Team:UANL Mty-Mexico/team/glosarioJ

From 2014.igem.org

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Wet-Lab</center></div>
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Project</div></center>
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Protocols</center>
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Abstract</center>
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<p><b>Thermonator III: The crop guardian</b></p>
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<p> <b>General Rules:</b>
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<p align="justify">Our project is about a genetic engineered machine in <i>E. coli</i> with the ability to produce Vip3ca3 which acts as a pesticide protein. Vip3ca3 production will be regulated by specific temperatures in order to avoid overproduction and it will show activity against target organisms Coleoptera and Lepidoptera, which are related to a local problem concerning potato crops. The Vip3ca3 production is regulated with a constitutive promoter and a riboswitch that initiate translation around 32°C. Since we want to produce the Vip3ca3 below the 32°C, we use a set of promoter-repressors in order to invert the activation of the protein production. This model may be used as a regulator in future transgenic plant generations for the production of substances against plagues, avoiding pesticide overproduction, thus reducing the effect in Non-Target Organisms and bioaccumulation and can be used with different temperature ranges and/or different proteins to attack other target organisms.</p>
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<br>1. - Always use your lab coat
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<br>2. - Always pick up the used material
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<br>3. - Never take any material to your mouth while you’re working with reactants
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<br>4. - Keep your working area clean
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<br>5. - All non-reusable material must be placed in the correct trash can
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<br>6. - Check for oppen gas and water valves, depending on your work
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<br>7. - Wash your hands before and after every session
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<br>8. - Always wear gloves when working with Ethidium bromide
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<br>9. - Every question, accident, etc. tell the instructor</p>
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<p><b>Recommendations for Microbiology</b>
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<br>10. - Sterilize with heat any crop material before and after using it.
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<br>11. - Don’t talk while you are working with sterilized material
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<br>12. - When you start and finish, sterilize the surface with 70% alcohol
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<br>13. - Al testing tubes, must be vertical 
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<br>14. - Never deposit a living cultivation in the sink
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<br>15. - Sterilize every used material</p></td>
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<p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/d/da/Imagen2.jpg" width="202px" height="203px" /> </p>
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<img src="https://static.igem.org/mediawiki/2013hs/d/d3/D9b5b18226b32476e3e16622aae4da9e.jpg" height="300px" />
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<p> <b>Laboratory Protocol </b></p>
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<p><b> Handling of lyophilized DNA’s plates.</b> </p>
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<p> This step is for searching and obtaining the desired piece.
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<br>Procedure:
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<br>1.- Search for the BioBrick in the Parts Registry 
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<br>2.- Drill the plate, and add 10ul of mQ water
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<br>3.- Mix very well, and take the resusupended DNA to an Eppendorf tube </p>
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<p> <b>Preparation for competent cells and their transformation</b></p>
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<p>This is to prepare the plasmid in the bacterium with the thermal shock
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<br>Transformation Procedure:
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<br>1.- Take 100ul of the bacteria to an Eppendorf tube
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<br>2.- Add 2ul of DNA and mix them
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<br>3.- Let in ice from 20 to 30 minutes
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<br>4.- Dive the Eppendorfs with 42°C water for 1 minute
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<br>5.- Take it to ice for 2 minutes
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<br>6.- Add LB and incubate them for 20 min, then put them into petri dish at 37°C all the night. </p></td>
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<p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/2/2a/Imagen1.jpg" width="266px" height="265px" /> </p>
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<p><b> Inoculation in Petri Dish and Test Tube </b></p>
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<p>Inoculate bacteria in dishes with agar and medium tubes with their antibiotics
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<br>Procedure:
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<br><b>For planting</b>
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<br>1.- Take the colonies in a test tube with LB and the antibiotic
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<br>2.- Take the handle sterilized and spread the colonies in a Petri dish.
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<br>3.- Incubate for 37°C FOR 16 hours
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<br>Reseeding
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<br>1.- Add the antibiotic to a test tube previously half filled with Lb medium
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<br>2.- With a handle take a colony
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<br>3.- Then agitate the handle in the Tube, finally incubate at 37°C </p>
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<td><p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/8/85/Imagen4.jpg" width="224px" height="227px" /> </p></td></tr></table>
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<p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/b/b4/Imagen3.jpg" width="500px" height="200px" /> </p>
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<p><b>Plasmid DNA miniprep</p></b>
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<p>This is to obtain the DNA plasmid of a colony
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<br>Procedure:
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<br>1.- Add 1.5ml of colonies to an Eppendorf, centrifuge 30 seconds and throw the liquid  in the trash with soap.
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<br>2.- Add  200ul of solution I, II and III in times of 5 minutes respectively and then centrifuge the new solution.
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<br>3.- Pass the liquid to and Eppendorf containing 1ml of alcohol at 100%
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<br>4.- Incubate at -20°C for 10 minutes
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<br>5.- Centrifuge and throw the liquid.
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<br>6.- Add 200ul of Ethanol 70% and mix very well
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<br>7.- Centrifuge again and throw the liquid
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<br>8.- Let the precipitate incubate at 37°C, then add 200ul of mQ water with RNase and mix it
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<br>9.- Check in the gel or save it at 4°C </p></td>
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<td><p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/e/e6/Imagen_1.jpg" width="132px" height="285px" /> </p></td></tr></table>
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<p><b> DNA quantification by UV spectrophotometer</b> </p>
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<p>This is for quantify the plasmid DNA extracted
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<br> Procedure:
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<br>1.- Take 1ml of mQ water in an Eppendorf
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<br>2.- Add 1ul of plasmid DNA
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<br>3.- Prepare the spectrophotometer
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<br>4.- Pass the DNA to the spectrophotometer
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<br>5.- Select the option (DNA or RNA)
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<br>6.- Write the lecture of the spectro at 260, 280 and 320, relation 260/280 and the concentration.</p></td></tr></table>
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<p> <b>Plasmid DNA characterization </b></p>
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<p> This is for identify the piece we have
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<br>Procedure:
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<br>1.- Prepare the mix
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<br>2.- Deal the mix in equal parts
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<br>3.- Add the DNA and mix
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<br>4.- Incubate the reactions at 37°C for 1 hour
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<br>5.- Use the gel for check the result </p>
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<td><p align="center"> <img src="https://static.igem.org/mediawiki/2013hs/a/af/Imagen6.jpg" width="282px" height="232px" /> </p></td></tr></table>
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<p><b>BioBrick parts assembly</b></p>
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<p> Procedure:
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<br>In this step we are going to cut the plasmid with the appropriate restriction enzymes for deliver the desired fragment and to paste into another bacterium.
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<br>1.- Prepare the mix
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<br>2.- Lead the mix in equal parts and the add DNA, mix it.
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<br>3.- Incubate the reactions for 1 hour
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<br>4.- Check in the gel
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<br>5.- Save it for its ligation</p>
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<p><b>Ligation of genetic parts</b></p>
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<p>This step is for paste the desired piece in the bacterium we are going to use.
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<br>Procedure:
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<br>1.- Take in count the concentration of each one of the DNA’s.
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<br>2.- Use the calculator for obtain the amounts for preparing the ligation mix at 20ul
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<br>3.- Prepare the mix in the next order: mQ water, Buffer, and the plasmid
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<br>4.- Lead the mix and add the appropriate ligase
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<br>5.- Incubate the reactions at 25°C</p>
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Revision as of 21:45, 17 October 2014

Project
Abstract

Thermonator III: The crop guardian

Our project is about a genetic engineered machine in E. coli with the ability to produce Vip3ca3 which acts as a pesticide protein. Vip3ca3 production will be regulated by specific temperatures in order to avoid overproduction and it will show activity against target organisms Coleoptera and Lepidoptera, which are related to a local problem concerning potato crops. The Vip3ca3 production is regulated with a constitutive promoter and a riboswitch that initiate translation around 32°C. Since we want to produce the Vip3ca3 below the 32°C, we use a set of promoter-repressors in order to invert the activation of the protein production. This model may be used as a regulator in future transgenic plant generations for the production of substances against plagues, avoiding pesticide overproduction, thus reducing the effect in Non-Target Organisms and bioaccumulation and can be used with different temperature ranges and/or different proteins to attack other target organisms.
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