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<div class = "day" id="725"> For InterLab, we counted our grown colonies on our 36 plates. We then performed the CFU/mL calculations and submitted InterLab Form IV. <br> Members: Kaylee and Brittany <br><br> We reran our digested plasmid 1 with the restriction enzyme BamHI on a 0.7% gel. We redigested plasmid 1 with DpnI and began making more pSB1C3 backbone in case our gel does not work correctly or give us the expected results. <br> Members: Elisa </div> | <div class = "day" id="725"> For InterLab, we counted our grown colonies on our 36 plates. We then performed the CFU/mL calculations and submitted InterLab Form IV. <br> Members: Kaylee and Brittany <br><br> We reran our digested plasmid 1 with the restriction enzyme BamHI on a 0.7% gel. We redigested plasmid 1 with DpnI and began making more pSB1C3 backbone in case our gel does not work correctly or give us the expected results. <br> Members: Elisa </div> | ||
<div class = "date" id="72618"> 7/26/18 </div> | <div class = "date" id="72618"> 7/26/18 </div> | ||
− | <div class = "day" id="726"> We re-amplified and re-ran the plasmid 2 blocks. <br> Members: Liz <br><br> We made a new pSB1C3 backbone from 23-O in the 2018 kit 7. We also redid PCR overlap extension for 2/3 and 4/5 and ran it on a 1% gel. We extracted the correct sequences from the gel and reran the new pSB1C3 on a 1% gel. <br> Members: Elisa <br><br> We transformed and cultured 4 different pSB1C3 plasmid backbones to try to put our parts into. We put them in the incubator to grow overnight. <br> Members: Kaylee and Elisa | + | <div class = "day" id="726"> We re-amplified and re-ran the plasmid 2 blocks. <br> Members: Liz <br><br> We made a new pSB1C3 backbone from 23-O in the 2018 kit 7. We also redid PCR overlap extension for 2/3 and 4/5 and ran it on a 1% gel. We extracted the correct sequences from the gel and reran the new pSB1C3 on a 1% gel. <br> Members: Elisa <br><br> We transformed and cultured 4 different pSB1C3 plasmid backbones to try to put our parts into. We put them in the incubator to grow overnight. <br> Members: Kaylee and Elisa </div> |
− | + | ||
− | </div> | + | |
<div class = "date" id="72718"> 7/27/18 </div> | <div class = "date" id="72718"> 7/27/18 </div> | ||
<div class = "day" id="727"> We retransformed and cultured 4 different pSB1C3 plasmid backbones.<br> Members: Kaylee and Elisa </div> | <div class = "day" id="727"> We retransformed and cultured 4 different pSB1C3 plasmid backbones.<br> Members: Kaylee and Elisa </div> | ||
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<div class = "date" id="8218"> 8/2/18 </div> | <div class = "date" id="8218"> 8/2/18 </div> | ||
<div class = "day" id="82"> We recultured plates with PETase and glycolate oxidase, reamplified our PETase and ran it on a 1% gel, and inoculated our cultures that had glycolate oxidase and PETase. <br> Members: Elisa <br><br> For plasmid 2, we performed the NEBuilder protocol on the plasmid 2 parts and transformed it into competent 5-alpha cells. We are incubating the results overnight. <br> Members: Liz </div> | <div class = "day" id="82"> We recultured plates with PETase and glycolate oxidase, reamplified our PETase and ran it on a 1% gel, and inoculated our cultures that had glycolate oxidase and PETase. <br> Members: Elisa <br><br> For plasmid 2, we performed the NEBuilder protocol on the plasmid 2 parts and transformed it into competent 5-alpha cells. We are incubating the results overnight. <br> Members: Liz </div> | ||
+ | <div class = "date" id="8318"> 8/3/18 </div> | ||
+ | <div class = "day" id="83"> We inoculated cultures of plasmid 2. <br> Members:Liz </div> | ||
<div class = "week"> Week 10 </div> | <div class = "week"> Week 10 </div> | ||
+ | <div class = "date" id="8618"> 8/6/18 </div> | ||
+ | <div class = "day" id="86"> We found the new Glycolate Oxidase sequences because we realized we only had taken the sequence for the D subunit. We designed primers for the D, E, and F subunits for submission as well as insertion into the pETDuet vector. We made more PETase composite, made more pSB1C3 linearized backbone, and ran PETase on 0.7% gel. <br>Members: | ||
+ | Elisa and Kaylee <br><br> We performed plasmid mini prep on the inoculated cells with plasmid 2 for both backbones pSB3K3 and pSB1C3. We also inoculated more colonies to make glycerol stocks of each and let them grow overnight. <br> Members: Liz </div> | ||
+ | <div class = "date" id="8718"> 8/7/18 </div> | ||
+ | <div class = "day" id="87"> We digested more PETase, ran it on a 0.7% gel, and extracted it. We digested pSB1C3 backbone and PETase with EcoRI and PstI and digested the NEBuilder positive control. We ran the digested pSB1C3 and PETase on a 1% gel and extracted pSB1C3. The PETase sequence did not work correctly on the gel. <br> Members: Elisa and Kaylee <br><br> | ||
+ | We ran restriction digest on plasmid 2 using EcoRI and PstI enzymes. We also made glycerol stocks of the plasmid 2 for each backbone. <br> Members: Liz </div> | ||
+ | <div class = "date" id="8818"> 8/8/18 </div> | ||
+ | <div class = "day" id="88"> We made more PETase, redigested PETase with XbaI and PstI, transformed pSB1C3+Red fluorescent protein into 5Alpha cells, and plated the transformed cells on chlor. plates. <br> Members: Elisa and Ariel <br><br> We assembled plasmid 2 with a pSB1C3 backbone. We also transformed the plasmid into 5Alpha cells using the NEBuilder protocol. We inoculated the cells and plated them on LB+chlor plates to grow overnight.<br> Members: Liz and Kaylee </div> | ||
+ | <div class = "date" id="8918"> 8/9/18 </div> | ||
+ | <div class = "day" id="89"> We ran the new PETase and digested PETase on a 1% gel, inoculated colonies of RFP in LB+Chlor., rehydrated primers, performed colony PCR of glycolate oxidase, and started the amplification of just PETase, instead of PETase + pelB/RBS/promoter. <br> Members: Elisa and Ariel <br><br> | ||
+ | We picked and inoculated 5 cultures from plasmid 2 with the newest pSB1C3 backbone. <br> Members: Liz and Kaylee </div> | ||
+ | <div class = "date" id="81018"> 8/10/18 </div> | ||
+ | <div class = "day" id="810"> We ran PETase and glycolate oxidase on a 0.7% gel, miniprepped the RFP plasmid from 5Alpha cells, and inoculated more pET-Duet vector from the plate. We digested our miniprepped pET-Duet vector and the old pET-Duet backbone, extracted PETase and glycolate oxidase from the gel, ran the digested pET-Duet on 0.7% gel, and extracted it. <br> Members: Elisa <br><br> We performed plasmid miniprep on the 5 chosen cultures of plasmid 2. <br> Members: Liz and Kaylee </div> | ||
+ | |||
<div class = "week"> Week 11 </div> | <div class = "week"> Week 11 </div> | ||
+ | <div class = "date" id="81318"> 8/13/18 </div> | ||
+ | <div class = "day" id="813"> We digested pSB1C3 backbone with EcoRI and PstI, pET-Duet backbone with BamHI and XhoI, and our isolated glycolate oxidase parts with EcoRI and PstI for the one isolated with primers to be put into pSB1C3 and with BamHI and XhoI for the one isolated with primers to be put into pET-Duet. We ran all our digested parts and uncut versions of them on a 0.7% gel, and were able to extract the pET-Duet backbone and the glycolate oxidase to go into pSB1C3. <br> Members: | ||
+ | Kaylee and Ariel <br><br> We also digested our 5 plasmid 2 chosen colonies with EcoRI and PstI and ran them on a 1% gel. Unfortunately, the bands did not appear where we expected them to. <br> Members: Liz and Kaylee </div> | ||
+ | <div class = "date" id="81418"> 8/14/18 </div> | ||
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<br> | <br> | ||
<p style="font-weight:bold">Procedure</p> | <p style="font-weight:bold">Procedure</p> | ||
− | Fill a large bottle with lid with 570 mL of DI H2O. Insert a stir bar and place the bottle on the stir plate. Mix in 6 g Tryptone, 3 g yeast extract, 9 g agar, and 6 g NaCl. Ensure the solution has been mixed thoroughly and standardize the pH meter with the neutral standard solution. Add 2M NaOH to the solution dropwise until the pH reaches 7. <br> <br> Check the water level in the autoclave and put autoclave tape on the bottle. Ensure the lid is on but not tightened. Put the bottle in the autoclave and tighten the door. Set it for the liquid cycle and wait until the cycle is over and the pressure reaches 0 to open the door.<br><br> Put the bottle back on the stir plate and turn it to a low setting to prevent bubbles forming. Let the mixture cool until you can touch the bottle for several seconds comfortably. Add 600 µL of the chosen antibiotic (at 50 mg/mL) to the bottle. Label all plates before you begin to pour. Wearing a glove, use the aseptic technique to pour the media into the plates and leave them to solidify. <br><br> To make liquid media stock, the same procedure can be followed without adding agar or antibiotic and then storing in a 4°C fridge.</div> | + | Fill a large bottle with lid with 570 mL of DI H2O. Insert a stir bar and place the bottle on the stir plate. Mix in 6 g Tryptone, 3 g yeast extract, 9 g agar, and 6 g NaCl. Ensure the solution has been mixed thoroughly and standardize the pH meter with the neutral standard solution. Add 2M NaOH to the solution dropwise until the pH reaches 7. <br> <br> Check the water level in the autoclave and put autoclave tape on the bottle. Ensure the lid is on but not tightened. Put the bottle in the autoclave and tighten the door. Set it for the liquid cycle and wait until the cycle is over and the pressure reaches 0 to open the door.<br><br> Put the bottle back on the stir plate and turn it to a low setting to prevent bubbles forming. Let the mixture cool until you can touch the bottle for several seconds comfortably. Add 600 µL of the chosen antibiotic (at 50 mg/mL) to the bottle. Label all plates before you begin to pour. Wearing a glove, use the aseptic technique to pour the media into the plates and leave them to solidify. <br><br> To make liquid media stock, the same procedure can be followed without adding agar or antibiotic and then storing in a 4°C fridge. <br><br> <em>Adapted from Dr. Irene Reizman and Prather Labs, MIT</em></div> |
<div class = "prot" id="prot2"> Dry Ice Baths </div> | <div class = "prot" id="prot2"> Dry Ice Baths </div> | ||
<div class = "num" id="2"> There are a few uses for dry ice baths in biology. They are typically made of 70% | <div class = "num" id="2"> There are a few uses for dry ice baths in biology. They are typically made of 70% | ||
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Add the dry ice a LITTLE at a time, so the ethanol doesn't bubble over and make a mess. | Add the dry ice a LITTLE at a time, so the ethanol doesn't bubble over and make a mess. | ||
Do not handle dry ice without a glove or something to protect your skin or breathe it in. | Do not handle dry ice without a glove or something to protect your skin or breathe it in. | ||
− | Put the lid back on the dewar to keep the solution cool and prevent evaporation. </div> | + | Put the lid back on the dewar to keep the solution cool and prevent evaporation. <br><br> <em>Adapted from Dr. Irene Reizman and Prather Labs, MIT</em></div> |
<div class = "prot" id="prot3"> Gel Electrophoresis </div> | <div class = "prot" id="prot3"> Gel Electrophoresis </div> | ||
<div class = "num" id="3"> Gel electrophoresis is a quick way to determine the relative sizes of DNA pieces. When compared to a known ladder, gel electrophoresis can be used to determine if restriction enzymes cut in the predicted places or if separate DNA pieces combined together correctly during PCR. <br> <p style="font-weight:bold"> Materials </p> | <div class = "num" id="3"> Gel electrophoresis is a quick way to determine the relative sizes of DNA pieces. When compared to a known ladder, gel electrophoresis can be used to determine if restriction enzymes cut in the predicted places or if separate DNA pieces combined together correctly during PCR. <br> <p style="font-weight:bold"> Materials </p> | ||
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To make 1.5%: use .75 g Agarose to 50 mL TBE Buffer<br> | To make 1.5%: use .75 g Agarose to 50 mL TBE Buffer<br> | ||
To make 2%: use 1 g Agarose to 50 mL TBE Buffer<br> | To make 2%: use 1 g Agarose to 50 mL TBE Buffer<br> | ||
− | <img src = "https://static.igem.org/mediawiki/2018/e/ee/T--RHIT--AgaroseProtocol.jpg" style="width:60%"> <br>This table was adapted from http://schepartzlab.yale.edu/intranet/protocols/AgaroseGelElectrophores.pdf<br><br> | + | <img src = "https://static.igem.org/mediawiki/2018/e/ee/T--RHIT--AgaroseProtocol.jpg" style="width:60%"> <br> This table was adapted from: http://schepartzlab.yale.edu/intranet/protocols/AgaroseGelElectrophores.pdf <br><br> |
Add 1x TBE buffer and microwave until it begins to boil. If the solution is not totally clear (i.e. still has swirls in it) heat it again. Let the solution cool for a couple minutes and add 1 µL of Gel Red to the flask. Ensure the mold is turned so the walls are keeping the gel from running out. Pour the solution into the mold and add the well maker. <br> | Add 1x TBE buffer and microwave until it begins to boil. If the solution is not totally clear (i.e. still has swirls in it) heat it again. Let the solution cool for a couple minutes and add 1 µL of Gel Red to the flask. Ensure the mold is turned so the walls are keeping the gel from running out. Pour the solution into the mold and add the well maker. <br> | ||
<br> | <br> | ||
Running Gels <br> | Running Gels <br> | ||
− | Rotate the "submarine" (the piece holding the gel) so that the wells are closer to the black end ("run to red"). Fill the apparatus with 1x TBE buffer until it barely covers the top of the gel. Fill the wells with 10 µL of the desired ladder(s) and 10 µL of the desired DNA, mixed with 4 µL of loading dye. Put the lid on and hook up the red and black cables, then turn on the electricity to let the gels run at an appropriate voltage. Let the gels run out, but do not let the dye go all the way to the other end of the gel. | + | Rotate the "submarine" (the piece holding the gel) so that the wells are closer to the black end ("run to red"). Fill the apparatus with 1x TBE buffer until it barely covers the top of the gel. Fill the wells with 10 µL of the desired ladder(s) and 10 µL of the desired DNA, mixed with 4 µL of loading dye. Put the lid on and hook up the red and black cables, then turn on the electricity to let the gels run at an appropriate voltage. Let the gels run out, but do not let the dye go all the way to the other end of the gel.<br><br> <em>Adapted from Dr. Irene Reizman and Prather Labs, MIT </em> |
<br> | <br> | ||
</div> | </div> | ||
<div class = "prot" id="prot4"> PCR Overlap </div> | <div class = "prot" id="prot4"> PCR Overlap </div> | ||
− | <div class="num" id="4"> We followed the protocol from OpenWetWare for our PCR Overlap <br> <a href = "https://openwetware.org/wiki/PCR_Overlap_Extension"> https://openwetware.org/wiki/PCR_Overlap_Extension </a> | + | <div class="num" id="4"> We followed the protocol from OpenWetWare for our PCR Overlap. <br> <a href = "https://openwetware.org/wiki/PCR_Overlap_Extension"> https://openwetware.org/wiki/PCR_Overlap_Extension </a> |
</div> | </div> | ||
<div class = "prot" id="prot5"> Colony PCR </div> | <div class = "prot" id="prot5"> Colony PCR </div> | ||
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<img style="width:60%" src="https://static.igem.org/mediawiki/2018/6/65/T--RHIT--ColonyPCR.jpg"> | <img style="width:60%" src="https://static.igem.org/mediawiki/2018/6/65/T--RHIT--ColonyPCR.jpg"> | ||
</center> | </center> | ||
+ | <br><br> <em>Adapted from Dr. Irene Reizman and Prather Labs, MIT </em> | ||
</div> | </div> | ||
<div class = "prot" id="prot6"> NEBuilder Kit </div> | <div class = "prot" id="prot6"> NEBuilder Kit </div> | ||
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</div> | </div> | ||
+ | <div class = "prot" id="prot7"> Glycolate Oxidase Assay </div> | ||
+ | <div class = "num" id="7"> | ||
+ | <ul><li>Mix 0.94mL of mono-potassium phosphate with 0.6mL of di-potassium phosphate with 90 mL DI H2O to generate 100mL stock solution of potassium phosphate buffer at 8pH. </li> | ||
+ | <li>Follow Freeze/Thaw lyse protocol instructions. Note best results happen when using fresh lysate. If not possible follow step and aliquote out smaller volumes to freeze. | ||
+ | <img src = "https://static.igem.org/mediawiki/2018/6/69/T--RHIT--assay1.jpg"><img src = "https://static.igem.org/mediawiki/2018/d/da/T--RHIT--assay2.jpg"> | ||
+ | </li> | ||
+ | <li>Create Enzyme Assay master mixture 10mL:<br> | ||
+ | 666.7 micromoles of potassium phosphate<br> | ||
+ | 0.67 micromoles of DCIP <br> | ||
+ | 0.33 mL of 1% PMS <br> | ||
+ | 0.033 - 0.33 mL of cell extract <br> | ||
+ | = final volume should be ~10 mL<br> | ||
+ | Aliquote out 1 mL volumes for enzyme assay | ||
+ | </li> | ||
+ | <li>Create Positive Control 1 mL:<br> | ||
+ | 0.067 micromoles of DCIP <br> | ||
+ | 0.033 mL of 1% PMS <br> | ||
+ | 0.0033 - 0.033 mL of cell extract<br> | ||
+ | Top off with potassium phosphate buffer for 1 mL volume<br> | ||
+ | </li> | ||
+ | <li>Create Negative Control 1 mL:<br> | ||
+ | 0.067 micromoles of DCIP <br> | ||
+ | 0.033 mL of 1% PMS <br> | ||
+ | Top off with potassium phosphate buffer for 1 mL volume | ||
+ | </li> | ||
+ | <li>Make up a solution of potassium glycolate by mixing Add 10 micromoles of glycolic acid (MW: 76.05 g/mol) to enough buffer to neutralize it to glycolate (this should be enough for three 1 mL cuvette)</li> | ||
+ | <li>Measure 600 nm time-lapse spectrophotometer for positive control over 5 mins (Absorbance start at or be under 1) </li> | ||
+ | <li>Add 3.33 micromoles of potassium glycolate to the negative control mixture </li> | ||
+ | <li>Measure 600 nm time-lapse spectrophotometer for negative control over 5 mins (Absorbance start at or be under 1) </li> | ||
+ | <li>Add 3.33 micromoles of potassium glycolate to the enzyme mixture</li> | ||
+ | <li>Measure a decrease in extinction at 600 nm time-lapse spectrophotometer over 5 mins (Absorbance start at or be under 1)</li></ul> | ||
+ | Analysis: A decrease in extinction of 0.01 corresponds to oxidation of 1.8 nmoles of glycolate<br> | ||
+ | Adapted from J.M. Lord 1971 | ||
+ | |||
+ | </div> | ||
</div> | </div> | ||
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<div class = "event" id="event8"> Skype Call with Yale Team - 8/6/18 </div> | <div class = "event" id="event8"> Skype Call with Yale Team - 8/6/18 </div> | ||
<div class = "entry" id="entry8"> Kaylee, Brittany, and Ariel skyped with the Yale iGEM team to talk about our projects. Both of our teams are making cells that secrete the enzymes PETase and MHETase to break down PET plastic. They have had more success in the lab than we have, so we were able to ask some questions about how they got their plasmid working. They also suggested we try Gibson Assembly and ligation independent cloning for our plasmid. Additionally, they told us they were finding more success with the yebF secretion tag than some of the others they had tried, like our pelB secretion tag. We were able to answer some questions for them about the modeling we had done, our outreach plans, and other work on our wiki. </div> | <div class = "entry" id="entry8"> Kaylee, Brittany, and Ariel skyped with the Yale iGEM team to talk about our projects. Both of our teams are making cells that secrete the enzymes PETase and MHETase to break down PET plastic. They have had more success in the lab than we have, so we were able to ask some questions about how they got their plasmid working. They also suggested we try Gibson Assembly and ligation independent cloning for our plasmid. Additionally, they told us they were finding more success with the yebF secretion tag than some of the others they had tried, like our pelB secretion tag. We were able to answer some questions for them about the modeling we had done, our outreach plans, and other work on our wiki. </div> | ||
+ | <div class = "event" id="event9"> End of Summer Presentation at Research Symposium - 8/10/18 </div> | ||
+ | <div class = "entry" id="entry9"> Liz, Kaylee, Elisa, and Ariel did a 7 minute presentation at the Rose-Hulman Undergraduate Research Symposium on the basic premise of our iGEM project, focusing mainly on the development of the plasmids, the problem it addresses, and predictions made by the system’s model. Following the presentation, we answered questions from students and faculty about the project, most of which were geared toward how this could be implemented safely. It was a great opportunity to educate the campus community about our project and get live feedback about areas of concern. One of the biggest concerns people shared was how we planned to keep our bacteria from escaping loosely into the environment. One concerned student asked, “what if your bacteria ate my milk jug and my milk spilled everywhere?” We can use the feedback from this experience to better inform our implementation methods. | ||
+ | </div> | ||
+ | <div class = "event" id="event10"> Tribune-Star Phone Interview and Article - 8/13/18 </div> | ||
+ | <div class = "entry" id="entry10"> Ariel contacted recycling advocate and local newspaper columnist Jane Santucci as part of reaching the community with our survey. She not only agreed to share our survey link with her social media followers, she offered to write a column in the Tribune-Star on our team, project, and survey. We were very grateful, and following the phone interview, a column in the Sunday newspaper appeared. This was a wonderful opportunity to reach the community about current recycling, synthetic biology, and our team’s efforts to involve the community with our implementation plans. </div> | ||
+ | <div class = "event" id="event11"> Terre Haute Children’s Museum Exhibition - 9/1/18 </div> | ||
+ | <div class = "entry" id="entry11"> Kaylee, Liz, Ariel, and Elisa did a hands-on exhibit at our local children’s museum to teach about DNA structure and our project. We worked with the children to create color coded “DNA” bracelets, where they would match the bases, or beads, on two strings. Those were then made into bracelets and necklaces. They also had the opportunity to read our PEBBLE comic book. The parents were involved by helping the bead matching and asking questions about our project. The team got to talk face-to-face with the community about the project, hear their thoughts, and teach the future generations about cool biology. </div> | ||
+ | <div class = "event" id="event12"> Modeling Lecture for a Rose-Hulman Biomathematic Class - 9/13/18 </div> | ||
+ | <div class = "entry" id="entry12"> The iGEM team was invited to present on modeling aspects of the project to the Rose-Hulman BMTH 295 class. Ariel created the presentation and gave a lecture to students and professors on the development and results of our team’s project and the modeling predictions. This gave us the opportunity to inform the larger campus of iGEM and its opportunities for interested students. </div> | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
Latest revision as of 23:55, 15 October 2018