the complexity of its sequence. In the case of mammalian DNA, up to 1.0 μg is used per reaction. Typical amounts of yeast, bacterial, and plasmid DNAs used per reaction are 10 ng, <br>1 ng, and 10 pg, respectively.<br />
<br>2.If the thermal cycler is not fitted with a heated lid, overlay the reaction mixtures with 1 drop (approx. 50 μl) of light mineral oil. Alternatively, place a bead of wax into the tube if using a hot start protocol. Place the tubes or the micro titer plate in the thermal cycler.<br />
<br>3.Amplify the nucleic acids using the denaturation, annealing, and polymerization times and temperatures listed below.<br />
<br>4.Withdraw a sample (5-10 μl) from the test reaction mixture and the four control reactions, analyze them by electrophoresis through an agarose gel, and stain the gel with ethidium bromide or SYBR Gold to visualize the DNA.<br />
<br>A successful amplification reaction should yield a readily visible DNA fragment of the expected size. The identity of the band can be confirmed by DNA sequencing, Southern hybridization and/or restriction mapping. If all is well, lanes of the gel containing samples of the two positive controls (Tubes 1& 2) and the template DNA under test should contain a prominent band of DNA of the appropriate molecular weight. This band should be absent from the lanes containing samples of the negative controls (Tubes 3 & 4).<br />
<br>5. If mineral oil was used to overlay the reaction (Step 2), remove the oil from the sample by extraction with 150 μl of chloroform. The aqueous phase, which contains the amplified DNA, will form a micelle near the meniscus. The micelle can be transferred to a fresh tube with an automatic micropipette.<br />
<br>Do not attempt chloroform extractions in micro titer plates. The plastic used in these plates is not resistant to organic solvents.<br />
<br>Recipes<br />
<br>Amplification Buffer:<br />
<br>500 mM KCl.<br />
<br>100 mM Tris-Cl (pH 8.3 at room temperature).<br />
<br>15 mM MgCl2.<br />
<br>Autoclave the 10x buffer for 10 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Divide the sterile buffer into aliquots and store them at -20oC.<br />
<br>KCl<br />
<br>Dissolve an appropriate amount of solid KCl in H2O, autoclave for 20 minutes on liquid cycle and store at room temperature. Ideally, this 4 M solution should be divided into small (approx. 100 μl) aliquots in sterile tubes and each aliquot thereafter used one time.<br />
<br>Tris-Cl<br />
<br>Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl.<br />
<br>pH HCl<br />
<br>7.4 70 ml<br />
<br>7.6 60 ml<br />
<br>8.0 42 ml<br />
<br>(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 litre with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases approx. 0.03 pH units for each 1oC increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5oC, 25oC, and 37oC, respectively.<br />
<br>dNTP Solution<br />
<br>Dissolve each dNTP (deoxyribonucleoside triphosphates) in H2O at an approximate concentration of 100 mM. Use 0.05 M Tris base and a micropipette to adjust the pH of each of the solutions to 7.0 (use pH paper to check the pH). Dilute an aliquot of the neutralized dNTP appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each dNTP. Dilute the solutions with H2O to a final concentration of 50 mM dNTP. Store each separately at 70oC in small aliquots. For polymerase chain reactions (PCRs), adjust the dNTP solution to pH 8.0 with 2 N NaOH. Commercially available solutions of PCR-grade dNTPs require no adjustment.<br />
<br>Base wavelength(nm) Extinction Coefficient(E) (M-1cm-1)<br />
<br>A 259 1.54 x 104<br />
<br>G 253 1.37 x 104<br />
<br>C 271 9.10 x 103<br />
<br>T 267 9.60 x 103<br />
<br>For a cuvette with a path length of 1 cm, absorbance = EM. 100 mM stock solutions of each dNTP are commercially available .<br />
<br>Precautions<br />
<br>Chloroform<br />
<br>Chloroform CHCl3 is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile. Avoid breathing the vapours. Wear appropriate gloves and safety glasses. Always wear a chemical fume hood.<br />
<br>PREPARATION OF GENOMIC DNA FROM BACTERIA<br />
<br>Solutions required for this protocol<br />
<br>• TE buffer <br />
<br>• 10% (w/v) sodium dodecyl sulfate (SDS) <br />
<br>• 20 mg/ml proteinase K <br />
<br>• Phenol/chloroform <br />
<br>• Isopropanol <br />
<br>• 70% ethanol <br />
<br>• 3M sodium acetate ph 5.2 <br />
<br>• Phase Lock geltm (5 Prime, 3 Prime, Inc)<br />
<br>1. Transfer 1.5 ml to a micro centrifuge tube and spin 2 min. Decant the supernatant. Drain well onto a Kimwipe.<br />
<br>2. Resuspend the pellet in 467 µl TE buffer by repeated pipetting. Add 30 µl of 10% SDS and 3 µl of 20 mg/ml proteinase K, mix, and incubate 1 hr at 37°C.<br />
<br>3. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed. CAUTION: PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB COAT AND KEEP TUBES CAPPED TIGHTLY. Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin 2 min.<br />
<br>4. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a new Phase Lock GelTM tube and spin 2 min. Transfer the upper aqueous phase to a new tube.<br />
<br>5. Add 1/10 volume of sodium acetate.<br />
<br>6. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.<br />
<br>7. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end). <br />
Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec. <br />
Resuspend DNA in 100-200 µl TE buffer.<br />
<br>8. After DNA has dissolved, measure the concentration by diluting 10 µl of DNA into 1 ml of TE (1:100 dilution) and measure absorbance at 260 nm.<br><br />
Concentration of original DNA solution in µg/ml = Abs x 100 x 50 µg/ml.<br />
<br><br />
Protocol of Genomic DNA Isolation from a Gram-Negative Bacterium<br />
<br>The G NOME® kit (http://www.qbiogene.com) is used to quickly and efficiently isolate high molecular weight genomic DNA from Gram-negative bacterial cultures. Each preparation with the G NOME® kit yields up to 100 mg of genomic DNA.<br> The DNA isolated by the G NOME® procedure is suitable for restriction enzyme digestion or PCR amplification in as little as 1 hour after cell lysis.<br />
<br>Protocol<br />
<br>1. Bring cells*/tissues to a final volume of 1.85ml in Cell Suspension Solution. (Use a 15 ml clear plastic tube for efficient mixing). Mix until the solution appears homogeneous.<br />
<br>2. Add 50ml of RNase Mixx, mix thoroughly.<br />
<br>3. Add 100ml of Cell Lysis/Denaturing Solution**, mix well.<br />
<br>4. Incubate at 55°C for 15 minutes.<br />
<br>5. Add 25ml Protease Mixx, mix thoroughly. (Note: If precipitate is visible in Protease Mixx suspension, pulse spin and use 25 ml of supernatant.)<br />
<br>6. Incubate at 55°C for 30 to 120 minutes. (The longer time will result in minimal protein carry over and will also allow for substantial reduction in residual protease activity.)<br />
<br>7. Add 500ml “Salt-Out” Mixture, mix gently yet thoroughly. Divide sample into 1.5ml tubes. Refrigerate at 4°C for 10 minutes.<br />
<br>8. Spin for 10 minutes at maximum speed in a microcentrifuge (at least 10,000 x g). Carefully collect the supernatant, avoid the pellet. If a precipitate remains in the supernatant, spin again until it is clear. Pool the supernatants in a 15 ml (or larger) clear plastic tube.<br />
<br>9. To this supernatant, add 2 ml TE buffer and mix. Then add 8mls of 100% ethanol. If spooling the DNA, add the ethanol slowly and spool the DNA at the interphase with a clean glass rod. If centrifuging the DNA, add the ethanol and gently mix the solution by inverting the tube. Spin for 15 minutes at 1000-1500xg. Eliminate the excess ethanol by blotting or air drying the DNA.<br />
<br>10. Dissolve the genomic DNA in TE (10mM Tris pH 7.5, 1mM EDTA).<br />
<hr><br />
<br>Experiment 1 : Plasmid DNA Preparation<br />
<br>Objective: Isolation of plasmid DNA by Alkaline Lysis with SDS: Minipreparation<br />
<br>Principle: Bacterial plasmids are self replicating, circular extra chromosomal, DNA molecules. The most convenient method for preparing plasmid DNA is alkaline lysis method. In this procedure, cells are lysed by SDS at high pH and then neutralized. The plasmid DNA reanneals rapidly while most of the chromosomal DNA and bacterial proteins precipitate as a protein- DNA-SDS complex. <br />
<br>Materials Required:<br />
<br>Reagents and Buffers<br />
<br>Alkaline Lysis Solution I<br />
<br>50 mM glucose<br />
<br>25 mM Tris-Cl (pH 8.0)<br />
<br>10 mM EDTA (pH 8.0)<br />
<br>Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C.<br />
<br>Alkaline Lysis Solution II<br />
<br>0.2 N NaOH (freshly diluted from a 10 N stock)<br />
<br>1% (w/v) SDS<br />
<br>Prepare Solution II fresh and use at room temperature.<br />
<br>Alkaline Lysis Solution III<br />
<br>5 M potassium acetate, 60.0 ml<br />
<br>Glacial acetic acid, 11.5 ml<br />
<br>H2O, 28.5 ml<br />
<br>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.<br />
<br><br />
Alkaline Lysis Solution IV<br />
<br>Isopropanol<br />
<br>Step-wise Methodology: <br />
<br>1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.<br />
<br>2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.<br />
<br>3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.<br />
<br>4. Resuspend the bacterial pellet in 100 μl of ice-cold alkaline lysis solution I by vigorous vortexing.<br />
<br>5. Add 200 μl of freshly prepared alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice.<br />
<br>6. Add 150 μl of ice-cold alkaline lysis solution III. Close the tube and disperse alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.<br />
<br>7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube.<br><br />
8. (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.<br />
<br>9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.<br />
<br>10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.<br />
<br>11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.<br />
<br>12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.<br />
<br>13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube.<br />
<br>14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).<br />
<br>15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C.<br />
<br>Precautions:<br />
<br>• Dispose of used reagents according to local ordinances. <br />
<br>• Wear latex gloves and goggles during the procedure. <br />
<br>• The laboratory surfaces should be very clean during all procedures used in this activity. <br />
<br>• Use thoroughly clean instruments and glassware. Rinse all equipment with isopropyl alcohol or acetone. <br />
<br>• Ethanol is highly flammable; use caution.<br><br />
Observations: Plasmid DNA will migrate in 3 forms: supercoiled closed circular (superciled) DNA, single-strand nicked open circle DNA, and linear DNA. The exact mobilities of the 3 forms will vary based on conditions, but closed circular DNA will usually migrate faster than the other two because of its supercoiling. This means that if the DNA band farthest from your well on your uncut plasmid sample is the largest, you have prepared a plasmid DNA sample without excessively damaging your DNA. (Note that your gel may also contain a diffuse band of partially degraded RNA <br>– which will migrate faster than your DNA bands.)<br />
<br>Interpretations & Conclusions:<br />
<br>Experiment -2<br />
<br>Aim: Restriction of given plasmid or λ DNA with the restriction enzyme EcoRI and HindIIIand estimation of fragment size by comparison with 1KB ladder.<br><br />
Principle: Restriction enzymes are group of enzymes isolated from bacterial species which recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Each restriction enzyme recognizes specific sequences and cuts at specific sites Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA. Some Examples of restriction enzyme along with their, recognition sequence and cutting sites are given below.<br />
<br>Hind III A|AGCTT<br />
<br>Bam HI G|GATCC<br />
<br>Eco RI G|AATTC<br />
<br>PstI CTGCA|G<br />
<br>Eco RV GAT|ATC<br />
<br>Material required: Sterile water distilled water, 10X restriction enzyme buffer, plasmid DNA or λ DNA<br />
<br>Procedure:<br />
<br>1. Add the following in a 1.5ml centrifuge tube: 15 µl Sterile distilled water, 2.0 µl, 10X enzyme buffer, 1.0 µl Restriction enzyme, 2.0 µl Plasmid DNA\ λ DNA. <br />
<br>2. Maintain enzyme tube and plasmid DNA on ice. Do not leave on the table. <br />
<br>3. Mix by tapping the tube with your finger.<br />
<br>4. Briefly centrifuge to remove bubbles (DNA will adhere surface and becomes inaccessible to the enzymes).<br />
<br>5. Incubate at 37 C in a water bath for 45 minutes.<br />
<br>6. If the DNA is to be used for another manipulation, Heat inactivates the restriction enzyme by incubating the tubes at 65 C for 15 minutes.<br />
<br>7. Load the restriction enzymes cut and uncut plasmid on an agarose gel. Electrophorase the samples at 50-100 V for 1-2 hrs<br />
<br>8. Record the no. of fragments and their size in restriction enzyme digested samples.<br />
<br>Precautions:<br />
<br>1. Use fresh tip after each pipetting in order to avoid cross contamination of the chemicals and if you are using enzyme twice, change the pipet tip.<br />
<br>2. Enzymes should always be carried in a -20 mini cooler and work quickly. Do not expose the enzyme to warm temperature any longer than necessary.<br />
<br>3. Always wear gloves while handling enzymes.<br />
<br>Observations<br />
<br>The EcoR I digest of λ DNA yields the following 6 discrete fragments (in base pairs): 21226*, 7421, 5804, 5643, 4878, 3530* <br />
<br>The Hind III digest of λ DNA yields the following 8 discrete fragments (in base pairs): 23130*, 9416, 6557, 4361*, 2322, 2027, 564, 125.<br />
<br>Aim-To insert the PCR product into T vector by TA-cloning <br />
<br>Principle<br />
<br>"TA cloning" is a popular method of cloning without the use of restriction enzymes; instead, the fragment to be cloned, is amplified with only Taq DNA polymerase as PCR product. TA Cloning exploits the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. The PCR products with dA overhang are mixed with this vector in high proportion. The complementary overhangs of "T" vector and PCR product will be ligated under the action of T4 DNA ligase.<br />
<br>Advantages<br />
<br>1. Eliminate any enzymatic modification of PCR product<br />
<br>2. Does no require the use of PCR primer tthat contain restriction sites<br />
<br />
<br>RBC T&A cloning kit (cat.no. RC001)<br />
<br>RBC TA cloning system is ideal for rapid cloning PCR product generated using a thermos table DNA polymerase, which <br>adds a single 3’dA nucleotide overhang. Following the ligation the mixture may be used directly to transform competent cell or purified to achieve a higher efficiency of transformation.<br />
<br>Product components<br />
<br>1. T&A (25ng/μl) cloning vector : 40μl<br />
<br>2. Control Insert DNA (10ng/μl) : 10μl<br />
<br>3. T4 DNA ligase(3U/μl) : 20μl<br />
<br>4. T4 DNA ligase Buffer A : 100μl<br />
<br>5. T4 DNA ligase Buffer B : 100μl<br />
<br>6. forward primer(M13-F)(10μM)(50μl)<br />
<br>7. reverse primer(M13-R)(10μM)(50μl)<br />
<br>Procedure: <br />
<br>1. Centrifuge T&A cloning vector and/or PCR DNA tubes to collect contents at the bottom of the tubes.<br />
<br>2. Vortex the ligation buffer vigorously before use.<br />
<br>3. Set up the following items as described below:<br />
<br />
<br>3. Mix the reactions by pipetting.<br />
<br>4. Incubate the reactions for 5 to 15 min at 22oC. Alternatively, if the maximum of transformants is required, incubate the reactions overnight at 4oC.<br />
<br />
<br>Suggestions<br />
<br>1. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes by making single-use aliquots of Ligase Buffer.<br />
<br>2. Pfu DNA polymerase possesses proofreading activity; it does not have the terminal transferase-like activity demonstrated by Taq DNA Polymerase. Ligation reactions using non-tailed amplified DNA resulted in no positive colonies.<br />
<br>Aim: Transformation of E.coli. DH5 α cells with recombinant T- vector.<br />
<br>Principal <br><br />
Genetic transformation occurs when a host organism takes in foreign DNA and expresses the foreign gene. In this experiment, we will introduce recombinant T vector carrying genes for resistance to the antibiotic penicillin into competent cells of DH5 α which are otherwise sensitive to it. If the bacterial cells incorporate the foreign DNA, they will become penicillin resistant. The CaCl2 treated competent cell of E.coli and plasmid DNA is mixed and then briefly heated to 42C for a brief period of 45- 90 sec to give a heat shock to the cells. CaCI2treatment promotes binding of DNA to E. coli cell wall. It is taken up by the cell when temperature is raised to 42°C. <br />
MATERIALS and Equipments: LB medium (Liq.), 1.5 ml centrifuge tube, micro tips, centrifuge, Water bath, Recombinant vector, LB plates containing penicillin and X-gall<br />
<br>Procedure<br />
<br>1. Take the competent cells stored at -80C and thaw on ice.<br />
<br>2. Add 10µl of recombinant T vector to the 200µl of competent cells. Mix the content by swirly the tube gently. Store the tube on ice for 30 minutes<br />
<br>3. Transfer the tubes to a rack placed in a preheated 42C circulating water bathwater bath for 90 seconds<br />
<br>4. Rapidly transfer the tubes on ice. Allow the cells to chill for 1-2 minutes<br />
<br>5. Add 800µl of LB medium. Incubate the culture for 2-3 hours at 37C.Transfer 200µl of heat shock given cells on LB agar plate 100ug/µl ampicillin and X-gal <br />
<br>6. Incubate the plates at 37C in inverted position. Transformed colonies should appear in 12-16 hours.<br />
µl<br />
<br />
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ProjectTeam:JUIT-India/Project2012-09-26T18:48:09Z<p>Antresh kumar: </p>
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<h1>ABSTRACT</h1><br />
<br />
<p>“Global warming is too serious for the world any longer to ignore its danger or split into opposing factions on it”, quoted Tony Blair back in 2005. Well how much concerning this appears, since then, a lot more similar quotes can be added in its reference. We have come together as a team to have a different insight in dealing with this problem through genetic engineering. Rice, which is the staple diet of India and many other countries around the world, is believed to engender many potential green house gases or global warming gases per se like carbon dioxide, methane and nitrous oxide. Nitrous oxide, again, is released due to the inevitable use of nitrogen fertilizers which are added in the paddy fields. We are dealing with the conversion of this highly potential global warming gas, nitrous oxide into nitrate form using synthetic biology tools to insert two genes into a bacterial cassette along with its detection systems. This nitrate, as we know, can in turn, be utilized by the plant itself, solving our purpose and adding a new dimension to this diversion and in turn being beneficial for the farmers reducing the compromise factor that would, otherwise, have been done. <br />
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== '''Overall project''' ==<br />
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Synthetic biology aims to design and construct new biological functions and system that are not found in nature. We have given the name ‘Captain Green’ to our organism M.capsulatus who plays ‘hero’ like figure in providing the greener and healthy environment.<br />
Global warming has become an alarming issue in 21st century and we aim to take action against it. The tremendous increase in methane, nitrous oxide and carbon dioxide emission has become a great concern. While rice has the third-highest worldwide production and a staple crop for nearly half the world's population with the worldwide consumption of ~367 million metric ton per year but anoxic conditions in the wetland soils of rice paddies are ideal for microbes that produce methane, which trails only carbon dioxide in terms of its greenhouse effect. Rice agriculture is a big source of atmospheric methane, possibly the biggest of man-made methane sources. With an increasing world population, reductions in rice agriculture remain largely untenable as on Methane emission reduction strategy. Methane emission from paddy field makes up 29% of the total of Methane and Nitrous Oxide emission from agricultural land makes up 55%. So, greenhouse gas emissions from rice paddy fields are considered as one of the most important emission sources. The average concentration of nitrous oxide in the atmosphere is now increasing at a rate of 0.2 to 0.3% per year. Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. <br />
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=='''Complete Description'''==<br />
<br />
'''Project Description'''<br />
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We are going to use the nif (From M.capsulatus) and nos (from Pseudomonas) genes. The mmo genes are present in to forms in M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO). MMO enzyme catalyzes the conversion of methane to methanol. Once methanol is converted into formaldehyde it enters various biochemical cycles in the cell. Our system involves the utilization of the methanol as an inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for nitrogen fixation. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase. Nif genes then converts the nitrogen into nitrate, which can be easily be taken up by the plant. The main reason for addition of fertilizer is to increase the inorganic nutrients in the soil. By utilizing NosZ and Nif genes we are converting the nitrous oxide into nitrate, thus reducing the need of fertilizers in the soil by increasing the nitrogen available to the plants. This is a small step towards a more environment friendly future.<br />
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Details:<br />
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'''MxaF''':<br />
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Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C1) substrates more reduced than carbon dioxide as their sole source of carbon and energy.Methanotrophs possesses native methanol-inducible promoters, notably promoters which are located upstream of genes that encode methanol dehydrogenase and other proteins required for its activity and enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone. Of these, the promoter PmxaF has been thoroughly scrutinized both biochemically and in expression studies. In its native form in the chromosome, this strong promoter is methanol inducible. However, when this promoter is cloned in expression vectors, it acts essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide. Our system involves the utilization of the methanol as an inducer for MxaF promoter. This would result in the diversion of the flux thus leading to a faster degradation of methane for the cell to survive.<br />
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'''NosZ:'''<br />
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The complete denitrification of nitrate by bacteria to dinitrogen (N,) is generally an anaerobic respiratory process. The last step involves the dissimilatory reduction of nitrous oxide (N,O), the free energy change of which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck, 1990). nosZ is the structural gene for the periplasmic N,O reductase which is required to the conversion of nitrous oxide into free nitrogen. . The nitrous oxide that is emitted in the paddy fields is caused due to the excessive use of fertilizers. The microbes naturally present in the paddy field lead to the emission of nitrous oxide by utilizing these chemical fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for denitrification.<br />
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'''NifA:'''<br />
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The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. Expression of the nif genes is regulated at the transcriptional level by the products of nifA in response to molecular oxygen or ammonia. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase holoenzyme containing the alternative sigma factor, sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA, also known as upstream activation sequence (UAS), that is located more than 100 bp upstream of nif promoters. The NifA protein has three arbitrarily designated domains (20): an amino-terminal domain which is implicated in regulatory function, a catalytic domain that interacts with the sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif which recognizes the UAS on the nif promoters.<br />
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'''SacB:'''<br />
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Expression from sacB confers sensitivity to sucrose in a wide variety of gram positive and negative bacteria and thus has been used extensively for the last twenty years as a negative selectable marker. The Bacillus subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which is secreted by B. subtilis cells into the culture medium. Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules. In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose<br />
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<td colspan="5"><h1>Meet the team</h1></td><br />
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<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
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<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
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Pankaj<br><br />
<p>&nbsp;</p></td><br />
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<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
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<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasanth </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
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<td><h1>Where we're from</h1></td><br />
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<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
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</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-IndiaTeam:JUIT-India2012-09-26T18:46:10Z<p>Antresh kumar: </p>
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== ''' Project Overview ''' ==<br />
<br />
Captain Green<br />
<br />
Synthetic biology aims to design and construct new biological functions and system that are not found in nature. We have given the name ‘Captain Green’ to our organism M.capsulatus who plays ‘hero’ like figure in providing the greener and healthy environment.<br />
<br />
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<br />
== Project Details ==<br />
<br />
Global warming has become an alarming issue in 21st century and we aim to take action against it. The tremendous increase in methane, nitrous oxide and carbon dioxide emission has become a great concern. While rice has the third-highest worldwide production and a staple crop for nearly half the world's population with the worldwide consumption of ~367 million metric ton per year but anoxic conditions in the wetland soils of rice paddies are ideal for microbes that produce methane, which trails only carbon dioxide in terms of its greenhouse effect. Rice agriculture is a big source of atmospheric methane, possibly the biggest of man-made methane sources. With an increasing world population, reductions in rice agriculture remain largely untenable as on Methane emission reduction strategy. Methane emission from paddy field makes up 29% of the total of Methane and Nitrous Oxide emission from agricultural land makes up 55%. So, greenhouse gas emissions from rice paddy fields are considered as one of the most important emission sources. The average concentration of nitrous oxide in the atmosphere is now increasing at a rate of 0.2 to 0.3% per year. Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. <br />
<br />
=== Abstract ===<br />
<br />
The summer heat of 2012 is enough proof towards the increasing global warming. Our project focuses on the reduction of emission of greenhouse gases from the rice paddy fields. We are focusing on reducing methane emission and as well as converting nitrous oxide into nitrate, thereby increasing the fertility of the soil. By genetically modifying Methylococcus capsulatus we plan on converting methane into carbon dioxide. Methane is proven to be a more dangerous greenhouse gas than carbon dioxide. At the same time we are also going to be focusing on the most dangerous global warming gas i.e. nitrous oxide. We plan on converting nitrous oxide into nitrate using genes from Pseudomonas and Methylococcus.<br />
<br />
=== Objective ===<br />
<br />
1. The reduction in the greenhouse gases being emitted from the rice paddy fields.<br />
<br><br />
2. To increase the fertility of the soil by increasing the nitrate content in the soil. <br />
<br />
=== Methodology ===<br />
<br />
Our system involves the conversion of methane to methanol, then using the methanol as an inducer for homologous of Aox promoter, the target genes are used to convert nitrous oxide to nitrate, which easily be taken up by the plant and increase the yield also.<br />
<br />
For Complete Details visit our Project Description Page<br />
<br />
== Expected Outcomes ==<br />
As expected, the above project will efficiently reduce the emission rate of greenhouse gases to a significant level (which is an immediate concern of the Global warming) in Paddy crop along with increasing the fertility of the soil and thus increasing the rice yield<br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
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<!--- The Mission,</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/Human_PracticesTeam:JUIT-India/Human Practices2012-09-26T18:38:35Z<p>Antresh kumar: </p>
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<br><br />
<hr><br />
<br />
<h1>SURVEY:</h1><br><br />
<h3>Our team conducted a survey in & around our college. We came up with a lot of interesting answers and ideas. We explained synthetic biology to them in great details and shared their inputs regarding how it can be made safer for the environment and how to make public more open to the idea of synthetic biotechnology. </h3><br />
<hr><br />
<h2>These were the certain questions on which we focused?</h2><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/1/16/Sur.jpg" width="965" height="500"></div><br />
<br />
<br><br />
<h2>Some Unique answers :</h2><br />
<ol><br />
<li>Biotechnologists are people who repair hospital equipment.</li><br />
<li>Biotechnology and Electronics are the similar</li><br />
</ol><br />
<hr><br />
<h2>Next Question</h2><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/9/9c/Sur2.jpg" width="965" height="500"></div><br />
<br><br><br />
<h2>Some interesting ideas put forward by people :</h2><br />
<br>1. Creation of a superhero<br />
<br>2. Growing plants in air<br />
<br>3. Building homes on the moon<br />
<br>4. Jumping bacteria<br />
<br>5. Create fairies<br />
<br>6. Microbes as servants<br />
<br>7. Synthetic Biofuels<br />
<br>8. Flying Cars<br />
<br>9. Green Chemicals form agricultural wastes<br />
<br><br />
<br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/e/ec/Sur3.jpg" width="965" height="500"></div><br />
<h2>How to make Synthetic Biology safer??</h2><br />
<br><br />
Spreading awareness regarding the ethical issues.<br />
<br>1. Use of negative control<br />
<br>2. Prevent horizontal gene transfer<br />
<br>3. Prevent biomagnification<br />
<br>4. Inhibit use of synthetic biology by terrorists.<br />
<br>5. Spreading awareness regarding the ethical issues.<br />
<br />
<br />
<br />
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/File:Survey.jpgFile:Survey.jpg2012-09-26T18:37:35Z<p>Antresh kumar: </p>
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<div></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T18:26:46Z<p>Antresh kumar: </p>
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<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasanth <br><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasanth </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/2012/a/a3/Nidhi.jpg" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
<br />
<br />
<br />
<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</div><br />
</div><br />
</body><br />
<br />
<br />
<br />
<script>if (window.runOnloadHook) runOnloadHook();</script><br />
</div><br />
<!-- Served in 0.209 secs. --></body><br />
<br />
</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T18:26:20Z<p>Antresh kumar: </p>
<hr />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasanth <br><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasanth </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/2012/a/a3/Nidhi.jpg" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
<br />
<br />
<br />
<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</div><br />
</div><br />
</body><br />
<br />
<br />
<br />
<script>if (window.runOnloadHook) runOnloadHook();</script><br />
</div><br />
<!-- Served in 0.209 secs. --></body><br />
<br />
</html></div>Antresh kumarhttp://2012.igem.org/File:Nidhi.jpgFile:Nidhi.jpg2012-09-26T18:24:21Z<p>Antresh kumar: </p>
<hr />
<div></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/PartsTeam:JUIT-India/Parts2012-09-26T18:17:24Z<p>Antresh kumar: </p>
<hr />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/e/e8/Parts.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
</html><br />
<h1>We have added 4 parts in the registry :-</h1><br />
<ol><br />
<li>'''BBa_K730001''' :- Mxa F which act as a promoter </li><br />
<li>'''BBa_K730002''' :- NosZ which is a protein Coding part</li><br />
<li>'''BBa_K730003''' :- NifA which is a protein Coding part</li><br />
<li>'''BBa_K730005''' :- Composite Part</li><br />
</ol><br />
<p>For checking full information regarding parts visit Parts Registry page for our team.<br />
<br><br />
PARTS PAGE:-<br />
<br>http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2012&group=JUIT-India&Done=1<br />
<html><br />
<br><br />
<br />
<center><br />
</center><br />
</html><br />
<br />
<br />
=='''JUIT-India 2012 iGEM Team Parts Sandbox'''==<br />
<br />
<br>'''BBa_K730001'''<br />
<br>'''BBa_K730002''' <br />
<br>'''BBa_K730003''' <br />
<br>'''BBa_K730005''' <br />
<hr><br />
<br />
<html><br />
<br><br />
<h1>Our Title Sponsor :</h1><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/PartsTeam:JUIT-India/Parts2012-09-26T18:07:02Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
<style> <br />
#contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {<br />
display:none;}<br />
#top-section {<br />
border: 0px;<br />
height: 0px;}<br />
#content {<br />
border: none;}<br />
/* Removes "teams" from the menubar */<br />
#menubar > ul > li:last-child {<br />
display: none;}<br />
/* Resizes the menubar to fik the links (default is 400px) */<br />
#menubar {<br />
width: auto;}<br />
body {<br />
margin: 10px 0 0 0;<br />
padding: 5;}<br />
#top-section {<br />
width: 965px;<br />
height: 0;<br />
margin: 0 auto;<br />
padding: 0;<br />
border: none;}<br />
#menubar {<br />
font-size: 65%;<br />
top: -14 px;}<br />
.left-menu:hover {<br />
background-color: transparent;}<br />
#menubar li a {<br />
background-color: transparent;}<br />
#menubar:hover {<br />
color: white;}<br />
#menubar li a {<br />
color: transparent;}<br />
#menubar:hover li a {<br />
color: white;}<br />
<br />
<br />
img<br />
{<br />
opacity:1.0;<br />
filter:alpha(opacity=100); <br />
}<br />
img:hover<br />
{<br />
opacity:1.0;<br />
filter:alpha(opacity=100); <br />
}<br />
.cssmenu{<br />
border:none;<br />
border:0px;<br />
margin:0px;<br />
padding:0px;<br />
font: 67.5% 'Lucida Sans Unicode', 'Bitstream Vera Sans', 'Trebuchet Unicode MS', 'Lucida Grande', Verdana, Helvetica, sans-serif;<br />
font-size:14px;<br />
font-weight:bold;<br />
}<br />
.cssmenu ul{<br />
background:#333000;<br />
height:35px;<br />
list-style:none;<br />
margin:0;<br />
padding:0;<br />
}<br />
.cssmenu li{<br />
float:left;<br />
padding: 0px;<br />
}<br />
.cssmenu li a{<br />
background:#333333 url('https://static.igem.org/mediawiki/2012/3/3f/Seperator.gif') bottom right no-repeat;<br />
color:#cccccc;<br />
display:block;<br />
font-weight:normal;<br />
line-height:35px;<br />
margin:0px;<br />
padding:0px 25px;<br />
text-align:center;<br />
text-decoration:none;<br />
}<br />
.cssmenu li a:hover, .cssmenu ul li:hover a{<br />
background: #2580a2 url('https://static.igem.org/mediawiki/2012/e/e6/Hover.gif') bottom center no-repeat;<br />
<br />
text-decoration:none;<br />
}<br />
.cssmenu li ul{<br />
background:#333333;<br />
display:none;<br />
height:auto;<br />
padding:0px;<br />
margin:0px;<br />
border:0px;<br />
position:absolute;<br />
width:225px;<br />
z-index:200;<br />
/*top:1em;<br />
/*left:0;*/<br />
}<br />
.cssmenu li:hover ul{<br />
display:block;<br />
<br />
}<br />
.cssmenu li li {<br />
background:url('https://static.igem.org/mediawiki/2012/3/3f/Sub_sep.gif') bottom left no-repeat;<br />
display:block;<br />
float:none;<br />
margin:0px;<br />
padding:0px;<br />
width:225px;<br />
}<br />
.cssmenu li:hover li a{<br />
background:none;<br />
<br />
}<br />
.cssmenu li ul a{<br />
display:block;<br />
height:35px;<br />
font-size:12px;<br />
font-style:normal;<br />
margin:0px;<br />
padding:0px 10px 0px 15px;<br />
text-align:left;<br />
}<br />
.cssmenu li ul a:hover, .cssmenu li ul li:hover a{<br />
background:#2580a2 url('https://static.igem.org/mediawiki/2012/a/a8/Hover_sub.gif') center left no-repeat;<br />
border:0px;<br />
<br />
text-decoration:none;<br />
}<br />
.cssmenu p{<br />
clear:left;<br />
} <br />
</style><br />
<div class='cssmenu' z-index:5000><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/e/e8/Parts.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
</html><br />
<br />
<html><br />
<br><br />
<br />
<center><br />
</center><br />
</html><br />
<br />
<br />
=='''JUIT-India 2012 iGEM Team Parts Sandbox'''==<br />
<br />
<br>'''BBa_K730001'''<br />
<br>'''BBa_K730002''' <br />
<br>'''BBa_K730003''' <br />
<br>'''BBa_K730005''' <br />
<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/PartsTeam:JUIT-India/Parts2012-09-26T17:17:59Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
<style> <br />
#contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {<br />
display:none;}<br />
#top-section {<br />
border: 0px;<br />
height: 0px;}<br />
#content {<br />
border: none;}<br />
/* Removes "teams" from the menubar */<br />
#menubar > ul > li:last-child {<br />
display: none;}<br />
/* Resizes the menubar to fik the links (default is 400px) */<br />
#menubar {<br />
width: auto;}<br />
body {<br />
margin: 10px 0 0 0;<br />
padding: 5;}<br />
#top-section {<br />
width: 965px;<br />
height: 0;<br />
margin: 0 auto;<br />
padding: 0;<br />
border: none;}<br />
#menubar {<br />
font-size: 65%;<br />
top: -14 px;}<br />
.left-menu:hover {<br />
background-color: transparent;}<br />
#menubar li a {<br />
background-color: transparent;}<br />
#menubar:hover {<br />
color: white;}<br />
#menubar li a {<br />
color: transparent;}<br />
#menubar:hover li a {<br />
color: white;}<br />
<br />
<br />
img<br />
{<br />
opacity:1.0;<br />
filter:alpha(opacity=100); <br />
}<br />
img:hover<br />
{<br />
opacity:1.0;<br />
filter:alpha(opacity=100); <br />
}<br />
.cssmenu{<br />
border:none;<br />
border:0px;<br />
margin:0px;<br />
padding:0px;<br />
font: 67.5% 'Lucida Sans Unicode', 'Bitstream Vera Sans', 'Trebuchet Unicode MS', 'Lucida Grande', Verdana, Helvetica, sans-serif;<br />
font-size:14px;<br />
font-weight:bold;<br />
}<br />
.cssmenu ul{<br />
background:#333000;<br />
height:35px;<br />
list-style:none;<br />
margin:0;<br />
padding:0;<br />
}<br />
.cssmenu li{<br />
float:left;<br />
padding: 0px;<br />
}<br />
.cssmenu li a{<br />
background:#333333 url('https://static.igem.org/mediawiki/2012/3/3f/Seperator.gif') bottom right no-repeat;<br />
color:#cccccc;<br />
display:block;<br />
font-weight:normal;<br />
line-height:35px;<br />
margin:0px;<br />
padding:0px 25px;<br />
text-align:center;<br />
text-decoration:none;<br />
}<br />
.cssmenu li a:hover, .cssmenu ul li:hover a{<br />
background: #2580a2 url('https://static.igem.org/mediawiki/2012/e/e6/Hover.gif') bottom center no-repeat;<br />
<br />
text-decoration:none;<br />
}<br />
.cssmenu li ul{<br />
background:#333333;<br />
display:none;<br />
height:auto;<br />
padding:0px;<br />
margin:0px;<br />
border:0px;<br />
position:absolute;<br />
width:225px;<br />
z-index:200;<br />
/*top:1em;<br />
/*left:0;*/<br />
}<br />
.cssmenu li:hover ul{<br />
display:block;<br />
<br />
}<br />
.cssmenu li li {<br />
background:url('https://static.igem.org/mediawiki/2012/3/3f/Sub_sep.gif') bottom left no-repeat;<br />
display:block;<br />
float:none;<br />
margin:0px;<br />
padding:0px;<br />
width:225px;<br />
}<br />
.cssmenu li:hover li a{<br />
background:none;<br />
<br />
}<br />
.cssmenu li ul a{<br />
display:block;<br />
height:35px;<br />
font-size:12px;<br />
font-style:normal;<br />
margin:0px;<br />
padding:0px 10px 0px 15px;<br />
text-align:left;<br />
}<br />
.cssmenu li ul a:hover, .cssmenu li ul li:hover a{<br />
background:#2580a2 url('https://static.igem.org/mediawiki/2012/a/a8/Hover_sub.gif') center left no-repeat;<br />
border:0px;<br />
<br />
text-decoration:none;<br />
}<br />
.cssmenu p{<br />
clear:left;<br />
} <br />
</style><br />
<div class='cssmenu' z-index:5000><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/e/e8/Parts.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
</html><br />
<br />
<html><br />
<br><br />
<center><br />
</center><br />
</html><br />
<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/File:Parts.jpgFile:Parts.jpg2012-09-26T17:17:12Z<p>Antresh kumar: </p>
<hr />
<div></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ExperimentTeam:JUIT-India/Experiment2012-09-26T17:12:27Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
<style> <br />
#contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {<br />
display:none;}<br />
#top-section {<br />
border: 0px;<br />
height: 0px;}<br />
#content {<br />
border: none;}<br />
/* Removes "teams" from the menubar */<br />
#menubar > ul > li:last-child {<br />
display: none;}<br />
/* Resizes the menubar to fik the links (default is 400px) */<br />
#menubar {<br />
width: auto;}<br />
body {<br />
margin: 10px 0 0 0;<br />
padding: 5;}<br />
#top-section {<br />
width: 965px;<br />
height: 0;<br />
margin: 0 auto;<br />
padding: 0;<br />
border: none;}<br />
#menubar {<br />
font-size: 65%;<br />
top: -14 px;}<br />
.left-menu:hover {<br />
background-color: transparent;}<br />
#menubar li a {<br />
background-color: transparent;}<br />
#menubar:hover {<br />
color: white;}<br />
#menubar li a {<br />
color: transparent;}<br />
#menubar:hover li a {<br />
color: white;}<br />
<br />
<br />
img<br />
{<br />
opacity:1.0;<br />
filter:alpha(opacity=100); <br />
}<br />
img:hover<br />
{<br />
opacity:1.0;<br />
filter:alpha(opacity=100); <br />
}<br />
.cssmenu{<br />
border:none;<br />
border:0px;<br />
margin:0px;<br />
padding:0px;<br />
font: 67.5% 'Lucida Sans Unicode', 'Bitstream Vera Sans', 'Trebuchet Unicode MS', 'Lucida Grande', Verdana, Helvetica, sans-serif;<br />
font-size:14px;<br />
font-weight:bold;<br />
}<br />
.cssmenu ul{<br />
background:#333000;<br />
height:35px;<br />
list-style:none;<br />
margin:0;<br />
padding:0;<br />
}<br />
.cssmenu li{<br />
float:left;<br />
padding: 0px;<br />
}<br />
.cssmenu li a{<br />
background:#333333 url('https://static.igem.org/mediawiki/2012/3/3f/Seperator.gif') bottom right no-repeat;<br />
color:#cccccc;<br />
display:block;<br />
font-weight:normal;<br />
line-height:35px;<br />
margin:0px;<br />
padding:0px 25px;<br />
text-align:center;<br />
text-decoration:none;<br />
}<br />
.cssmenu li a:hover, .cssmenu ul li:hover a{<br />
background: #2580a2 url('https://static.igem.org/mediawiki/2012/e/e6/Hover.gif') bottom center no-repeat;<br />
<br />
text-decoration:none;<br />
}<br />
.cssmenu li ul{<br />
background:#333333;<br />
display:none;<br />
height:auto;<br />
padding:0px;<br />
margin:0px;<br />
border:0px;<br />
position:absolute;<br />
width:225px;<br />
z-index:200;<br />
/*top:1em;<br />
/*left:0;*/<br />
}<br />
.cssmenu li:hover ul{<br />
display:block;<br />
<br />
}<br />
.cssmenu li li {<br />
background:url('https://static.igem.org/mediawiki/2012/3/3f/Sub_sep.gif') bottom left no-repeat;<br />
display:block;<br />
float:none;<br />
margin:0px;<br />
padding:0px;<br />
width:225px;<br />
}<br />
.cssmenu li:hover li a{<br />
background:none;<br />
<br />
}<br />
.cssmenu li ul a{<br />
display:block;<br />
height:35px;<br />
font-size:12px;<br />
font-style:normal;<br />
margin:0px;<br />
padding:0px 10px 0px 15px;<br />
text-align:left;<br />
}<br />
.cssmenu li ul a:hover, .cssmenu li ul li:hover a{<br />
background:#2580a2 url('https://static.igem.org/mediawiki/2012/a/a8/Hover_sub.gif') center left no-repeat;<br />
border:0px;<br />
<br />
text-decoration:none;<br />
}<br />
.cssmenu p{<br />
clear:left;<br />
} <br />
</style><br />
<div class='cssmenu' z-index:5000><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
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<h1>Engineered Vector </h1><br><br />
<br />
<hr><br />
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<br />
<br />
<br />
<br />
<br />
</html><br />
=='''Complete Description'''==<br />
<br />
'''Project Description'''<br />
<br />
We are going to use the nif (From M.capsulatus) and nos (from Pseudomonas) genes. The mmo genes are present in to forms in M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO). MMO enzyme catalyzes the conversion of methane to methanol. Once methanol is converted into formaldehyde it enters various biochemical cycles in the cell. Our system involves the utilization of the methanol as an inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for nitrogen fixation. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase. Nif genes then converts the nitrogen into nitrate, which can be easily be taken up by the plant. The main reason for addition of fertilizer is to increase the inorganic nutrients in the soil. By utilizing NosZ and Nif genes we are converting the nitrous oxide into nitrate, thus reducing the need of fertilizers in the soil by increasing the nitrogen available to the plants. This is a small step towards a more environment friendly future.<br />
<br />
Details:<br />
<br />
'''MxaF''':<br />
<br />
Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C1) substrates more reduced than carbon dioxide as their sole source of carbon and energy.Methanotrophs possesses native methanol-inducible promoters, notably promoters which are located upstream of genes that encode methanol dehydrogenase and other proteins required for its activity and enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone. Of these, the promoter PmxaF has been thoroughly scrutinized both biochemically and in expression studies. In its native form in the chromosome, this strong promoter is methanol inducible. However, when this promoter is cloned in expression vectors, it acts essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide. Our system involves the utilization of the methanol as an inducer for MxaF promoter. This would result in the diversion of the flux thus leading to a faster degradation of methane for the cell to survive.<br />
<br />
'''NosZ:'''<br />
<br />
The complete denitrification of nitrate by bacteria to dinitrogen (N,) is generally an anaerobic respiratory process. The last step involves the dissimilatory reduction of nitrous oxide (N,O), the free energy change of which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck, 1990). nosZ is the structural gene for the periplasmic N,O reductase which is required to the conversion of nitrous oxide into free nitrogen. . The nitrous oxide that is emitted in the paddy fields is caused due to the excessive use of fertilizers. The microbes naturally present in the paddy field lead to the emission of nitrous oxide by utilizing these chemical fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for denitrification.<br />
<br />
'''NifA:'''<br />
<br />
The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. Expression of the nif genes is regulated at the transcriptional level by the products of nifA in response to molecular oxygen or ammonia. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase holoenzyme containing the alternative sigma factor, sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA, also known as upstream activation sequence (UAS), that is located more than 100 bp upstream of nif promoters. The NifA protein has three arbitrarily designated domains (20): an amino-terminal domain which is implicated in regulatory function, a catalytic domain that interacts with the sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif which recognizes the UAS on the nif promoters.<br />
<br />
<br />
'''SacB:'''<br />
<br />
Expression from sacB confers sensitivity to sucrose in a wide variety of gram positive and negative bacteria and thus has been used extensively for the last twenty years as a negative selectable marker. The Bacillus subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which is secreted by B. subtilis cells into the culture medium. Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules. In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose<br />
<br />
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ProjectTeam:JUIT-India/Project2012-09-26T17:10:20Z<p>Antresh kumar: </p>
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<h1>ABSTRACT</h1><br />
<br />
<p>“Global warming is too serious for the world any longer to ignore its danger or split into opposing factions on it”, quoted Tony Blair back in 2005. Well how much concerning this appears, since then, a lot more similar quotes can be added in its reference. We have come together as a team to have a different insight in dealing with this problem through genetic engineering. Rice, which is the staple diet of India and many other countries around the world, is believed to engender many potential green house gases or global warming gases per se like carbon dioxide, methane and nitrous oxide. Nitrous oxide, again, is released due to the inevitable use of nitrogen fertilizers which are added in the paddy fields. We are dealing with the conversion of this highly potential global warming gas, nitrous oxide into nitrate form using synthetic biology tools to insert two genes into a bacterial cassette along with its detection systems. This nitrate, as we know, can in turn, be utilized by the plant itself, solving our purpose and adding a new dimension to this diversion and in turn being beneficial for the farmers reducing the compromise factor that would, otherwise, have been done. <br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
Synthetic biology aims to design and construct new biological functions and system that are not found in nature. We have given the name ‘Captain Green’ to our organism M.capsulatus who plays ‘hero’ like figure in providing the greener and healthy environment.<br />
Global warming has become an alarming issue in 21st century and we aim to take action against it. The tremendous increase in methane, nitrous oxide and carbon dioxide emission has become a great concern. While rice has the third-highest worldwide production and a staple crop for nearly half the world's population with the worldwide consumption of ~367 million metric ton per year but anoxic conditions in the wetland soils of rice paddies are ideal for microbes that produce methane, which trails only carbon dioxide in terms of its greenhouse effect. Rice agriculture is a big source of atmospheric methane, possibly the biggest of man-made methane sources. With an increasing world population, reductions in rice agriculture remain largely untenable as on Methane emission reduction strategy. Methane emission from paddy field makes up 29% of the total of Methane and Nitrous Oxide emission from agricultural land makes up 55%. So, greenhouse gas emissions from rice paddy fields are considered as one of the most important emission sources. The average concentration of nitrous oxide in the atmosphere is now increasing at a rate of 0.2 to 0.3% per year. Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. <br />
<br />
=='''Complete Description'''==<br />
<br />
'''Project Description'''<br />
<br />
We are going to use the nif (From M.capsulatus) and nos (from Pseudomonas) genes. The mmo genes are present in to forms in M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO). MMO enzyme catalyzes the conversion of methane to methanol. Once methanol is converted into formaldehyde it enters various biochemical cycles in the cell. Our system involves the utilization of the methanol as an inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for nitrogen fixation. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase. Nif genes then converts the nitrogen into nitrate, which can be easily be taken up by the plant. The main reason for addition of fertilizer is to increase the inorganic nutrients in the soil. By utilizing NosZ and Nif genes we are converting the nitrous oxide into nitrate, thus reducing the need of fertilizers in the soil by increasing the nitrogen available to the plants. This is a small step towards a more environment friendly future.<br />
<br />
Details:<br />
<br />
'''MxaF''':<br />
<br />
Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C1) substrates more reduced than carbon dioxide as their sole source of carbon and energy.Methanotrophs possesses native methanol-inducible promoters, notably promoters which are located upstream of genes that encode methanol dehydrogenase and other proteins required for its activity and enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone. Of these, the promoter PmxaF has been thoroughly scrutinized both biochemically and in expression studies. In its native form in the chromosome, this strong promoter is methanol inducible. However, when this promoter is cloned in expression vectors, it acts essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide. Our system involves the utilization of the methanol as an inducer for MxaF promoter. This would result in the diversion of the flux thus leading to a faster degradation of methane for the cell to survive.<br />
<br />
'''NosZ:'''<br />
<br />
The complete denitrification of nitrate by bacteria to dinitrogen (N,) is generally an anaerobic respiratory process. The last step involves the dissimilatory reduction of nitrous oxide (N,O), the free energy change of which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck, 1990). nosZ is the structural gene for the periplasmic N,O reductase which is required to the conversion of nitrous oxide into free nitrogen. . The nitrous oxide that is emitted in the paddy fields is caused due to the excessive use of fertilizers. The microbes naturally present in the paddy field lead to the emission of nitrous oxide by utilizing these chemical fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for denitrification.<br />
<br />
'''NifA:'''<br />
<br />
The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. Expression of the nif genes is regulated at the transcriptional level by the products of nifA in response to molecular oxygen or ammonia. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase holoenzyme containing the alternative sigma factor, sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA, also known as upstream activation sequence (UAS), that is located more than 100 bp upstream of nif promoters. The NifA protein has three arbitrarily designated domains (20): an amino-terminal domain which is implicated in regulatory function, a catalytic domain that interacts with the sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif which recognizes the UAS on the nif promoters.<br />
<br />
<br />
'''SacB:'''<br />
<br />
Expression from sacB confers sensitivity to sucrose in a wide variety of gram positive and negative bacteria and thus has been used extensively for the last twenty years as a negative selectable marker. The Bacillus subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which is secreted by B. subtilis cells into the culture medium. Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules. In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose<br />
<br />
<br />
<html><br />
<br><br />
<h1>Our Title Sponsor :</h1><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T17:06:17Z<p>Antresh kumar: </p>
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<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasanth <br><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasanth </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
<br />
<br />
<br />
<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</div><br />
</div><br />
</body><br />
<br />
<br />
<br />
<script>if (window.runOnloadHook) runOnloadHook();</script><br />
</div><br />
<!-- Served in 0.209 secs. --></body><br />
<br />
</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T17:06:01Z<p>Antresh kumar: </p>
<hr />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Projec'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasanth <br><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasanth </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
<br />
<br />
<br />
<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</div><br />
</div><br />
</body><br />
<br />
<br />
<br />
<script>if (window.runOnloadHook) runOnloadHook();</script><br />
</div><br />
<!-- Served in 0.209 secs. --></body><br />
<br />
</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T17:02:35Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasanth <br><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasanth </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
<br />
<br />
<br />
<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</div><br />
</div><br />
</body><br />
<br />
<br />
<br />
<script>if (window.runOnloadHook) runOnloadHook();</script><br />
</div><br />
<!-- Served in 0.209 secs. --></body><br />
<br />
</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T14:34:13Z<p>Antresh kumar: </p>
<hr />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasant <br><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasant </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
<br />
<br />
<br />
<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</div><br />
</div><br />
</body><br />
<br />
<br />
<br />
<script>if (window.runOnloadHook) runOnloadHook();</script><br />
</div><br />
<!-- Served in 0.209 secs. --></body><br />
<br />
</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T14:33:43Z<p>Antresh kumar: </p>
<hr />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<div id="contentArea"><br />
<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and two Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasant </p><br />
Pankaj<br><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/92/Eshi.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasant </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
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<br />
<br />
<!-- Where we're from table starts here --> <br />
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<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
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</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/TeamTeam:JUIT-India/Team2012-09-26T14:30:08Z<p>Antresh kumar: </p>
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<li><a href='#'><span>Team</span></a><br />
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<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/c/c8/Team_d.jpg" width="965" height="300"></div><br />
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<br />
<br />
<br />
<!-- Team table starts here --> <br />
<br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and One Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasant </p><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/23/Sri.JPG" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristhi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/0/07/Eshita.JPG" height="110"/></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/c/c1/Vasant.JPG" height="110" /></td><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasant </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
</tr><br />
<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
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<br />
<br />
<!-- Where we're from table starts here --> <br />
<br />
<br />
<br />
<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
</table><br />
<br />
<!-- Where we're from table ends here --> <br />
<br />
<br />
<hr> <br />
<br />
Our Title sponsor- <br />
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</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/SafetyTeam:JUIT-India/Safety2012-09-26T10:08:05Z<p>Antresh kumar: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/58/Safety_path-to-perfect-safety.jpg" width="965" height="300"></div><br />
<br><br />
<hr><br />
<br />
<h1>Safety Protocols</h1><br />
<br><br>Experiments were conducted in the Undergraduate lab of the Dept. Of Biotechnology at Jaypee University Of Information Technology. The lab is confirmed to the standards for a bio-safety 1 lab according to the Center for Disease Control and Prevention. Bio-safety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. <br />
<br><br>Standard Microbiological Practices<br />
<br />
<br>a. Statement of Purpose (SOP) have been developed and are kept for easy access which enlists all the safety measures each experiment should be abided for.<br />
<br>b. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.<br />
<br>c. People wash their hands after handling viable materials, after removing gloves, and before leaving the laboratory.<br />
<br>d. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. People who wear contact lenses in laboratories must wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.<br />
<br>e. Mouth pipetting is prohibited .Instead mechanical pipetting devices are to be used.<br />
<br>f. Policies for the safe handling of sharp instruments are clearly instructed.<br />
<br>g. All procedures are performed carefully to minimize the creation of splashes or aerosols.<br />
<br>h. Work surfaces are decontaminated atleast once a day and after any spill of viable material.<br />
<br>i. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method for example Autoclaving. Materials to be decontaminated outside the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. These materials are packaged in accordance with applicable local, state, and federal regulations before removing from the facility.<br />
<br />
<br />
<br><br>Safety Equipment (Primary Barriers)<br />
<br />
<br>a. Special containment devices or equipment such as a biological safety cabinet is generally not required for manipulations of agents assigned to Bio-safety Level 1.<br />
<br>b. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.<br />
<br>c. Gloves are must especially if there is a cut on the hand or if a rash is present. Alternatives to powdered latex gloves should be available.<br />
<br>d. Protective eyewear should be worn while conducting experiments in which splashes of microorganisms or other hazardous materials are anticipated.<br />
<br />
<br><br>Laboratory Facilities (Secondary Barriers)<br />
<br />
<br>a. Laboratory has doors for access control.<br />
<br>b. A sink is provided in the Laboratory for washing hands.<br />
<br>c. The Laboratory is designed such that it can be easily cleaned.<br />
<br>d. Bench tops are impervious to water and are resistant to moderate heat, organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.<br />
<br>e. Laboratory furniture is capable of supporting anticipated loadings and uses. Spaces between benches, cabinets, and equipments are accessible for cleaning.<br />
<br>f. It is made sure that the Laboratory is cut-off from the external environment.<br />
<br />
<br />
Would any of your project ideas raise safety issues in terms of:<br />
<br> researcher safety,<br />
<br> public safety, or<br />
<br> environmental safety?<br />
<br />
<br><br>Researcher Safety<br />
<br>Working in a laboratory environment is not without its risks, so we took a variety of steps to ensure the safety of our team during our lab work. We worked in a laboratory environment certified for Biosafety Level 1 work.<br />
Advanced synthetic biology research does at times require interaction with potentially dangerous substances. Listed below are those substances which posed the most significant hazards to researcher safety. Again, however, it should be noted that our use of these substances was consistent with Harvard's stringent safety standards. These substances included:<br />
<br> Ethidium Bromide (EtBr) was used to stain DNA for gel electrophoresis. Although toxic and a suspected mutagen, the harmful effects of ethidium bromide can be avoided by avoiding direct skin contact and inhalation, which can in turn be avoided by proper observance of safety precautions. In order to minimize primary contact, ethidium bromide is directly contacted only with micropipette tips. Skin contact when handling ethidium bromide-stained gels was avoided by the use of nitrile gloves that were promptly discarded for a fresh pair when switching from the task of pouring gels to other lab work. Secondary contact was avoided by the disposal of ethidium bromide-contaminated gloves and micropipette tips, as well as the designation of a specific bench and set of micropipettes exclusively for use with ethidium bromide.<br />
<br />
<br><br>• Ultraviolet (UV) light was used in visualizing stained DNA in gel electrophoresis. To avoid exposure to UV radiation when reading gels, protective, UV-blocking shields were used at all times. When performing gel extractions, exposure was avoided by wearing protective clothing to cover any exposed skin and safety glasses with UV-protection lenses.<br />
<br><br>• The laboratory strains of E. coli, Methylococcus capsulatus used were non-pathogenic and therefore not a threat to researcher safety. We have conferred various basic antibiotic resistances (including kanamycin resistance, all common features of synthetic biology lab work) to our strains. However, these strains are unlikely to survive in the wild, or in humans specifically—where they would be outcompeted by naturally-occuring bacteria—and thus this resistance does not present a significant problem for researcher safety. In addition, all used materials that contacted the bacteria were decontaminated with 10% bleach, ideally for a 20 minute minimum contact time, before disposal in designated biohazard receptacles.<br />
<br><br>Public Safety<br />
<br><br>• All work with live E.coli , Methylococcus capsulatus cells is carried out in the laminar hood that eliminates the possibility of any GMO(Genetically Modified Organisms) from being released out.<br />
<br>• If toxic, flammable, or chemically reactive substances used in our experiments are released, they may post threat to the general public. These include physical hazards or health hazards. <br />
<br>• It is made sure that appropriate measures for waste disposal are taken. Waste is not washed down the sink or thrown into public dust-bins.<br />
<br />
<br><br>Environmental Safety<br />
<br>Our project poses no identifiable threat to environmental safety. The microbes used in our project are not able to survive outside the lab, and all cells were disposed of safely after disinfection with 10% bleach. Bio-hazardous and flammable chemicals were disposed of by following the proper regulations. No gloves were allowed to leave the laboratory, so chemical and biological hazards were restricted to the laboratory.<br />
<br />
<br><br>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,<br />
<br> Did you document these issues in the Registry?<br />
<br> How did you manage to handle the safety issue?<br />
<br> How could other teams learn from your experience?<br />
<br />
<br>No. All bio-bricks made are according to the safety guidelines provided by the Center for Disease Control and Prevention.<br />
<br />
<br><br>Is there a local biosafety group, committee, or review board at your institution?<br />
<br> If yes, what does your local biosafety group think about your project?<br />
<br> If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
<br />
<br><br>Yes, our project safety is governed by the Committee on Bio Safety, Department of Biotech/Bioinformatics, JUIT. We have presented our project proposal to the same committee as mentioned above , and after a review of our materials and procedures, the biosafety office has approved our project and deemed our practices consistent with biosafety regulations.<br />
<br />
<br />
<br />
<br><br>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
<br />
<br><br>Antibiotic resistance is a public health problem of increasing magnitude, and finding effective solutions to address this problem is a critical focus of CDC activities.<br />
<br>1. Decreasing the susceptibility of recombinant cells to antibiotics through Antibiotic resistance genes proves to be a high risk to the environment.<br />
<br>2. Our new idea is to use Sac B gene insertion as a tool for selecting transformed mutants. We have designed a new plasmid, that does not contain antibiotic resistant gene. Instead it has Sac B whose expression in the presence of sucrose is lethal to Methylococcus capsulatus. Our results imply that the sacB gene can be used as a positive selection system in Methylococcus capsulatus.<br />
<br />
<br>Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.<br />
The link national biosafety regulations in India [http://dbtbiosafety.nic.in/].<br />
<br />
<br />
<br />
</html><br />
<br />
<br />
<br />
<br />
<!--- The Mission,</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ReferencesTeam:JUIT-India/References2012-09-26T09:57:08Z<p>Antresh kumar: </p>
<hr />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/55/Idea.jpg" width="965" height="300"></div><br />
<br />
<hr><br />
<h1>BRAINSTOTMING IDEAS </h1><br />
<hr><br />
<h2><br />
<ol><br />
<li>Magnetic Microbes</li><br />
<li>Microbes that can conduct electricity</li><br />
<li>Microbes with high metabolism that can help degrade metal at high altitudes like Siachen Lake</li><br />
<li>Microbes that can help plant survive in air without soil</li><br />
<li>Dyes that can stop greying of hair</li><br />
<br />
<br />
<br />
<br />
</html><br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/WetLabWorkTeam:JUIT-India/WetLabWork2012-09-26T09:56:41Z<p>Antresh kumar: </p>
<hr />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg" width="965" height="300"></div><br />
<br />
<r><br />
<hr><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
Diary: <br />
<br />
== ''' June ''' ==<br />
<br />
• Brainstorming various ideas<br />
<br><br />
• Decide the project to work on for this year<br />
<br />
<br />
==July:==<br />
<br>• Week 1<br />
<br>o Research on the project thoroughly<br />
<br>o Primers Designing<br />
• Week 2<br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of MxaF <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of MxaF<br />
<br>Agarose Gel Electrophoresis<br />
Result: Non-Specific Binding<br><br />
Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
Agarose Gel Electrophoresis<br><br />
Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br />
<br>• Week 3 <br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of NifA <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Results: NO band was observed (due to less duration in the -200c freezer<br />
<br> Wednesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NifA<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br>• Week 4<br />
<br>o Culturing of B.subtilis<br />
<br>o Isolation of genomic DNA from B.subtilis<br />
<br>o PCR reaction for amplification of SacB<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured B.subtilis<br />
<br> Tuesday : Isolated Genomic DNA from B.subtilis<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of SacB<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Non-Specific Binding<br />
<br> Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
==August: ==<br />
<br>• Week 1<br />
<br>o Culturing of P. aeruginosa<br />
<br>o Isolation of genomic DNA from P. aeruginosa<br />
<br>o PCR Reaction for amplification of NosZ<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured P. aeruginosa<br />
<br> Tuesday : Isolated Genomic DNA from P. aeruginosa<br />
Agarose Gel Electrophoresis<br />
Results: NO band was observed <br />
<br> Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method<br />
Agarose Gel Electrophoresis<br />
Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NosZ<br />
<br>Agarose Gel Electrophoresis<br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
Agarose Gel Electrophoresis<br />
Result: No bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
<br />
<br>• Week 2<br />
<br>o PCR reactions for amplification of MxaF & NifA<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture<br />
<br> Monday : PCR reaction performed for MxaF & NifA<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of MxaF into TA Vector<br />
<br> Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: Failed(Contamination)<br />
<br> Thursday : Ligation of MxaF into TA Vector <br />
<br> Friday: Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
<br> Saturday: Ligation of NifA into TA Vector <br />
<br> Sunday: : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
o <br />
<br />
<br>• Week 3<br />
<br>o PCR reactions for amplification of NosZ & SacB<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture\<br />
<br> Monday : PCR reaction performed for NosZ & SacB<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of NosZ into TA Vector<br><br />
Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured <br />
Thursday : Ligation of SacB into TA Vector<br> <br />
Friday: Transformation using MgCl2 & CaCl2 method<br><br />
Result: No colonies were observed<br><br />
Saturday: Ligation of SacB into TA Vector <br><br />
Sunday: : Transformation using MgCl2 & CaCl2 method<br><br />
Result: White Colonies were selected and cultured<br><br />
<br />
• Week 4<br><br />
o Culture transformed Cells<br><br />
o Plasmid Isolation<br><br />
o PCR reactions to confirm the insertion of genes<br><br />
o Agarose Gel Electrophoresis<br><br />
Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells<br><br />
Tuesday : Plasmid Isolation for MxaF & NifA transformed cells<br />
Restriction Digestion of the plasmids<br><br />
Wednesday : PCR reaction performed for amplification of MxaF & NifA<br />
Agarose Gel Electrophoresis. <br><br />
Results: Sharp Bands were observed.<br><br />
Thursday : Plasmid Isolation for NosZ & SacB transformed cells<br />
Restriction Digestion of the plasmids.<br><br />
Agarose Gel Electrophoresis<br><br />
Friday: PCR reaction performed for amplification of MxaF & NifA <br><br />
Saturday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
==September: ==<br />
• Week 1<br><br />
o Restriction Digestion of MxaF containing plasmids<br><br />
o Ligation of MxaF into psb1c3<br><br />
o Restriction Digestion of NosZcontaining plasmids<br><br />
o Ligation of NosZ into psb1c3 containg MxaF<br><br />
Monday : Restriction Digestion of MxaF containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 using EcoR1<br />
Ligation of MxaF into psb1c3<br><br />
Thursday : Restriction Digestion of NosZ containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br> <br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of NosZ into psb1c3 containing MxaF<br><br />
<br />
• <b>Week 2:</b><br><br />
o Restriction Digestion of NifA<br><br />
o Ligation of NifA into psb1c3 containg NifA & SacB<br><br />
o Restriction Digestion of SacB<br><br />
o Ligation of SacB into psb1c3 containg all the other genes<br><br />
Monday : Restriction Digestion of NifA containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1<br />
Ligation of NifA into psb1c3<br><br />
Thursday : Restriction Digestion of SacB containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br><br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of SacB into psb1c3 containing all the other genes.<br><br />
<br />
• <b>Week 3:</b><br><br />
o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes<br><br />
o Growth of transformed cells on selective media.<br><br />
o Plasmid Isolation <br><br />
o PCR reaction to confirm the insertion of genes<br><br />
Monday: Cultures M.Capsulatus Cells<br><br />
Tuesday : Preparation of M.Capsulatus Competent Cells<br><br />
Wednesday: Transformation of competent cells using the prepared plasmids<br><br />
Thursday : Growth of plates on LB plates containg chloramphenicol<br><br />
Friday: Cultured transformed cells in LB agar<br><br />
Saturday: Plasmid Isolation <br><br />
Restriction Digestion<br><br />
Sunday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
<br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/NotebookTeam:JUIT-India/Notebook2012-09-26T09:55:37Z<p>Antresh kumar: </p>
<hr />
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<div z-index:100><img src="https://static.igem.org/mediawiki/2012/2/20/Lg.jpg" width="965" height="100"></div><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg" width="965" height="300"></div><br />
<br />
<r><br />
<hr><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
Diary: <br />
<br />
== ''' June ''' ==<br />
<br />
• Brainstorming various ideas<br />
<br><br />
• Decide the project to work on for this year<br />
<br />
<br />
==July:==<br />
<br>• Week 1<br />
<br>o Research on the project thoroughly<br />
<br>o Primers Designing<br />
• Week 2<br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of MxaF <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of MxaF<br />
<br>Agarose Gel Electrophoresis<br />
Result: Non-Specific Binding<br><br />
Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
Agarose Gel Electrophoresis<br><br />
Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br />
<br>• Week 3 <br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of NifA <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Results: NO band was observed (due to less duration in the -200c freezer<br />
<br> Wednesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NifA<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br>• Week 4<br />
<br>o Culturing of B.subtilis<br />
<br>o Isolation of genomic DNA from B.subtilis<br />
<br>o PCR reaction for amplification of SacB<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured B.subtilis<br />
<br> Tuesday : Isolated Genomic DNA from B.subtilis<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of SacB<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Non-Specific Binding<br />
<br> Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
==August: ==<br />
<br>• Week 1<br />
<br>o Culturing of P. aeruginosa<br />
<br>o Isolation of genomic DNA from P. aeruginosa<br />
<br>o PCR Reaction for amplification of NosZ<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured P. aeruginosa<br />
<br> Tuesday : Isolated Genomic DNA from P. aeruginosa<br />
Agarose Gel Electrophoresis<br />
Results: NO band was observed <br />
<br> Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method<br />
Agarose Gel Electrophoresis<br />
Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NosZ<br />
<br>Agarose Gel Electrophoresis<br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
Agarose Gel Electrophoresis<br />
Result: No bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
<br />
<br>• Week 2<br />
<br>o PCR reactions for amplification of MxaF & NifA<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture<br />
<br> Monday : PCR reaction performed for MxaF & NifA<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of MxaF into TA Vector<br />
<br> Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: Failed(Contamination)<br />
<br> Thursday : Ligation of MxaF into TA Vector <br />
<br> Friday: Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
<br> Saturday: Ligation of NifA into TA Vector <br />
<br> Sunday: : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
o <br />
<br />
<br>• Week 3<br />
<br>o PCR reactions for amplification of NosZ & SacB<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture\<br />
<br> Monday : PCR reaction performed for NosZ & SacB<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of NosZ into TA Vector<br><br />
Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured <br />
Thursday : Ligation of SacB into TA Vector<br> <br />
Friday: Transformation using MgCl2 & CaCl2 method<br><br />
Result: No colonies were observed<br><br />
Saturday: Ligation of SacB into TA Vector <br><br />
Sunday: : Transformation using MgCl2 & CaCl2 method<br><br />
Result: White Colonies were selected and cultured<br><br />
<br />
• Week 4<br><br />
o Culture transformed Cells<br><br />
o Plasmid Isolation<br><br />
o PCR reactions to confirm the insertion of genes<br><br />
o Agarose Gel Electrophoresis<br><br />
Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells<br><br />
Tuesday : Plasmid Isolation for MxaF & NifA transformed cells<br />
Restriction Digestion of the plasmids<br><br />
Wednesday : PCR reaction performed for amplification of MxaF & NifA<br />
Agarose Gel Electrophoresis. <br><br />
Results: Sharp Bands were observed.<br><br />
Thursday : Plasmid Isolation for NosZ & SacB transformed cells<br />
Restriction Digestion of the plasmids.<br><br />
Agarose Gel Electrophoresis<br><br />
Friday: PCR reaction performed for amplification of MxaF & NifA <br><br />
Saturday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
==September: ==<br />
• Week 1<br><br />
o Restriction Digestion of MxaF containing plasmids<br><br />
o Ligation of MxaF into psb1c3<br><br />
o Restriction Digestion of NosZcontaining plasmids<br><br />
o Ligation of NosZ into psb1c3 containg MxaF<br><br />
Monday : Restriction Digestion of MxaF containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 using EcoR1<br />
Ligation of MxaF into psb1c3<br><br />
Thursday : Restriction Digestion of NosZ containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br> <br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of NosZ into psb1c3 containing MxaF<br><br />
<br />
• <b>Week 2:</b><br><br />
o Restriction Digestion of NifA<br><br />
o Ligation of NifA into psb1c3 containg NifA & SacB<br><br />
o Restriction Digestion of SacB<br><br />
o Ligation of SacB into psb1c3 containg all the other genes<br><br />
Monday : Restriction Digestion of NifA containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1<br />
Ligation of NifA into psb1c3<br><br />
Thursday : Restriction Digestion of SacB containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br><br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of SacB into psb1c3 containing all the other genes.<br><br />
<br />
• <b>Week 3:</b><br><br />
o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes<br><br />
o Growth of transformed cells on selective media.<br><br />
o Plasmid Isolation <br><br />
o PCR reaction to confirm the insertion of genes<br><br />
Monday: Cultures M.Capsulatus Cells<br><br />
Tuesday : Preparation of M.Capsulatus Competent Cells<br><br />
Wednesday: Transformation of competent cells using the prepared plasmids<br><br />
Thursday : Growth of plates on LB plates containg chloramphenicol<br><br />
Friday: Cultured transformed cells in LB agar<br><br />
Saturday: Plasmid Isolation <br><br />
Restriction Digestion<br><br />
Sunday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
<br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
<html><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Juit_logo.jpeg" ><br />
</center><br />
</html><br />
<br />
<br />
{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/PartsTeam:JUIT-India/Parts2012-09-26T09:54:46Z<p>Antresh kumar: </p>
<hr />
<div><html><br />
<style> <br />
#contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {<br />
display:none;}<br />
#top-section {<br />
border: 0px;<br />
height: 0px;}<br />
#content {<br />
border: none;}<br />
/* Removes "teams" from the menubar */<br />
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display: none;}<br />
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/ProtocolTeam:JUIT-India/Protocol2012-09-26T09:54:25Z<p>Antresh kumar: </p>
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<h1>Protocols :-</h1><br />
<h2>Objective:</h2><br />
<hr><br />
To perform restriction digestion of DNA with EcoR I and BamHI enzymes.<br />
<br><br />
Principle:<br />
<br>Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of DNA, found in bacteria. As they cut within the molecule, they are commonly called restriction endonucleases. They specifically cleave the nucleic acids at specific nucleotide sequence called Restriction sites to generate a set of smaller fragments .<br />
Restriction enzymes form part of the restriction-modification system of bacterial cells that provides protection against invasion of the cell by foreign DNA – especially bacteriophage DNA. But the cells own DNA is not cleaved by these Restriction enzymes. This self protection is achieved by the help of the specific DNA methyltransferase enzyme which will methylates the specific DNA sequence for its respective restriction enzymes by transferring methyl groups to adenine or cytosine residues to produce N6-methyladenine or 5-methylcytosine. An interesting feature of restriction endonuclease is that they commonly recognize recognition sequences that are mostly palindromes - they shows the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom strand) sequences. In other words, they are nucleotide sequences or complimentary strands that read the same in opposite direction.<br />
<hr><br />
Materials Required<br />
<ul> <br />
<li> Microcentrifuge tubes</li><br />
<li> Vial stand</li><br />
<li> 10µl pipette</li><br />
<li> Pipette tips</li><br />
<li> Beaker</li><br />
<li> Table top mini centrifuge</li><br />
<li> Incubator</li><br />
<li> Reagents</li><br />
</ul><br />
<hr><br />
Procedure<br />
<ol><br />
<li> Transfer the following solutions in a micro centrifuge tube.</li><br />
<br />
<br />
<li>Incubate the mixture at 37 o C for 1 h to overnight. Keep the tubes in -4o C freezer or in -20o C freezer, after the incubation.</li><br />
</ol><br />
<hr><br />
Precaution<br />
<ul><br />
<li>Make sure that the restriction enzyme does not exceed more than 10% of the total reaction volume, Otherwise the glycerol and the EDTA in the enzyme storage buffer may inhibit digestion process.</li><br />
</ul><br />
<br><br />
Differences Encountered in Real Laboratory<br />
<ul><br />
<li> After performing the experiment, confirm the Digestion of DNA by running a small amount of it in agarose gel with an undigested standard DNA. </li><br />
<li>Some restriction enzymes require BSA. In such cases make sure that, it is added to the reaction mixture. Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction.</li><br />
<li>Before performing the experiment, check whether the restriction enzymes have star activity or not. <br />
</li><br />
<br><br />
<h4>Objectives</h4><br />
<hr><br />
To understand the basic procedures involved in the isolation process of DNA from various sources such as blood, tissue, bacteria etc.<br />
<br />
Requirements for Plasmid Isolation:<br />
<ul><br />
<li>Micro centrifuge.</li><br />
<li> Water bath (37°C).</li><br />
<li> Automatic micropipettes with tips.</li><br />
<li>95-100% isopropanol Ice.</li><br />
</ul><br><br><br />
Buffers and Solutions:<br><br />
1. Alkaline lysis solution I.<br><br />
2.Alkaline lysis solution II.<br><br />
3.Alkaline lysis solution III.<br><br />
4.Antibiotic for plasmid selection.<br><br />
5.Ethanol.<br><br />
6.Phenol: chloroform (1:1, v/v).<br><br />
7.STE.<br><br />
8.TE (pH 8.0) containing 20 μg/ml RNAse A.<br><br />
<br>Media:<br />
<br> <br />
1.Rich medium.<br><br />
Procedure:<br />
<hr> <br />
<br>1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.<br />
<br>2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.<br />
<br>3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.<br />
<br>4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.<br />
<br>5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents well by inverting the tube . Do not vortex! Store the tube in ice.<br />
<br>6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube in ice for 3-5 minutes.<br />
<br>7. Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C in a microfuge. Collect the supernatant to a fresh tube.<br />
<br>8. (Optional) Add equal volume of phenol: chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.<br />
<br>9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.<br />
<br>10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.<br />
<br>11. Discard the supernatant by aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kim wipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.<br />
<br>12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.<br />
<br>13. Remove all of the supernatant by aspiration. Take care with this step, as the pellet sometimes does not adhere tightly to the tube.<br />
<br>14. Remove any beads of ethanol from the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).<br />
<br>15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds and store the DNA at -20°C.<br />
<br><br> <br />
Recipes for Buffers, Solutions and Media:<br />
<br>Alkaline Lysis Solution I :<br />
<br>50 mM glucose.<br />
<br>25 mM Tris-Cl (pH 8.0).<br />
<br>10 mM EDTA (pH 8.0).<br />
<br>Prepare Solution I from standard stocks in batches of approx. 100 ml, sterilize by autoclaving and store at 4°C.<br />
(For plasmid preparation.)<br />
<br>Alkaline Lysis Solution II:<br />
<br>0.2 N NaOH (freshly diluted from a 10 N stock).<br />
<br>1% (w/v) SDS.<br />
<br>Prepare Solution II fresh and use at room temperature.<br />
(For plasmid preparation.)<br />
<br>Alkaline Lysis Solution III:<br />
<br>5 M potassium acetate, 60.0 ml.<br />
<br>Glacial acetic acid, 11.5 ml.<br />
<br>H2O, 28.5 ml.<br />
<br><br>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.<br />
<br>(For plasmid preparation.)<br />
<br><br>EDTA:<br />
<br>To prepare 0.5 M EDTA (pH 8.0): Dissolve 186.1 g of disodium EDTA•2H2O in 800 ml of Distilled 2H2O. Stir well on a magnetic stirrer. EDTA will not dissolve into solution until the pH of the solution is reached to ~ 8.0 . So the pH should adjust to 8.0 with NaOH (~ 20 g of NaOH pellets) and make up the final volume to 1000ml with distilled water. Prepare the aliquots and sterilize by autoclaving.<br />
<br><br>Glycerol:<br />
<br>To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology grade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a pre rinsed 0.22-μm filter. Store in 200-ml aliquots at 4°C.<br />
<br><br>LB Media<br />
<br>Deionized H2O, to 950 ml.<br />
<br>Tryptone, 10 g.<br />
<br>Yeast extract, 5 g.<br />
<br><br>NaCl, 10 g.<br />
<br>To prepare LB (Luria-Bertani) medium, shake the ingredients , mentioned above with Distilled water until the solutes have dissolved. Adjust pH to 7.0 with 5 N NaOH and make up the final volume of the solution to 1 liter with deionized H2O. Then sterilize it for 20 minutes by autoclaving at 15 psi .<br />
<br><br>NaCl:<br />
<br>To prepare 5 M NaCl : Dissolve 292 g of NaCl in 800 ml of sterile H2O and the volume is make up to to 1 liter with deionized H2O. Prepare the aliquots and sterilize it by autoclaving.<br />
<br><br>NaOH:<br />
<br> To 800 ml of H2O, add 400g of NaOH pellets slowly, stirring continuously. After dissolving the pellets,completely, make up the final volume to 1 liter with sterile H2O. Store the solution at room temperature.<br />
<br><br>Potassium Acetate:<br />
<br> 5 M potassium acetate, 60 ml.<br />
<br>Glacial acetic acid, 11.5 ml.<br />
<br>H2O, 28.5 ml.<br />
<br>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at room temperature<br />
<br><br>SDS:<br />
<br>Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of SDS in 900 ml of H2O. Heat to a temperature of 68°C and stir with a magnetic stirrer to help dissolution. Adjust the volume to 1 liter with distilled H2O. Store at room temperature. Autoclaving not necessary.<br />
<br><br>STE:<br />
<br>10 mM Tris-Cl (pH 8.0).<br />
<br>0.1 M NaCl.<br />
<br>1 mM EDTA (pH 8.0).<br />
<br>Sterilize the solution by autoclaving and store at 4°C.<br />
<br><br>TE:<br />
<br>100 mM Tris-Cl (desired pH).<br />
<br>10 mM EDTA (pH 8.0).<br />
<br>(10x Tris EDTA) Sterilize the buffer by autoclaving and store at room temperature.<br />
<br><br>Tris-Cl:<br />
<br>Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH by adding concentrated HCl , to the desired value.The volume of the solution is make up to 1 liter with distilled H2O. Prepare the aliquots and sterilize by autoclaving. <br />
<br>Procedure for Operating the Virtual Lab:<br />
<br>Check whether you have done all the steps listed below:<br />
<br>• Prepare the culture containing the desired plasmid.<br />
<br>• Incubate the culture for 24 hours at 37°C.<br />
<br>• Take the culture from the incubator.<br />
<br>• Transfer 1.5ml of the culture to a microfuge tube.<br />
<br>• Centrifuge the tube for 30seconds at maximum speed (4°C).<br />
<br>• Remove the supernatant.<br />
<br>• Add 100μl alkaline lysis solution I.<br />
<br>• Vortex the sample.<br />
<br>• Add 200μl of alkaline lysis solution II.<br />
<br>• Mix the sample by inverting the tube.<br />
<br>• Store in ice for 1 minute.<br />
<br>• Add alkaline lysis solution III.<br />
<br>• Mix the contents by inverting the tube.<br />
<br>• Store in ice for 3-5 minutes.<br />
<br>• Centrifuge the solution at maximum speed (4°C) for 5 minutes .<br />
<br>• Collect the supernatant to a fresh tube.<br />
<br>• Precipitate the nucleic acid by adding 2 volumes of ethanol.<br />
<br>• Mix by vortexing.<br />
<br>• Stand the tubes for 2 minutes.<br />
<br>• Centrifuge for 5 minutes.<br />
<br>• Collect the precipitated DNA.<br />
<br>• Discard the supernatant by aspiration.<br />
<br>• Stand the tube as inverted to drain the fluid away.<br />
<br>• Add 1ml 70% ethanol.<br />
<br>• Mix by inverting.<br />
<br>• Centrifuge the mixture for 2 minutes.<br />
<br>• Discard the supernatant by aspiration.<br />
<br>• Allow to dry for 3-5 minutes.<br />
<br>• Add TE buffer with RNAse.<br />
<br>• Mix by flickering.<br />
<br>• Detect the plasmid by doing agarose gel electrophoresis.<br><br />
<br><hr><br />
Objective<br />
<br> To separate the DNA fragments based on their Molecular weight.<br />
<br>Materials Required:<br />
<br> Buffers and Solutions:<br />
<br> Agarose solutions.<br />
<br>Ethidium bromide.<br />
<br>Electrophoresis buffer.<br />
<br> Nucleic Acids and Oligonucleotides:<br />
<br> DNA samples.<br />
<br>DNA Ladders.<br />
<br> (Samples of DNAs of known size are typically generated by restriction enzyme digestion of a plasmid or bacteriophage DNA of known sequence).<br />
<br><br>The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:<br />
<br>• An electrophoresis chamber and power supply.<br />
<br>• Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.<br />
<br>• Sample combs, around which molten agarose is poured to form sample wells in the gel.<br />
<br>• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).<br />
<br>• Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.<br />
<br>• Ethidium bromide, a fluorescent dye used for staining nucleic acids.<br />
<br>• Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.<br />
<br> NOTE: Always wear protective eyewear when observing DNA on a Transilluminator to prevent damage to the eyes from UV light.<br />
<br>1. Prepare a 50x stock solution of TAE buffer in 1000m of distilled H2O:<br />
<br><br>For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.<br />
<br>Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml distilled water. EDTA is insoluble and it can be made soluble by adding sodium hydroxide pellets. Check the pH using pH meter. Make the solution 100ml by adding distilled water.<br />
<br>Pipette out 57.1 ml of glacial acetic acid.<br />
<br>Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the volume to 1000ml<br />
<br>2. Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the electrophoresis tank and to cast the gel:<br />
<br>For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA<br />
<br>It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the gel preparation.<br />
<br>3. Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:<br />
<br>For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.<br />
<br><br>Agarose Concentration in Gel (% [w/v]) Range of Separation of Linear DNA Molecules (kb)<br />
<br>0.3 5-60<br />
<br>0.6 1-20<br />
<br>0.7 0.8-10<br />
<br>0.9 0.5-7<br />
<br>1.2 0.4-6<br />
<br>1.5 0-2-3<br />
<br>2.0 0.1-2<br />
<br>4. Loosely plug the neck of the Erlenmeyer flask. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves. The agarose solution can boil over very easily so keep checking it. It is good to stop it after 45 seconds and give it a swirl. It can become superheated and NOT boil until you take it out whereupon it boils out all over you hands. So wear gloves and hold it at arm's length. You can use a Bunsen burner instead of a microwave <br>- just remember to keep watching it.<br />
<br>5. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling. <br />
<br>(For the preparation of ethidium bromide adds 1 g of ethidium bromide to 100 ml of H2O. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room temperature.)<br />
<br>6.While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel.<br />
<br>7.Pour the warm agarose solution into the mold.<br />
<br>(The gel should be between 3 - 5 mm thick. Check that no air bubbles are under or between the teeth of the comb.)<br />
<br>8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer. Mount the gel in the electrophoresis tank.<br />
<br>9.Add just enough electrophoresis buffers to cover the gel to a depth of approx. 1mm.<br />
<br>10. Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.<br />
<br>11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette or an automatic micropipettor or a drawn-out Pasteur pipette or a glass capillary tube. Load size standards into slots on both the right and left sides of the gel.<br />
<br>12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the electrodes are 10cm apart then run the gel at 50V. It is fine to run the gel slower than this but do not run it any faster. Above 5V/cm the agarose may heat up and begin to melt with disastrous effects on your gel's resolution. If the leads have been attached correctly, bubbles should be generated at the anode and cathode.<br />
<br>13. Run the gel until the bromophenol blue and xylenecyanol FF have migrated an appropriate distance through the gel.<br />
<br> (The presence of ethidium bromide allows the gel to be examined by UV illumination at any stage during electrophoresis).<br />
<br>14. The gel tray may be removed and placed directly on a transilluminator. When the UV is switched on we can see orange bands of DNA. <br />
<br><br>Procedure for operating the virtual lab:<br />
<br>Check whether you have done all the steps listed below:<br />
<br>• Prepare TAE buffer.<br />
<br>• Transfer 100ml of the buffer to a conical flask.<br />
<br>• Weigh 2grams of agarose and add to the 100ml buffer solution.<br />
<br>• Keep in oven.<br />
<br>• Take the solution from oven.<br />
<br>• Add ethidium bromide.<br />
<br>• Pour the solution to a gel caster.<br />
<br>• Place the comb.<br />
<br>• Pour the 100ml buffer solution to the electrophoretic chamber.<br />
<br>• Place the gel in the caster in the electrophoretic chamber.<br />
<br>• Connect the electrodes and switch on the current.<br />
<br>• Switch off the power supply.<br />
<br>• Remove the gel from the electrophoretic chamber.<br />
<br>• Place the gel in the UV Transilluminator.<br />
<br>• Switch on the Transilluminator.<br />
<br><br>CAUTION:<br />
<br>• Ethidium bromide is a mutagen and should be handled as a hazardous chemical (so wear gloves while handling)<br />
<br><br>DIFFERENCES ENCOUNTERED IN REAL LABORATORY:<br />
<br>1. Make sure that the Agarose is fully dissolved in the buffer. If it is not dissolved well, again melt it some more time to dissolve completely.<br />
<br>2. Before casting the gel, the tray and comb should wipe with ethanol.<br />
<br>3. Make sure that the gel in the Chamber is immersed in the TAE Buffer.<br />
<br>4. Labelings should be proper.<br />
<br>5. Ensure that the connections should be proper.<br />
<br>6. Before the incubation step, ensure that the water bath is set at the correct temperature that we required or not.<br><br />
<hr><br />
Objective: <br><br />
To familiarize with how cells are made competent which is the primary step for transformation.<br />
<br><br>Materials Required:-<br />
<br>• LB broth<br />
<br>• Culture plates<br />
<br>• Ice cold CaCl2.2H2O (1 M)<br />
<br>• Ice cold MgCl2 CaCl2 solution <br />
<br>• Shaking incubator<br />
<br>• Vortex mixer<br />
<br>• Centrifuge<br />
<br>• Water bath<br />
<br>• Inoculation loop<br />
<br>• Microfuge tubes<br />
<br>• Polypropylene tubes<br />
<br>• Micro pipettes and tips<br />
<br><br>Procedure:-<br />
<br>1. Pick a single bacterial colony from a culture plate which is incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml LB broth a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH 5 alpha contains approx. 109 bacteria/ml.<br />
<br>2. Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes. Cool the cultures to 0°C by storing them on ice for 10 minutes.<br />
<br>3. Recover the cells by centrifugation at 2700g at 4°C for 10 minutes .<br />
<br>4. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for<br> 1 minute to allow the last traces of media to drain away.<br />
<br>5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.<br />
<br>6. Recover the cells by centrifugation at 2700g at 4°C for 10 minutes .<br />
<br>7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.<br />
<br>8. Resuspend the pellet in 2 ml of ice-cold 0.1 M CaCl2 (or TFB) by gentle vortexing for each 50ml of original culture. Standard TFB may be used instead of CaCl2 for most strains of E. coli.<br />
<br>9. At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.<br />
<hr><br />
Objectives <br />
<br>1) To develop an understanding about the transformation.<br />
<br>2) Transform bacteria cells with a foreign DNA.<br />
<br>Materials Required<br />
<br>• LB broth<br />
<br>• LA- Amp plate<br />
<br>• Micropipette and tips<br />
<br>• Incubator<br />
<br>• Water bath<br />
<br>• Microfuge tube<br />
<br>• Calcium chloride treated competent cells<br />
<br>• L-rod<br />
<br>Reagents Required<br />
<br>• X Gal: Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. The X-gal tube should wrap with aluminium foil in order to prevent the damage caused by light and store at -20°C.<br />
<br>• IPTG: Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG with 8 ml of distilled H2O. Prepare the aliquots and store them at -20°C.<br />
<br>Procedure<br />
<br>1. To transform the CaCl2- treated cells directly, transfer 200 µl of each suspension of competent cells to a sterile, chilled polypropylene tube using a chilled micropipette tip.<br />
<br>2. Add DNA (<50ng in a volume of 10 µl) to each tube. Mix the contents of the tubes by gentle swirling. Keep the tubes in ice for 30 minutes. <br />
<br>3. Transfer the tubes to rack placed in a preheated 42 ºC circulating water bath. Keep the tubes in rack for 90 seconds.<br />
<br>4. Transfer the tubes to an ice bath immediately. Allow the cells to chill for 1-2 minutes.<br />
<br>5. Add 800 µl of LB medium to each tube. Incubate the cultures for 45 minutes in a water bath at 37 ºC .<br />
<br>6. After incubation, add 40 µl of X –Gal and 7 µl of IPTG to the LA-Amp plate.<br />
<br>7. Add appropriate volume of transformed competent cells into the plate.<br />
<br>8. Spread all the contents uniformly using an L-rod. Keep the plates at room temperature until the liquid has been absorbed.<br />
<br>9. Invert the plates and keep for incubation at 37 ºC. Transformed colonies will appear in 12-16 hours of incubation. <br />
<br>Differences Encountered in Real Laboratory<br />
<br>1. After taking the competent cells from the freezer, it should thaw at room temperature.<br />
<br>2. There should not be much time lag between adding and spreading of EDTA ,Beta-gal and Transformed cells. If so, the components will not spread uniformly in the plate.<br />
<br>3. Make sure that the samples are uniformly spread in to the plate(should care the spreading).<br />
<br>4. Make sure that the plates with transformed cells should be in inverted position while incubation.<br />
Objective<br />
<hr> <br />
To extract specific bands of DNA from agarose gels in which they are separated through electrophoresis.<br />
<br>Materials Required<br />
<br>• Elution buffer<br />
<br>• Scalpel blade<br />
<br>• UV transilluminator<br />
<br>• Agarose<br />
<br>• Micro pipettes<br />
<br>• Micro pipette tips<br />
<br>• Dry bath incubator<br />
<br>• Microfuge tubes<br />
<br>• Centrifuge<br />
<br>• N-Butanol<br />
<br>• Cryo box<br />
<br>• Cyclomixer<br />
<br>• 70% Ethanol<br />
<br>• 95% Ethanol<br />
<br>• TE buffer<br />
<br>• -20oC freezer<br />
<br>• -70oC freezer<br />
<br>Procedure<br />
<br>1. Visualize the low melting point agarose gel with DNA bands under a UV transilluminator and locate the desired DNA band to cut.<br />
<br>2. Carefully cut around the desired DNA band using a scalpel blade.<br />
<br>3. Transfer the gel piece into a microfuge tube.<br />
<br>4. Add elution buffer into the microfuge tube until the level of buffer is just above the level of gel slice.<br />
<br>5. Heat the gel slice at 65oC until it melts.<br />
<br>6. Freeze the melted gel with DNA by placing in a -70oC freezer for10minuts.<br />
<br>7. After freezing, centrifuge for 10minutes and transfer the supernatant into a new microfuge tube.<br />
<br>8. Again add half amount of elution buffer that you added in the previous step into the pellet.<br />
<br>9. Heat at 65oC until the agarose melts.<br />
<br>10. Freeze the melted gel with DNA by placing in a -70oC freezer for10minuts.<br />
<br>11. Centrifuge the tube again for 10 minutes and transfer (pool) the supernatant into the previous tube with supernatant.<br />
<br>12. Discard the tube with pellet.<br />
<br>13. Add an equal volume of n-Butanol to the supernatant and mix the contents well.<br />
<br>14. Vortex the tube for 15 minutes in order to remove the Ethidium bromide. <br />
<br>15. Discard the upper phase of butanol and repeat the process by adding n-butanol again for one or more times.<br />
<br>16. Add 2 times volume of 95% ethanol and mix thoroughly.<br />
<br>17. Keep for precipitation in -70oC freezer for 30minutes to overnight.<br />
<br>18. After precipitation, centrifuge for 15 minutes.<br />
<br>19. Discard the supernatant into a waste beaker and add 200µl of 70% ethanol to the pellet.<br />
<br>20. Centrifuge for 5minutes and discard the supernatant again.<br />
<br>21. Allow the pellets to dry well.<br />
<br>22. Suspend the pellets in 20µl of TE buffer. (If you want to confirm the recovered DNA, run (1µl) it on a gel.<br />
<br>23. The recovered DNA can be now used for further process of cloning otherwise can stored in -20oC freezer. <br />
<br>Objective:-<br />
<hr><br />
<br>To perform ligation reaction using T4 DNA ligase.<br />
<br>Materials Required:-<br />
<br>• Vials and Vial stand<br />
<br>• Micro pipette<br />
<br>• pipette tips<br />
<br>• Table top mini centrifuge<br />
<br>• Beaker<br />
<br>• Ice box<br />
<br>Reagents:-<br />
<br>Procedure:-<br />
<br>1. Take one clean fresh microfuge tube(Sample) from the rack.<br />
<br>2. In this microfuge tube(Vial), add 5µL water, 1µL Vector, 2.5µL insert and 1µL T4 DNA ligase buffer.<br />
<br>3. 0.5 µL of T4 DNA Ligase enzyme was added to the sample tube ( total reaction volume is 10 µl).<br />
<br>4. The vial is kept in the micro centrifuge and just spin for a few seconds.<br />
<br>5. Incubate the vial at room temperature (22˚C ) for 2 hours.<br />
<br>6. After 2 hours, the ligated mixture is taken for doing transformation.<br />
<br>1.REAL LAB VERSUS VIRTUAL LAB:<br />
<br />
<br>1. T4 DNA Ligase Buffer contains ATP, so repeated freeze thaw cycles can degrade ATP, thereby decreasing the efficiency of Ligation. <br />
<br>2. It is better to vortex or spin the T4 DNA ligase enzyme before pipetting to ensure that it is mixed well.<br />
<br>Objectives<br />
<hr>To amplify a given region of DNA(region of interest).<br />
<br>Procedure:<br />
<br>The protocol describes how to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase. It is the foundation for all subsequent variations of the polymerase chain reaction.<br />
<br>Materials<br />
<br>Buffers and Solutions<br />
<br>10x Amplification buffer<br />
<br>Chloroform<br />
<br>dNTP solution (20 mM) containing all four dNTPs (pH 8.0)<br />
<br>Enzymes and Buffers<br />
<br>Thermostable DNA polymerase<br />
<br>Nucleic Acids and Oligonucleotides<br />
<br>Forward primer (20 μM) in H2O<br />
<br>Reverse primer (20 μM) in H2O<br />
<br>Template DNA.<br />
<br>Dissolve template DNA in 10 mM Tris-Cl (pH 7.6) containing a low concentration of EDTA (<0.1 mM) at the following concentrations: mammalian genomic DNA, 100 μg/ml; yeast genomic DNA, 1 μg/ml; bacterial genomic DNA, 0.1 μg/ml; and plasmid DNA, 1-5 ng/ml.<br />
<br>Method<br />
<br>1. In a sterile 0.5-ml microfuge tube, mix in the following order:<br />
<br>REAGENTS AMOUNT(μl)<br />
<br>Deionized water 37.5 μl<br />
<br>Taq assay buffer(10x) 5 μl<br />
<br>Template DNA 1μl<br />
<br>dNTPs mix 2 μl<br />
<br>Forward primer 2 μl<br />
<br>Reverse primer 2 μl<br />
<br>Taq DNA polymerase 5 μl<br />
<br>The table below provides standard reaction conditions for PCR. Mg2+ (1.5 mM) ;KCl(50 mM) ;dNTPs (200 μM) ;Primers(1 μM );DNA polymerase (1-5 units); Template DNA(1 pg to 1 μg ).<br />
<br> The amount of template DNA required varies according to the complexity of its sequence. In the case of mammalian DNA, up to 1.0 μg is used per reaction. Typical amounts of yeast, bacterial, and plasmid DNAs used per reaction are 10 ng, <br>1 ng, and 10 pg, respectively.<br />
<br>2.If the thermal cycler is not fitted with a heated lid, overlay the reaction mixtures with 1 drop (approx. 50 μl) of light mineral oil. Alternatively, place a bead of wax into the tube if using a hot start protocol. Place the tubes or the micro titer plate in the thermal cycler.<br />
<br>3.Amplify the nucleic acids using the denaturation, annealing, and polymerization times and temperatures listed below.<br />
<br>4.Withdraw a sample (5-10 μl) from the test reaction mixture and the four control reactions, analyze them by electrophoresis through an agarose gel, and stain the gel with ethidium bromide or SYBR Gold to visualize the DNA.<br />
<br>A successful amplification reaction should yield a readily visible DNA fragment of the expected size. The identity of the band can be confirmed by DNA sequencing, Southern hybridization and/or restriction mapping. If all is well, lanes of the gel containing samples of the two positive controls (Tubes 1& 2) and the template DNA under test should contain a prominent band of DNA of the appropriate molecular weight. This band should be absent from the lanes containing samples of the negative controls (Tubes 3 & 4).<br />
<br>5. If mineral oil was used to overlay the reaction (Step 2), remove the oil from the sample by extraction with 150 μl of chloroform. The aqueous phase, which contains the amplified DNA, will form a micelle near the meniscus. The micelle can be transferred to a fresh tube with an automatic micropipette.<br />
<br>Do not attempt chloroform extractions in micro titer plates. The plastic used in these plates is not resistant to organic solvents.<br />
<br>Recipes<br />
<br>Amplification Buffer:<br />
<br>500 mM KCl.<br />
<br>100 mM Tris-Cl (pH 8.3 at room temperature).<br />
<br>15 mM MgCl2.<br />
<br>Autoclave the 10x buffer for 10 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Divide the sterile buffer into aliquots and store them at -20oC.<br />
<br>KCl<br />
<br>Dissolve an appropriate amount of solid KCl in H2O, autoclave for 20 minutes on liquid cycle and store at room temperature. Ideally, this 4 M solution should be divided into small (approx. 100 μl) aliquots in sterile tubes and each aliquot thereafter used one time.<br />
<br>Tris-Cl<br />
<br>Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl.<br />
<br>pH HCl<br />
<br>7.4 70 ml<br />
<br>7.6 60 ml<br />
<br>8.0 42 ml<br />
<br>(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 litre with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases approx. 0.03 pH units for each 1oC increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5oC, 25oC, and 37oC, respectively.<br />
<br>dNTP Solution<br />
<br>Dissolve each dNTP (deoxyribonucleoside triphosphates) in H2O at an approximate concentration of 100 mM. Use 0.05 M Tris base and a micropipette to adjust the pH of each of the solutions to 7.0 (use pH paper to check the pH). Dilute an aliquot of the neutralized dNTP appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each dNTP. Dilute the solutions with H2O to a final concentration of 50 mM dNTP. Store each separately at 70oC in small aliquots. For polymerase chain reactions (PCRs), adjust the dNTP solution to pH 8.0 with 2 N NaOH. Commercially available solutions of PCR-grade dNTPs require no adjustment.<br />
<br>Base wavelength(nm) Extinction Coefficient(E) (M-1cm-1)<br />
<br>A 259 1.54 x 104<br />
<br>G 253 1.37 x 104<br />
<br>C 271 9.10 x 103<br />
<br>T 267 9.60 x 103<br />
<br>For a cuvette with a path length of 1 cm, absorbance = EM. 100 mM stock solutions of each dNTP are commercially available .<br />
<br>Precautions<br />
<br>Chloroform<br />
<br>Chloroform CHCl3 is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile. Avoid breathing the vapours. Wear appropriate gloves and safety glasses. Always wear a chemical fume hood.<br />
<br>PREPARATION OF GENOMIC DNA FROM BACTERIA<br />
<br>Solutions required for this protocol<br />
<br>• TE buffer <br />
<br>• 10% (w/v) sodium dodecyl sulfate (SDS) <br />
<br>• 20 mg/ml proteinase K <br />
<br>• Phenol/chloroform <br />
<br>• Isopropanol <br />
<br>• 70% ethanol <br />
<br>• 3M sodium acetate ph 5.2 <br />
<br>• Phase Lock geltm (5 Prime, 3 Prime, Inc)<br />
<br>1. Transfer 1.5 ml to a micro centrifuge tube and spin 2 min. Decant the supernatant. Drain well onto a Kimwipe.<br />
<br>2. Resuspend the pellet in 467 µl TE buffer by repeated pipetting. Add 30 µl of 10% SDS and 3 µl of 20 mg/ml proteinase K, mix, and incubate 1 hr at 37°C.<br />
<br>3. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed. CAUTION: PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB COAT AND KEEP TUBES CAPPED TIGHTLY. Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin 2 min.<br />
<br>4. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a new Phase Lock GelTM tube and spin 2 min. Transfer the upper aqueous phase to a new tube.<br />
<br>5. Add 1/10 volume of sodium acetate.<br />
<br>6. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.<br />
<br>7. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end). <br />
Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec. <br />
Resuspend DNA in 100-200 µl TE buffer.<br />
<br>8. After DNA has dissolved, measure the concentration by diluting 10 µl of DNA into 1 ml of TE (1:100 dilution) and measure absorbance at 260 nm.<br><br />
Concentration of original DNA solution in µg/ml = Abs x 100 x 50 µg/ml.<br />
<br><br />
Protocol of Genomic DNA Isolation from a Gram-Negative Bacterium<br />
<br>The G NOME® kit (http://www.qbiogene.com) is used to quickly and efficiently isolate high molecular weight genomic DNA from Gram-negative bacterial cultures. Each preparation with the G NOME® kit yields up to 100 mg of genomic DNA.<br> The DNA isolated by the G NOME® procedure is suitable for restriction enzyme digestion or PCR amplification in as little as 1 hour after cell lysis.<br />
<br>Protocol<br />
<br>1. Bring cells*/tissues to a final volume of 1.85ml in Cell Suspension Solution. (Use a 15 ml clear plastic tube for efficient mixing). Mix until the solution appears homogeneous.<br />
<br>2. Add 50ml of RNase Mixx, mix thoroughly.<br />
<br>3. Add 100ml of Cell Lysis/Denaturing Solution**, mix well.<br />
<br>4. Incubate at 55°C for 15 minutes.<br />
<br>5. Add 25ml Protease Mixx, mix thoroughly. (Note: If precipitate is visible in Protease Mixx suspension, pulse spin and use 25 ml of supernatant.)<br />
<br>6. Incubate at 55°C for 30 to 120 minutes. (The longer time will result in minimal protein carry over and will also allow for substantial reduction in residual protease activity.)<br />
<br>7. Add 500ml “Salt-Out” Mixture, mix gently yet thoroughly. Divide sample into 1.5ml tubes. Refrigerate at 4°C for 10 minutes.<br />
<br>8. Spin for 10 minutes at maximum speed in a microcentrifuge (at least 10,000 x g). Carefully collect the supernatant, avoid the pellet. If a precipitate remains in the supernatant, spin again until it is clear. Pool the supernatants in a 15 ml (or larger) clear plastic tube.<br />
<br>9. To this supernatant, add 2 ml TE buffer and mix. Then add 8mls of 100% ethanol. If spooling the DNA, add the ethanol slowly and spool the DNA at the interphase with a clean glass rod. If centrifuging the DNA, add the ethanol and gently mix the solution by inverting the tube. Spin for 15 minutes at 1000-1500xg. Eliminate the excess ethanol by blotting or air drying the DNA.<br />
<br>10. Dissolve the genomic DNA in TE (10mM Tris pH 7.5, 1mM EDTA).<br />
<hr><br />
<br>Experiment 1 : Plasmid DNA Preparation<br />
<br>Objective: Isolation of plasmid DNA by Alkaline Lysis with SDS: Minipreparation<br />
<br>Principle: Bacterial plasmids are self replicating, circular extra chromosomal, DNA molecules. The most convenient method for preparing plasmid DNA is alkaline lysis method. In this procedure, cells are lysed by SDS at high pH and then neutralized. The plasmid DNA reanneals rapidly while most of the chromosomal DNA and bacterial proteins precipitate as a protein- DNA-SDS complex. <br />
<br>Materials Required:<br />
<br>Reagents and Buffers<br />
<br>Alkaline Lysis Solution I<br />
<br>50 mM glucose<br />
<br>25 mM Tris-Cl (pH 8.0)<br />
<br>10 mM EDTA (pH 8.0)<br />
<br>Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C.<br />
<br>Alkaline Lysis Solution II<br />
<br>0.2 N NaOH (freshly diluted from a 10 N stock)<br />
<br>1% (w/v) SDS<br />
<br>Prepare Solution II fresh and use at room temperature.<br />
<br>Alkaline Lysis Solution III<br />
<br>5 M potassium acetate, 60.0 ml<br />
<br>Glacial acetic acid, 11.5 ml<br />
<br>H2O, 28.5 ml<br />
<br>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.<br />
<br><br />
Alkaline Lysis Solution IV<br />
<br>Isopropanol<br />
<br>Step-wise Methodology: <br />
<br>1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.<br />
<br>2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.<br />
<br>3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.<br />
<br>4. Resuspend the bacterial pellet in 100 μl of ice-cold alkaline lysis solution I by vigorous vortexing.<br />
<br>5. Add 200 μl of freshly prepared alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice.<br />
<br>6. Add 150 μl of ice-cold alkaline lysis solution III. Close the tube and disperse alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.<br />
<br>7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube.<br><br />
8. (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.<br />
<br>9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.<br />
<br>10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.<br />
<br>11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.<br />
<br>12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.<br />
<br>13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube.<br />
<br>14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).<br />
<br>15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C.<br />
<br>Precautions:<br />
<br>• Dispose of used reagents according to local ordinances. <br />
<br>• Wear latex gloves and goggles during the procedure. <br />
<br>• The laboratory surfaces should be very clean during all procedures used in this activity. <br />
<br>• Use thoroughly clean instruments and glassware. Rinse all equipment with isopropyl alcohol or acetone. <br />
<br>• Ethanol is highly flammable; use caution.<br><br />
Observations: Plasmid DNA will migrate in 3 forms: supercoiled closed circular (superciled) DNA, single-strand nicked open circle DNA, and linear DNA. The exact mobilities of the 3 forms will vary based on conditions, but closed circular DNA will usually migrate faster than the other two because of its supercoiling. This means that if the DNA band farthest from your well on your uncut plasmid sample is the largest, you have prepared a plasmid DNA sample without excessively damaging your DNA. (Note that your gel may also contain a diffuse band of partially degraded RNA <br>– which will migrate faster than your DNA bands.)<br />
<br>Interpretations & Conclusions:<br />
<br>Experiment -2<br />
<br>Aim: Restriction of given plasmid or λ DNA with the restriction enzyme EcoRI and HindIIIand estimation of fragment size by comparison with 1KB ladder.<br><br />
Principle: Restriction enzymes are group of enzymes isolated from bacterial species which recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Each restriction enzyme recognizes specific sequences and cuts at specific sites Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA. Some Examples of restriction enzyme along with their, recognition sequence and cutting sites are given below.<br />
<br>Hind III A|AGCTT<br />
<br>Bam HI G|GATCC<br />
<br>Eco RI G|AATTC<br />
<br>PstI CTGCA|G<br />
<br>Eco RV GAT|ATC<br />
<br>Material required: Sterile water distilled water, 10X restriction enzyme buffer, plasmid DNA or λ DNA<br />
<br>Procedure:<br />
<br>1. Add the following in a 1.5ml centrifuge tube: 15 µl Sterile distilled water, 2.0 µl, 10X enzyme buffer, 1.0 µl Restriction enzyme, 2.0 µl Plasmid DNA\ λ DNA. <br />
<br>2. Maintain enzyme tube and plasmid DNA on ice. Do not leave on the table. <br />
<br>3. Mix by tapping the tube with your finger.<br />
<br>4. Briefly centrifuge to remove bubbles (DNA will adhere surface and becomes inaccessible to the enzymes).<br />
<br>5. Incubate at 37 C in a water bath for 45 minutes.<br />
<br>6. If the DNA is to be used for another manipulation, Heat inactivates the restriction enzyme by incubating the tubes at 65 C for 15 minutes.<br />
<br>7. Load the restriction enzymes cut and uncut plasmid on an agarose gel. Electrophorase the samples at 50-100 V for 1-2 hrs<br />
<br>8. Record the no. of fragments and their size in restriction enzyme digested samples.<br />
<br>Precautions:<br />
<br>1. Use fresh tip after each pipetting in order to avoid cross contamination of the chemicals and if you are using enzyme twice, change the pipet tip.<br />
<br>2. Enzymes should always be carried in a -20 mini cooler and work quickly. Do not expose the enzyme to warm temperature any longer than necessary.<br />
<br>3. Always wear gloves while handling enzymes.<br />
<br>Observations<br />
<br>The EcoR I digest of λ DNA yields the following 6 discrete fragments (in base pairs): 21226*, 7421, 5804, 5643, 4878, 3530* <br />
<br>The Hind III digest of λ DNA yields the following 8 discrete fragments (in base pairs): 23130*, 9416, 6557, 4361*, 2322, 2027, 564, 125.<br />
<br>Aim-To insert the PCR product into T vector by TA-cloning <br />
<br>Principle<br />
<br>"TA cloning" is a popular method of cloning without the use of restriction enzymes; instead, the fragment to be cloned, is amplified with only Taq DNA polymerase as PCR product. TA Cloning exploits the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. The PCR products with dA overhang are mixed with this vector in high proportion. The complementary overhangs of "T" vector and PCR product will be ligated under the action of T4 DNA ligase.<br />
<br>Advantages<br />
<br>1. Eliminate any enzymatic modification of PCR product<br />
<br>2. Does no require the use of PCR primer tthat contain restriction sites<br />
<br />
<br>RBC T&A cloning kit (cat.no. RC001)<br />
<br>RBC TA cloning system is ideal for rapid cloning PCR product generated using a thermos table DNA polymerase, which <br>adds a single 3’dA nucleotide overhang. Following the ligation the mixture may be used directly to transform competent cell or purified to achieve a higher efficiency of transformation.<br />
<br>Product components<br />
<br>1. T&A (25ng/μl) cloning vector : 40μl<br />
<br>2. Control Insert DNA (10ng/μl) : 10μl<br />
<br>3. T4 DNA ligase(3U/μl) : 20μl<br />
<br>4. T4 DNA ligase Buffer A : 100μl<br />
<br>5. T4 DNA ligase Buffer B : 100μl<br />
<br>6. forward primer(M13-F)(10μM)(50μl)<br />
<br>7. reverse primer(M13-R)(10μM)(50μl)<br />
<br>Procedure: <br />
<br>1. Centrifuge T&A cloning vector and/or PCR DNA tubes to collect contents at the bottom of the tubes.<br />
<br>2. Vortex the ligation buffer vigorously before use.<br />
<br>3. Set up the following items as described below:<br />
<br />
<br>3. Mix the reactions by pipetting.<br />
<br>4. Incubate the reactions for 5 to 15 min at 22oC. Alternatively, if the maximum of transformants is required, incubate the reactions overnight at 4oC.<br />
<br />
<br>Suggestions<br />
<br>1. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes by making single-use aliquots of Ligase Buffer.<br />
<br>2. Pfu DNA polymerase possesses proofreading activity; it does not have the terminal transferase-like activity demonstrated by Taq DNA Polymerase. Ligation reactions using non-tailed amplified DNA resulted in no positive colonies.<br />
<br>Aim: Transformation of E.coli. DH5 α cells with recombinant T- vector.<br />
<br>Principal <br><br />
Genetic transformation occurs when a host organism takes in foreign DNA and expresses the foreign gene. In this experiment, we will introduce recombinant T vector carrying genes for resistance to the antibiotic penicillin into competent cells of DH5 α which are otherwise sensitive to it. If the bacterial cells incorporate the foreign DNA, they will become penicillin resistant. The CaCl2 treated competent cell of E.coli and plasmid DNA is mixed and then briefly heated to 42C for a brief period of 45- 90 sec to give a heat shock to the cells. CaCI2treatment promotes binding of DNA to E. coli cell wall. It is taken up by the cell when temperature is raised to 42°C. <br />
MATERIALS and Equipments: LB medium (Liq.), 1.5 ml centrifuge tube, micro tips, centrifuge, Water bath, Recombinant vector, LB plates containing penicillin and X-gall<br />
<br>Procedure<br />
<br>1. Take the competent cells stored at -80C and thaw on ice.<br />
<br>2. Add 10µl of recombinant T vector to the 200µl of competent cells. Mix the content by swirly the tube gently. Store the tube on ice for 30 minutes<br />
<br>3. Transfer the tubes to a rack placed in a preheated 42C circulating water bathwater bath for 90 seconds<br />
<br>4. Rapidly transfer the tubes on ice. Allow the cells to chill for 1-2 minutes<br />
<br>5. Add 800µl of LB medium. Incubate the culture for 2-3 hours at 37C.Transfer 200µl of heat shock given cells on LB agar plate 100ug/µl ampicillin and X-gal <br />
<br>6. Incubate the plates at 37C in inverted position. Transformed colonies should appear in 12-16 hours.<br />
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<h1>ABSTRACT</h1><br />
<br />
<p>“Global warming is too serious for the world any longer to ignore its danger or split into opposing factions on it”, quoted Tony Blair back in 2005. Well how much concerning this appears, since then, a lot more similar quotes can be added in its reference. We have come together as a team to have a different insight in dealing with this problem through genetic engineering. Rice, which is the staple diet of India and many other countries around the world, is believed to engender many potential green house gases or global warming gases per se like carbon dioxide, methane and nitrous oxide. Nitrous oxide, again, is released due to the inevitable use of nitrogen fertilizers which are added in the paddy fields. We are dealing with the conversion of this highly potential global warming gas, nitrous oxide into nitrate form using synthetic biology tools to insert two genes into a bacterial cassette along with its detection systems. This nitrate, as we know, can in turn, be utilized by the plant itself, solving our purpose and adding a new dimension to this diversion and in turn being beneficial for the farmers reducing the compromise factor that would, otherwise, have been done. <br />
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== '''Overall project''' ==<br />
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Synthetic biology aims to design and construct new biological functions and system that are not found in nature. We have given the name ‘Captain Green’ to our organism M.capsulatus who plays ‘hero’ like figure in providing the greener and healthy environment.<br />
Global warming has become an alarming issue in 21st century and we aim to take action against it. The tremendous increase in methane, nitrous oxide and carbon dioxide emission has become a great concern. While rice has the third-highest worldwide production and a staple crop for nearly half the world's population with the worldwide consumption of ~367 million metric ton per year but anoxic conditions in the wetland soils of rice paddies are ideal for microbes that produce methane, which trails only carbon dioxide in terms of its greenhouse effect. Rice agriculture is a big source of atmospheric methane, possibly the biggest of man-made methane sources. With an increasing world population, reductions in rice agriculture remain largely untenable as on Methane emission reduction strategy. Methane emission from paddy field makes up 29% of the total of Methane and Nitrous Oxide emission from agricultural land makes up 55%. So, greenhouse gas emissions from rice paddy fields are considered as one of the most important emission sources. The average concentration of nitrous oxide in the atmosphere is now increasing at a rate of 0.2 to 0.3% per year. Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. <br />
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<br />
<!-- Team table starts here --> <br />
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<td colspan="5"><h1>Meet the team</h1></td><br />
</tr><br />
<tr><br />
<td colspan="2" rowspan="7" valign="top"><p>Hi! We are the JUIT-India iGEM Team, a collection of Biotehnology and Bioinformatics all focused on increase in yield of the rice and reducing greenhouse effect. </p><br />
<p>The team consists of seven Biotechnologist and One Bio-Informatics. The whole team have been inducted into the Department Of Biotechnology and Bio-Informatics at the Jaypee University This means everybody is cleared to work in the lab and all of us can contribute to the hands-on experiments.<br /><br />
</p><br />
<h1>Our Team</h1><br />
<p>Antresh Kumar - Faculty Incharge <br /><br />
Saurabh Bansal - Faculty Advisor <br /><br />
Eshita Varma <br /><br />
Mohak <br /><br />
Shilpa <br /><br />
Priya <br /><br />
Pranika <br /><br />
Shristi <br /><br />
Nidhi<br> <br />
Vasant </p><br />
<p>&nbsp;</p></td><br />
<td width="20%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/e/ec/Shrishti.jpg" width="110" height="122" /></td><br />
<td width="18%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/42/Pranika.JPG" width="109" height="121" /></td><br />
<td width="19%" align="center"><img src="https://static.igem.org/mediawiki/igem.org/6/66/Priya.JPG" width="84" height="120" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Shristi </h3></td><br />
<td align="center"><h3>Pranika </h3></td><br />
<td align="center"><h3>Priya </h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/0/07/Eshita.JPG" height="110"/></td><br />
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<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/2/22/Shilpa.JPG" height="110" /></td><br />
</tr><br />
<tr><br />
<td align="center"><h3>Eshita</h3></td><br />
<td align="center"><h3>Vasant </h3></td><br />
<td align="center"><h3>Shilpa</h3></td><br />
</tr><br />
<tr><br />
<td align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Mohak.JPG" height="110" /></td><br />
<br />
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<tr><br />
<td align="center"><h3>Mohak </h3></td><br />
<br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- Team table ends here --> <br />
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<!-- Where we're from table starts here --> <br />
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<table width="100%"><br />
<tr><br />
<td><h1>Where we're from</h1></td><br />
</tr><br />
<tr><br />
<td>We're from the Jaypee University of Information Technology located in India. The University was founded in 2002. The Department of Biotechnology and Bioinformatics ranks No. 1 department amongst the private Universities of India. </td><br />
</tr><br />
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</html></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-IndiaTeam:JUIT-India2012-09-26T09:52:44Z<p>Antresh kumar: </p>
<hr />
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== ''' Project Overview ''' ==<br />
<br />
Captain Green<br />
<br />
Synthetic biology aims to design and construct new biological functions and system that are not found in nature. We have given the name ‘Captain Green’ to our organism M.capsulatus who plays ‘hero’ like figure in providing the greener and healthy environment.<br />
<br />
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<br />
<br />
<br />
== Project Details ==<br />
<br />
Global warming has become an alarming issue in 21st century and we aim to take action against it. The tremendous increase in methane, nitrous oxide and carbon dioxide emission has become a great concern. While rice has the third-highest worldwide production and a staple crop for nearly half the world's population with the worldwide consumption of ~367 million metric ton per year but anoxic conditions in the wetland soils of rice paddies are ideal for microbes that produce methane, which trails only carbon dioxide in terms of its greenhouse effect. Rice agriculture is a big source of atmospheric methane, possibly the biggest of man-made methane sources. With an increasing world population, reductions in rice agriculture remain largely untenable as on Methane emission reduction strategy. Methane emission from paddy field makes up 29% of the total of Methane and Nitrous Oxide emission from agricultural land makes up 55%. So, greenhouse gas emissions from rice paddy fields are considered as one of the most important emission sources. The average concentration of nitrous oxide in the atmosphere is now increasing at a rate of 0.2 to 0.3% per year. Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. <br />
<br />
=== Abstract ===<br />
<br />
The summer heat of 2012 is enough proof towards the increasing global warming. Our project focuses on the reduction of emission of greenhouse gases from the rice paddy fields. We are focusing on reducing methane emission and as well as converting nitrous oxide into nitrate, thereby increasing the fertility of the soil. By genetically modifying Methylococcus capsulatus we plan on converting methane into carbon dioxide. Methane is proven to be a more dangerous greenhouse gas than carbon dioxide. At the same time we are also going to be focusing on the most dangerous global warming gas i.e. nitrous oxide. We plan on converting nitrous oxide into nitrate using genes from Pseudomonas and Methylococcus.<br />
<br />
=== Objective ===<br />
<br />
1. The reduction in the greenhouse gases being emitted from the rice paddy fields.<br />
<br><br />
2. To increase the fertility of the soil by increasing the nitrate content in the soil. <br />
<br />
=== Methodology ===<br />
<br />
Our system involves the conversion of methane to methanol, then using the methanol as an inducer for homologous of Aox promoter, the target genes are used to convert nitrous oxide to nitrate, which easily be taken up by the plant and increase the yield also.<br />
<br />
For Complete Details visit our Project Description Page<br />
<br />
== Expected Outcomes ==<br />
As expected, the above project will efficiently reduce the emission rate of greenhouse gases to a significant level (which is an immediate concern of the Global warming) in Paddy crop along with increasing the fertility of the soil and thus increasing the rice yield<br />
<br />
== Sponsors ==<br />
Our Title Sponsor :<br />
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<!--- The Mission,</div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/SponsorsTeam:JUIT-India/Sponsors2012-09-26T09:52:18Z<p>Antresh kumar: </p>
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{|align="justify"</div>Antresh kumarhttp://2012.igem.org/File:Juit_logo.pngFile:Juit logo.png2012-09-26T09:50:15Z<p>Antresh kumar: uploaded a new version of &quot;File:Juit logo.png&quot;: Reverted to version as of 09:48, 26 September 2012</p>
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<div></div>Antresh kumarhttp://2012.igem.org/Team:JUIT-India/NotebookTeam:JUIT-India/Notebook2012-09-26T09:38:15Z<p>Antresh kumar: </p>
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<div><html><br />
<style> <br />
#contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {<br />
display:none;}<br />
#top-section {<br />
border: 0px;<br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India'><span>Home</span></a></li><br />
<li><a href='#'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Team'><span> Team</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Sponsors'><span>Sponsors</span></a></li><br />
</ul><br />
</li><br />
<li><a href='#'><span>Project</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Project'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Protocol'><span>Protocol</span></a></li><br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Parts'><span>Parts</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Design'><span>Design</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Modeling'><span>Modeling</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Human_Practices'><span>Human Practices</span></a><br />
<ul><br />
</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Experiment'><span>Experimental Design</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/WetLabWork'><span>Diary</span></a></li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/References'><span>Brainstorming</span></a></li><br />
<br />
</ul><br />
</li><br />
<li><a href='https://2012.igem.org/Team:JUIT-India/Safety'><span>Safety</span></a></li><br />
<br />
</ul><br />
</div><br />
<div z-index:100><img src="https://static.igem.org/mediawiki/2012/5/5d/Diary.jpg" width="965" height="300"></div><br />
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Diary: <br />
<br />
== ''' June ''' ==<br />
<br />
• Brainstorming various ideas<br />
<br><br />
• Decide the project to work on for this year<br />
<br />
<br />
==July:==<br />
<br>• Week 1<br />
<br>o Research on the project thoroughly<br />
<br>o Primers Designing<br />
• Week 2<br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of MxaF <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of MxaF<br />
<br>Agarose Gel Electrophoresis<br />
Result: Non-Specific Binding<br><br />
Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br><br />
Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
Agarose Gel Electrophoresis<br><br />
Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br />
<br>• Week 3 <br />
<br>o Culturing of M.Capsulatus<br />
<br>o Isolation of Genomic DNA from M.Capsulatus<br />
<br>o PCR reaction for amplification of NifA <br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured M.Capsulatus<br />
<br> Tuesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Results: NO band was observed (due to less duration in the -200c freezer<br />
<br> Wednesday : Isolated Genomic DNA from M.Capsulatus<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NifA<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Sharp bands were observed<br />
<br>Gel Extraction.<br />
<br />
<br>• Week 4<br />
<br>o Culturing of B.subtilis<br />
<br>o Isolation of genomic DNA from B.subtilis<br />
<br>o PCR reaction for amplification of SacB<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured B.subtilis<br />
<br> Tuesday : Isolated Genomic DNA from B.subtilis<br />
<br>Agarose Gel Electrophoresis<br />
<br> Wednesday : PCR Reaction for amplification of SacB<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: Non-Specific Binding<br />
<br> Thursday : PCR reaction performed again<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: NO bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: light bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
<br>Result: bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
==August: ==<br />
<br>• Week 1<br />
<br>o Culturing of P. aeruginosa<br />
<br>o Isolation of genomic DNA from P. aeruginosa<br />
<br>o PCR Reaction for amplification of NosZ<br />
<br>o Agarose Gel Electrophoresis<br />
<br>o Optimization & Standardization of PCR<br />
<br> Monday : Cultured P. aeruginosa<br />
<br> Tuesday : Isolated Genomic DNA from P. aeruginosa<br />
Agarose Gel Electrophoresis<br />
Results: NO band was observed <br />
<br> Wednesday : Isolated Genomic DNA from P. aeruginosa using CTAB method<br />
Agarose Gel Electrophoresis<br />
Result: Sharp Bands were observed<br />
<br> Thursday : PCR reaction performed for amplification of NosZ<br />
<br>Agarose Gel Electrophoresis<br />
Result: NO bands were observed<br />
<br> Friday : PCR reaction performed<br />
Agarose Gel Electrophoresis<br />
Result: No bands were observed<br />
<br> Saturday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Nonspecific bands were observed<br />
<br> Sunday: PCR reaction performed<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br />
<br />
<br />
<br>• Week 2<br />
<br>o PCR reactions for amplification of MxaF & NifA<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture<br />
<br> Monday : PCR reaction performed for MxaF & NifA<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of MxaF into TA Vector<br />
<br> Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: Failed(Contamination)<br />
<br> Thursday : Ligation of MxaF into TA Vector <br />
<br> Friday: Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
<br> Saturday: Ligation of NifA into TA Vector <br />
<br> Sunday: : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured<br />
o <br />
<br />
<br>• Week 3<br />
<br>o PCR reactions for amplification of NosZ & SacB<br />
<br>o Gel Extraction for the genes<br />
<br>o Ligation of genes into TA Vector<br />
<br>o Transformation of E.Coli cells with TA Vector<br />
<br>o Culturing of Transformed Cells – Starter Culture\<br />
<br> Monday : PCR reaction performed for NosZ & SacB<br />
<br>Agarose Gel Electrophoresis<br />
Result: Sharp bands were observed<br />
<br>Gel Extraction was performed.<br />
<br> Tuesday : Ligation of NosZ into TA Vector<br><br />
Wednesday : Transformation using MgCl2 & CaCl2 method<br />
Result: White Colonies were selected and cultured <br />
Thursday : Ligation of SacB into TA Vector<br> <br />
Friday: Transformation using MgCl2 & CaCl2 method<br><br />
Result: No colonies were observed<br><br />
Saturday: Ligation of SacB into TA Vector <br><br />
Sunday: : Transformation using MgCl2 & CaCl2 method<br><br />
Result: White Colonies were selected and cultured<br><br />
<br />
• Week 4<br><br />
o Culture transformed Cells<br><br />
o Plasmid Isolation<br><br />
o PCR reactions to confirm the insertion of genes<br><br />
o Agarose Gel Electrophoresis<br><br />
Monday : Cultured MxaF, NifA, SacB & NosZ transformed Cells<br><br />
Tuesday : Plasmid Isolation for MxaF & NifA transformed cells<br />
Restriction Digestion of the plasmids<br><br />
Wednesday : PCR reaction performed for amplification of MxaF & NifA<br />
Agarose Gel Electrophoresis. <br><br />
Results: Sharp Bands were observed.<br><br />
Thursday : Plasmid Isolation for NosZ & SacB transformed cells<br />
Restriction Digestion of the plasmids.<br><br />
Agarose Gel Electrophoresis<br><br />
Friday: PCR reaction performed for amplification of MxaF & NifA <br><br />
Saturday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
==September: ==<br />
• Week 1<br><br />
o Restriction Digestion of MxaF containing plasmids<br><br />
o Ligation of MxaF into psb1c3<br><br />
o Restriction Digestion of NosZcontaining plasmids<br><br />
o Ligation of NosZ into psb1c3 containg MxaF<br><br />
Monday : Restriction Digestion of MxaF containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 using EcoR1<br />
Ligation of MxaF into psb1c3<br><br />
Thursday : Restriction Digestion of NosZ containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br> <br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of NosZ into psb1c3 containing MxaF<br><br />
<br />
• <b>Week 2:</b><br><br />
o Restriction Digestion of NifA<br><br />
o Ligation of NifA into psb1c3 containg NifA & SacB<br><br />
o Restriction Digestion of SacB<br><br />
o Ligation of SacB into psb1c3 containg all the other genes<br><br />
Monday : Restriction Digestion of NifA containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Gel Extraction was performed.<br><br />
Tuesday : Restriction Digestion of psb1c3 containing MxaF & NosZ using EcoR1<br />
Ligation of NifA into psb1c3<br><br />
Thursday : Restriction Digestion of SacB containing plasmids<br><br />
Agarose Gel Electrophoresis<br><br />
Result: Sharp bands were observed<br><br />
Friday: Transformation using MgCl2 & CaCl2 method <br><br />
Saturday: Restriction Digestion of psb1c3<br><br />
Ligation of SacB into psb1c3 containing all the other genes.<br><br />
<br />
• <b>Week 3:</b><br><br />
o Transformation of M.Capsulatus competent Cells using the plasmid containing all the genes<br><br />
o Growth of transformed cells on selective media.<br><br />
o Plasmid Isolation <br><br />
o PCR reaction to confirm the insertion of genes<br><br />
Monday: Cultures M.Capsulatus Cells<br><br />
Tuesday : Preparation of M.Capsulatus Competent Cells<br><br />
Wednesday: Transformation of competent cells using the prepared plasmids<br><br />
Thursday : Growth of plates on LB plates containg chloramphenicol<br><br />
Friday: Cultured transformed cells in LB agar<br><br />
Saturday: Plasmid Isolation <br><br />
Restriction Digestion<br><br />
Sunday: Agarose Gel Electrophoresis<br><br />
<br />
<br />
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== Sponsors ==<br />
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{|align="justify"</div>Antresh kumar