Team:Cornell/notebook

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wiki

|type=wiki

|text=We just started working on the wiki! Our first assignment: figuring out how wiki templates work. With templates the website would become much easier to edit, but for some reason HTML and wiki text don't seem to work very well together...

|text=We just started working on the wiki! Our first assignment: figuring out how wiki templates work. With templates the website would become much easier to edit, but for some reason HTML and wiki text don't seem to work very well together...

|author=Nupur

|author=Nupur

|type=wet

|type=wet

|head=April 20th

|head=April 20th

|author=Rafael

|author=Rafael

}}

}}

|type=dry

|type=dry

|head=June 17th

|head=June 17th

|author=Mac  

|author=Mac  

}}

}}

|tech=From the plasmid pUCATPH (pAh13G), we amplified the ''trpC'' promoter (P''trpC'', a constitutive fungal promoter) and the ''trpC'' terminator (T''trpC''). From pNG (pAg13H), we amplified ''nptII'', the geneticin (a plant/fungal antibiotic) resistance gene.

|tech=From the plasmid pUCATPH (pAh13G), we amplified the ''trpC'' promoter (P''trpC'', a constitutive fungal promoter) and the ''trpC'' terminator (T''trpC''). From pNG (pAg13H), we amplified ''nptII'', the geneticin (a plant/fungal antibiotic) resistance gene.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wiki

|type=wiki

|author=Nupur

|author=Nupur

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wiki

|type=wiki

|author=Nupur

|author=Nupur

}}

}}

|tech=We transformed T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) constructs into DH5α via electroporation.

|tech=We transformed T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) constructs into DH5α via electroporation.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

<h5 class="centered">(06/24 - 06/30)</h5>

<h5 class="centered">(06/24 - 06/30)</h5>

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</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|author=Rafael

|author=Rafael

}}

}}

|text=I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.

|text=I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.

|author=Nupur

|author=Nupur

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=June 30th

|head=June 30th

|author=Rafael

|author=Rafael

}}

}}

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{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|head=July 1st

|head=July 1st

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{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

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{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|head=July 1st

|head=July 1st

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{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=July 2nd

|head=July 2nd

|tech=''hph'' was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V.  

|tech=''hph'' was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V.  

|author=Rafael

|author=Rafael

|head=July 3rd

|head=July 3rd

|text=All transformations failed yet again, and were repeated once more. Four tears shed for the lost. ''hph'' was Gibson-assembled, and we ran a Q5 PCR of the ''bar'' gene, under the assumption it matches other published ''bar'' sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on ''nptII'' (pCg13R) to remove its internal PstI site.

|text=All transformations failed yet again, and were repeated once more. Four tears shed for the lost. ''hph'' was Gibson-assembled, and we ran a Q5 PCR of the ''bar'' gene, under the assumption it matches other published ''bar'' sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on ''nptII'' (pCg13R) to remove its internal PstI site.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|text=Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.

|text=Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.

|author=Nupur

|author=Nupur

}}

}}

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<h5 class="centered">(07/08 - 07/14)</h5>

<h5 class="centered">(07/08 - 07/14)</h5>

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</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=hprac

|type=hprac

|text= Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14.  

|text= Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14.  

|author=Hannah

|author=Hannah

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|text=After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop.  

|text=After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop.  

|author=Rebecca

|author=Rebecca

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=July 12th

|head=July 12th

|author=Rafael

|author=Rafael

}}

}}

|type=wet

|type=wet

|head=July 13th

|head=July 13th

|author=Rafael

|author=Rafael

}}

}}

|type=wet

|type=wet

|head=July 14th

|head=July 14th

|author=Rafael

|author=Rafael

}}

}}

|type=dry

|type=dry

|head=July 14th

|head=July 14th

|author=Mac  

|author=Mac  

}}

}}

<h5 class="centered">(07/15 - 07/21)</h5>

<h5 class="centered">(07/15 - 07/21)</h5>

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</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|head=July 15th

|head=July 15th

|author=Rebecca

|author=Rebecca

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

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{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=July 18th

|head=July 18th

|author=Danielle

|author=Danielle

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

<h5 class="centered">(07/22 - 07/28)</h5>

<h5 class="centered">(07/22 - 07/28)</h5>

</html>

</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|text=Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)

|text=Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)

|author=Hannah

|author=Hannah

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

<h5 class="centered">(07/29 - 08/04)</h5>

<h5 class="centered">(07/29 - 08/04)</h5>

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</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|text=Sara and Rebecca met up with our advisor Dr Bruce Land and he provided lots of helpful advice with regard to the design considerations (where the heater should be in our incubation chamber, convection currents, type of heating plate to use). He also suggested that we use a proportional-integral-derivative controller (PID controller) to regulate the heating.

|text=Sara and Rebecca met up with our advisor Dr Bruce Land and he provided lots of helpful advice with regard to the design considerations (where the heater should be in our incubation chamber, convection currents, type of heating plate to use). He also suggested that we use a proportional-integral-derivative controller (PID controller) to regulate the heating.

|author=Rebecca

|author=Rebecca

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|text= Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi.  

|text= Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi.  

|author=Hannah

|author=Hannah

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|text= Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.

|text= Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.

|author=Hannah

|author=Hannah

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|head=August 5th

|head=August 5th

|text= Our previous construction of pC13AY failed. We submitted pC13BF and pC13BG for  sequencing. We ran a colony PCR for pAK13AB, pAK13AC and pAK13BB. A few pAK13AC and pAK13BB colonies were successful.  

|text= Our previous construction of pC13AY failed. We submitted pC13BF and pC13BG for  sequencing. We ran a colony PCR for pAK13AB, pAK13AC and pAK13BB. A few pAK13AC and pAK13BB colonies were successful.  

|tech= We electroporated pA13AM into ''DH5α'' and plated on ampicillin infused plates. We will know if the transformation was successful by the presence of red colonies as a result of ''mRFP'' in the plasmid. We found that our construction of pC13AY failed after running a digest screen as none of the lanes displayed bands at around 3kb, which corresponds to inserted ''PtrpC'' and ''T7 polymerase'' and 2kb corresponding to the vector. However, the gel may have been showing unique banding patterns for the construct, so another gel will be run to check for any supercoiling or linearized plasmids. We also submitted our constructs pC13BF and pC13BG for sequencing. Colony PCRs were done for pAK13AB, pAK13AC, and pAK13BB. A few pAK13AC and pAK13BB colonies were successful.  

|tech= We electroporated pA13AM into ''DH5α'' and plated on ampicillin infused plates. We will know if the transformation was successful by the presence of red colonies as a result of ''mRFP'' in the plasmid. We found that our construction of pC13AY failed after running a digest screen as none of the lanes displayed bands at around 3kb, which corresponds to inserted ''PtrpC'' and ''T7 polymerase'' and 2kb corresponding to the vector. However, the gel may have been showing unique banding patterns for the construct, so another gel will be run to check for any supercoiling or linearized plasmids. We also submitted our constructs pC13BF and pC13BG for sequencing. Colony PCRs were done for pAK13AB, pAK13AC, and pAK13BB. A few pAK13AC and pAK13BB colonies were successful.  

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=hprac

|type=hprac

|head=August 5th

|head=August 5th

|author=Hannah

|author=Hannah

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 6th

|head=August 6th

|text= After the previous failure of pC13AY construction, we recreated it with successfully screened parts. We redid a colony PCR for pAK13AB, which failed again. We will need to reconstruct pAK13AB.  

|text= After the previous failure of pC13AY construction, we recreated it with successfully screened parts. We redid a colony PCR for pAK13AB, which failed again. We will need to reconstruct pAK13AB.  

|tech= Yesterday’s transformation of pA13AM yielded no transformants, so we retried the transformation today. We ran a digest screen of ''PtrpC'' and ''T7 polymerase'' parts and found that the ''PtrpC'' used for our previous construction of pC13AY was of poor quality. We then set up another ligation of ''PtrpC'' and ''T7 polymerase'' using successfully screened parts. For fluorescence characterization, we double digested pAK13D and pC13P with SpeI and PstI for subsequent ligation with ''GFP'' and ''mRFP'' inserts. We then quantified our construct containing the <i>G. lucidum</i> homologous regions in pSB1C3, but we had a low yield. We will wait for a new miniprep kit to grow new cultures and perform the miniprep again. We redid a colony PCR for pAK13AB for extra colonies. They all failed so we need to reconstruct pAK13AB.

|tech= Yesterday’s transformation of pA13AM yielded no transformants, so we retried the transformation today. We ran a digest screen of ''PtrpC'' and ''T7 polymerase'' parts and found that the ''PtrpC'' used for our previous construction of pC13AY was of poor quality. We then set up another ligation of ''PtrpC'' and ''T7 polymerase'' using successfully screened parts. For fluorescence characterization, we double digested pAK13D and pC13P with SpeI and PstI for subsequent ligation with ''GFP'' and ''mRFP'' inserts. We then quantified our construct containing the <i>G. lucidum</i> homologous regions in pSB1C3, but we had a low yield. We will wait for a new miniprep kit to grow new cultures and perform the miniprep again. We redid a colony PCR for pAK13AB for extra colonies. They all failed so we need to reconstruct pAK13AB.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 7th

|head=August 7th

|text= We received sequencing back for pC13BF and pC13BG and only the later was successful, so we restarted construction for pC13BF. We also ran a PCR to amplify ''GFP'' and ''mRFP'' inserts out of their respective plasmids.

|text= We received sequencing back for pC13BF and pC13BG and only the later was successful, so we restarted construction for pC13BF. We also ran a PCR to amplify ''GFP'' and ''mRFP'' inserts out of their respective plasmids.

|tech= We designed primers for ''lox'' Gibson assembly and appended cut sites to genes flanked by ''lox''. We also found a possible viable kill switch in the Holin/Chitinase system. Our second try at transforming pA13AM was successful as shown by the presence of red colonies and we set up LB cultures for subsequent cloning. Sequencing showed that construction of pC13BG was successful and cultures were made for further use. However, sequencing of pC13BF was unsuccessful and we reran the PCR of pC13F in order to append BamHI and KpnI cut sites for ligation with pA13BE. We ran another digest screen for pC13AY but many lanes did not have bands so we will retry it tomorrow. We also amplified ''GFP'' from pC13Z and ''mRFP'' from pC13AK using primers 23 and 24 in a Q5 PCR. Afterwards, a gel screen on PCR products was run to verify that the correct genes were amplified. The gel image indicated consistent bands with lengths of 0.7 kb, thus the PCR to obtain ''GFP'' and ''mRFP'' inserts was successful! We made a rough draft of our protoplasting protocol to attempt in the near future, too. We then rummaged through previous notebook entries to figure out the problems with ''crtB'' and ''crtI''.  

|tech= We designed primers for ''lox'' Gibson assembly and appended cut sites to genes flanked by ''lox''. We also found a possible viable kill switch in the Holin/Chitinase system. Our second try at transforming pA13AM was successful as shown by the presence of red colonies and we set up LB cultures for subsequent cloning. Sequencing showed that construction of pC13BG was successful and cultures were made for further use. However, sequencing of pC13BF was unsuccessful and we reran the PCR of pC13F in order to append BamHI and KpnI cut sites for ligation with pA13BE. We ran another digest screen for pC13AY but many lanes did not have bands so we will retry it tomorrow. We also amplified ''GFP'' from pC13Z and ''mRFP'' from pC13AK using primers 23 and 24 in a Q5 PCR. Afterwards, a gel screen on PCR products was run to verify that the correct genes were amplified. The gel image indicated consistent bands with lengths of 0.7 kb, thus the PCR to obtain ''GFP'' and ''mRFP'' inserts was successful! We made a rough draft of our protoplasting protocol to attempt in the near future, too. We then rummaged through previous notebook entries to figure out the problems with ''crtB'' and ''crtI''.  

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|author=Rebecca

|author=Rebecca

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 8th

|head=August 8th

|text= Cloning of the ''Cre'' gene and ''PpelA'' is going well, and we submitted ''PT7'' with ''GFP'' for sequencing, as well as pC13AS, pC13W, pAK13BB, and pAK13AC.  

|text= Cloning of the ''Cre'' gene and ''PpelA'' is going well, and we submitted ''PT7'' with ''GFP'' for sequencing, as well as pC13AS, pC13W, pAK13BB, and pAK13AC.  

|tech= We ran a PCR of pA13BW. The ''CRE'' gene amplified correctly and the sample was column purified, yielding 54.8ng/uL (with GC enhancer) and 49.9ng/uL (with non GC enhancer). We ran out of supplies to miniprep, so we pelleted pA13AM and pC13BG cultures and began to not so patiently await the arrival of a new miniprep kit. Our PCR of pC13F to append BamHI and KpnI cut sites yesterday failed completely, so we digested pC13F with KpnI to linearize it with an internal cut site in ''mRFP'' for another PCR to be run tomorrow. Another gel was run of the pC13AY digests and all lanes were blank. There may have been an issue in quantification by nanodrop that was reading over 1000 ng/μL that mislead us into using low volumes of DNA for digestions. We decided to electroporate with the remaining pC13AY to miniprep and redo gel screening. On a brighter note, our restriction cocktail digestion of pA13BE with BamHI, KpnI and SacI showed a band at 500bp, which is the approximate length of ''PpelA''. We also searched for a pAK13AN miniprep in the DNA storage box and for a glycerol stock of the same plasmid. We couldn’t find either. However, we did obtain a plate of colonies that had been transformed with pAK13AN. We picked colonies off this plate to miniprep and send off to sequencing. In addition, we column purified our inserts from the PCR run yesterday of pC13Z and pK13AL. We then quantified inserts after column purification. After, we digested backbones pAK13D and pC13P with SpeI and PstI and vector dephosphorylated both. The inserts, ''GFP'' in pC13Z and ''mRFP'' in pK13AL were then digested with XbaI and PstI. We ran a gel of backbones and inserts for confirmation of digests. Unfortunately, the results showed no bands from DNA. Perhaps there was not enough DNA from backbones for proper visualization. We also received materials for protoplasting today, and autoclaved or sterilized the supplies in whatever way we could. We made a filtration system such that all the filtering process during <i>G. lucidum</i> protoplasting will remain sterile. We made another stock of <i>G. lucidum</i> in CYM media, as well as 25 mL cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM. We made glycerol stocks of pC13M and pC13K from glycerol stocks. We submitted pAK13AC for sequencing and ran a Q5 PCR for amplifying insert for constructing pC13I from pC13A.   

|tech= We ran a PCR of pA13BW. The ''CRE'' gene amplified correctly and the sample was column purified, yielding 54.8ng/uL (with GC enhancer) and 49.9ng/uL (with non GC enhancer). We ran out of supplies to miniprep, so we pelleted pA13AM and pC13BG cultures and began to not so patiently await the arrival of a new miniprep kit. Our PCR of pC13F to append BamHI and KpnI cut sites yesterday failed completely, so we digested pC13F with KpnI to linearize it with an internal cut site in ''mRFP'' for another PCR to be run tomorrow. Another gel was run of the pC13AY digests and all lanes were blank. There may have been an issue in quantification by nanodrop that was reading over 1000 ng/μL that mislead us into using low volumes of DNA for digestions. We decided to electroporate with the remaining pC13AY to miniprep and redo gel screening. On a brighter note, our restriction cocktail digestion of pA13BE with BamHI, KpnI and SacI showed a band at 500bp, which is the approximate length of ''PpelA''. We also searched for a pAK13AN miniprep in the DNA storage box and for a glycerol stock of the same plasmid. We couldn’t find either. However, we did obtain a plate of colonies that had been transformed with pAK13AN. We picked colonies off this plate to miniprep and send off to sequencing. In addition, we column purified our inserts from the PCR run yesterday of pC13Z and pK13AL. We then quantified inserts after column purification. After, we digested backbones pAK13D and pC13P with SpeI and PstI and vector dephosphorylated both. The inserts, ''GFP'' in pC13Z and ''mRFP'' in pK13AL were then digested with XbaI and PstI. We ran a gel of backbones and inserts for confirmation of digests. Unfortunately, the results showed no bands from DNA. Perhaps there was not enough DNA from backbones for proper visualization. We also received materials for protoplasting today, and autoclaved or sterilized the supplies in whatever way we could. We made a filtration system such that all the filtering process during <i>G. lucidum</i> protoplasting will remain sterile. We made another stock of <i>G. lucidum</i> in CYM media, as well as 25 mL cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM. We made glycerol stocks of pC13M and pC13K from glycerol stocks. We submitted pAK13AC for sequencing and ran a Q5 PCR for amplifying insert for constructing pC13I from pC13A.   

|author=Jonlin  

|author=Jonlin  

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 9th

|head=August 9th

|text= Cloning of the Cre gene is going well, and we may have found a useful limonene synthase gene! We are continuing the previously problematic cloning of ''GFP'' with T7 promoter-hopefully it works this time! Sequencing of pAK13AC unfortunately failed.  

|text= Cloning of the Cre gene is going well, and we may have found a useful limonene synthase gene! We are continuing the previously problematic cloning of ''GFP'' with T7 promoter-hopefully it works this time! Sequencing of pAK13AC unfortunately failed.  

|tech= We digested both the PCR products of ''Cre'' and pC13F with EcoRI and SpeI. We may have found a limonene synthase gene we can use. We ran another PCR to append cut sites to the linearized pC13F that ultimately failed with no bands visible on a gel. As our previous construction of pC13AY likely failed since no colonies being present on the new plate, we redid digestions of pC13E and pC13P to redo the construction. We also redigested both pAK13D and pC13P minipreps with SpeI and PstI since the gel from yesterday was inconclusive. We then ligated pAK13D with ''GFP'', pAK13D with ''mRFP'', pC13P with ''GFP'' and pC13P with ''mRFP''. We prepared pellets from our cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM cells which were stored in the fridge as we wait for our miniprep kit to arrive. Sequencing of pAK13AC has failed, and we will resubmit a miniprep of it. We plated stocks of pC13M and pC13K.  

|tech= We digested both the PCR products of ''Cre'' and pC13F with EcoRI and SpeI. We may have found a limonene synthase gene we can use. We ran another PCR to append cut sites to the linearized pC13F that ultimately failed with no bands visible on a gel. As our previous construction of pC13AY likely failed since no colonies being present on the new plate, we redid digestions of pC13E and pC13P to redo the construction. We also redigested both pAK13D and pC13P minipreps with SpeI and PstI since the gel from yesterday was inconclusive. We then ligated pAK13D with ''GFP'', pAK13D with ''mRFP'', pC13P with ''GFP'' and pC13P with ''mRFP''. We prepared pellets from our cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM cells which were stored in the fridge as we wait for our miniprep kit to arrive. Sequencing of pAK13AC has failed, and we will resubmit a miniprep of it. We plated stocks of pC13M and pC13K.  

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=hprac

|type=hprac

|author=Hannah

|author=Hannah

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|text= We are starting cloning of limonene synthase and continuing cloning of the ''Cre'' gene.  

|text= We are starting cloning of limonene synthase and continuing cloning of the ''Cre'' gene.  

Cloning of fluorescent constructs is going well.  

Cloning of fluorescent constructs is going well.  

|author=Rafael

|author=Rafael

}}

}}

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|type=wet

|type=wet

|head=August 12th

|head=August 12th

|tech= We still didn’t receive a miniprep kit so we pelleted the pC13AY cultures. The PCRs we ran on pC13F to append homologous regions and append cut sites may have been successful. DNA was present but in low concentrations that we could not check on a gel. We had transformants for pC13BX and pC13CJ so we ran a colony PCR and made cultures. The colony PCRs showed little to nothing on the gel, perhaps because Taq polymerase lost activity. We will miniprep the culture and use Q5. We also attempted a restriction cocktail digest today for both ''mRFP'' and ''GFP'' inserts. We cut pC13Z and pC13AK with Xba, PstI, SacI, and ApaLI. We also performed a colony PCR of pC13AT cultures and pC13AU cultures using primer 23 and 24 to verify length of inserts. The lengths should be around .7kb for each insert. When we ran a gel of the PCR products, the colony PCR showed a .7kb insert for pC13AT colony 1 and a .3kb insert for pC13AT colony 2. The bands for pC13AU were inconclusive. We will miniprep the potential pC13AT colony 1 and send for sequencing once the miniprep kit arrives. We digested our plasmid pAh13G with SphI in order to create linear DNA for fungal transformation. We attempted to transform this into what <i>G. lucidum</i> protoplasts we had generated, but the electroporation lead to arcing. Arching most likely resulted from the sodium citrate in the cell solution which we were instructed to use by the literature. Nevertheless, we plated the cells on hygromycin cym agar.

|tech= We still didn’t receive a miniprep kit so we pelleted the pC13AY cultures. The PCRs we ran on pC13F to append homologous regions and append cut sites may have been successful. DNA was present but in low concentrations that we could not check on a gel. We had transformants for pC13BX and pC13CJ so we ran a colony PCR and made cultures. The colony PCRs showed little to nothing on the gel, perhaps because Taq polymerase lost activity. We will miniprep the culture and use Q5. We also attempted a restriction cocktail digest today for both ''mRFP'' and ''GFP'' inserts. We cut pC13Z and pC13AK with Xba, PstI, SacI, and ApaLI. We also performed a colony PCR of pC13AT cultures and pC13AU cultures using primer 23 and 24 to verify length of inserts. The lengths should be around .7kb for each insert. When we ran a gel of the PCR products, the colony PCR showed a .7kb insert for pC13AT colony 1 and a .3kb insert for pC13AT colony 2. The bands for pC13AU were inconclusive. We will miniprep the potential pC13AT colony 1 and send for sequencing once the miniprep kit arrives. We digested our plasmid pAh13G with SphI in order to create linear DNA for fungal transformation. We attempted to transform this into what <i>G. lucidum</i> protoplasts we had generated, but the electroporation lead to arcing. Arching most likely resulted from the sodium citrate in the cell solution which we were instructed to use by the literature. Nevertheless, we plated the cells on hygromycin cym agar.

|author=Rafael

|author=Rafael

|type=wet

|type=wet

|head=August 13th

|head=August 13th

|author=Rafael

|author=Rafael

}}

}}

|head=August 15th

|head=August 15th

|text= We ran a Gibson assembly to create pC13BH and transformed the construct.

|text= We ran a Gibson assembly to create pC13BH and transformed the construct.

|tech= We ran a Gibson assembly of pC13F and <i>G. lucidum</i> ''PgpdA'' to create pC13BH and then transformed and plated the new construct. As our previous attempts to append BamHI and KpnI cut sites to pC13F failed, we again ran another PCR, but tried using four times the amount of dNTPs. We ran a PCR of pC13CJ once again from the cultures. The gel looks good for colony 4 and okay for colony 3. We will miniprep colony 4 and send it in for sequencing. We also made five 5ml liquid LB cultures of pA13CZ and miniprepped pC13AQ. We ran a digest screen of select pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BL, pC13F+pc13BM colonies with EcoRI and SpeI. Our initial gel didn’t show inserts (lengths of 130 and 230 base pairs) so we ran a second gel with 2% agarose. We still couldn’t see inserts, so we ran digest screens on all colonies (1-5) and ran a new gel. We saw very faint bands for some of them, and nothing for others. Additionally, Arun created a device to use the shaking power of our vortex to agitate a sample in the waterbath. We made LB cultures of pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13AC and pAK13BB. We set up new overnight cultures of pC13AS, pC13W, and pC13Y.

|tech= We ran a Gibson assembly of pC13F and <i>G. lucidum</i> ''PgpdA'' to create pC13BH and then transformed and plated the new construct. As our previous attempts to append BamHI and KpnI cut sites to pC13F failed, we again ran another PCR, but tried using four times the amount of dNTPs. We ran a PCR of pC13CJ once again from the cultures. The gel looks good for colony 4 and okay for colony 3. We will miniprep colony 4 and send it in for sequencing. We also made five 5ml liquid LB cultures of pA13CZ and miniprepped pC13AQ. We ran a digest screen of select pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BL, pC13F+pc13BM colonies with EcoRI and SpeI. Our initial gel didn’t show inserts (lengths of 130 and 230 base pairs) so we ran a second gel with 2% agarose. We still couldn’t see inserts, so we ran digest screens on all colonies (1-5) and ran a new gel. We saw very faint bands for some of them, and nothing for others. Additionally, Arun created a device to use the shaking power of our vortex to agitate a sample in the waterbath. We made LB cultures of pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13AC and pAK13BB. We set up new overnight cultures of pC13AS, pC13W, and pC13Y.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|author=Rebecca

|author=Rebecca

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 16th

|head=August 16th

|text= The miniprep kit finally arrived so we were able to continue progress on our pC13BG, pC13AY and pC13AW constructs. To aid in our protoplasting, we decided to seek help from the Turgeon Lab.

|text= The miniprep kit finally arrived so we were able to continue progress on our pC13BG, pC13AY and pC13AW constructs. To aid in our protoplasting, we decided to seek help from the Turgeon Lab.

|tech= The miniprep kit arrived, so we finally were able to miniprep our pelleted pC13BG, pC13AY and pA13AM cultures. We got transformants for the Gibson assembly of pC13BH and we cultured them. The PCR to append cut sites to pC13F again failed, so we need to extensively troubleshoot this PCR. We made large cultures of pC13BX colony 1 and pC13CJ colony 4 to miniprep and sequence. We made electrocompetent stocks of BL21. The symbol is a purple star. We later met with Bradford Condon, a graduate student who works with Professor Turgeon. He gave us a few tips on our method, including using young mycelia, and avoiding the hydrophobic disc that forms on the top of our cultures for protoplasting. The advice was that we should blend <i>G. lucidum</i> which has been growing for an extended period of time, and make new cultures for a short period of time with the fungal "smoothie". We also miniprepped pC13BM, pC13BJ, pC13BL, and pC13BK 6 & 7. The LB stock of pAK13AC was made with the wrong antibiotic so we remade it. We also remade LB stock of pAK13AB. We miniprepped pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13BB, pC13AS, pC13W, and pC13Y. We plated pC13A, pC13D, pC13F, pC13K, pC13L.

|tech= The miniprep kit arrived, so we finally were able to miniprep our pelleted pC13BG, pC13AY and pA13AM cultures. We got transformants for the Gibson assembly of pC13BH and we cultured them. The PCR to append cut sites to pC13F again failed, so we need to extensively troubleshoot this PCR. We made large cultures of pC13BX colony 1 and pC13CJ colony 4 to miniprep and sequence. We made electrocompetent stocks of BL21. The symbol is a purple star. We later met with Bradford Condon, a graduate student who works with Professor Turgeon. He gave us a few tips on our method, including using young mycelia, and avoiding the hydrophobic disc that forms on the top of our cultures for protoplasting. The advice was that we should blend <i>G. lucidum</i> which has been growing for an extended period of time, and make new cultures for a short period of time with the fungal "smoothie". We also miniprepped pC13BM, pC13BJ, pC13BL, and pC13BK 6 & 7. The LB stock of pAK13AC was made with the wrong antibiotic so we remade it. We also remade LB stock of pAK13AB. We miniprepped pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13BB, pC13AS, pC13W, and pC13Y. We plated pC13A, pC13D, pC13F, pC13K, pC13L.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 17th

|head=August 17th

|tech=Today, we made glycerol stocks of pC13AT and prepared them for sequencing. We then miniprepped pAK13AC. However, the miniprep qualities are poor so we will redo with a larger stock of cells. We ran a PCR of pAK13AC, pAK13BB, pAK13A (for both ''RBS'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but pAK13A failed. Thus, we retried PCR of pAK13A inserts from a fresh batch of minipreps. We prepared cultures of pC13S, pC13P, pC13Q, and pC13BG.

|tech=Today, we made glycerol stocks of pC13AT and prepared them for sequencing. We then miniprepped pAK13AC. However, the miniprep qualities are poor so we will redo with a larger stock of cells. We ran a PCR of pAK13AC, pAK13BB, pAK13A (for both ''RBS'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but pAK13A failed. Thus, we retried PCR of pAK13A inserts from a fresh batch of minipreps. We prepared cultures of pC13S, pC13P, pC13Q, and pC13BG.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|head=August 18th

|head=August 18th

|text= Cloning of ''T7 promoter'' with ''mRFP'' has been problematic - but we will keep trying. Minipreps of large stocks of pAK13AB, pAK13AC and pAK13BB yielded better results!  

|text= Cloning of ''T7 promoter'' with ''mRFP'' has been problematic - but we will keep trying. Minipreps of large stocks of pAK13AB, pAK13AC and pAK13BB yielded better results!  

|tech= Today we miniprepped pC13AT along with pA13CZ. We also ran a gel of PCR products of pAK13AO (5 colonies) in pSB1C3. Insert lengths should be around .7kb, but no bands showed up. We will rerun a gel using more DNA from the same PCR. We ran digest screens of pC13BM, pC13BJ, pC13BL, and pC13BK minipreps 6 & 7, which appear to have all worked. We miniprepped pAK13AB, pAK13AC, pAK13BB, pC13A and plasmid concentration was much higher this time around. We miniprepped pC13S, pC13P, pC13Q, and pC13BG, to prepare for cloning.

|tech= Today we miniprepped pC13AT along with pA13CZ. We also ran a gel of PCR products of pAK13AO (5 colonies) in pSB1C3. Insert lengths should be around .7kb, but no bands showed up. We will rerun a gel using more DNA from the same PCR. We ran digest screens of pC13BM, pC13BJ, pC13BL, and pC13BK minipreps 6 & 7, which appear to have all worked. We miniprepped pAK13AB, pAK13AC, pAK13BB, pC13A and plasmid concentration was much higher this time around. We miniprepped pC13S, pC13P, pC13Q, and pC13BG, to prepare for cloning.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=anim

|type=anim

|type=wet

|type=wet

|head=August 19th

|head=August 19th

|tech= We ran a PCR of the pC13BH minipreps to verify the success of the Gibson assembly on a gel. We also digested pC13AY in order to gel screen it and check if it is successful. We began constructing pA13AW by digesting and ligating pA13AM and pC13E. We also reran a gel of a colony PCR of pAK13AO. Next, we prepped pC13AT and pC13AQ for sequencing. We sent in pC13BJ, pC13BK, pC13BL, pC13BM minipreps 6 & 7, pC13AS, pC13W, and pC13Y for sequencing. We ran a gel for the PCR products from Aug. 17 and Aug. 18. Everything is good: pAK13AB, pAK13AC, pAK13BB, pC13A (both ''rbs'' + ''crtE'' and ''crtE''). We ran Q5 PCR of pC13N, pC13L, pC13S, pC13Q, and pC13W for creating inserts. We digested pC13S, pC13Q, and pC13W with X and P, and digested pC13BG, pC13D, pC13Y, and pC13P with S and P. We dephosphorylated and set up the ligations of those inserts and vectors to make pC13BQ, pC13BR, pC13CC, and pC13CD.

|tech= We ran a PCR of the pC13BH minipreps to verify the success of the Gibson assembly on a gel. We also digested pC13AY in order to gel screen it and check if it is successful. We began constructing pA13AW by digesting and ligating pA13AM and pC13E. We also reran a gel of a colony PCR of pAK13AO. Next, we prepped pC13AT and pC13AQ for sequencing. We sent in pC13BJ, pC13BK, pC13BL, pC13BM minipreps 6 & 7, pC13AS, pC13W, and pC13Y for sequencing. We ran a gel for the PCR products from Aug. 17 and Aug. 18. Everything is good: pAK13AB, pAK13AC, pAK13BB, pC13A (both ''rbs'' + ''crtE'' and ''crtE''). We ran Q5 PCR of pC13N, pC13L, pC13S, pC13Q, and pC13W for creating inserts. We digested pC13S, pC13Q, and pC13W with X and P, and digested pC13BG, pC13D, pC13Y, and pC13P with S and P. We dephosphorylated and set up the ligations of those inserts and vectors to make pC13BQ, pC13BR, pC13CC, and pC13CD.

|author=Rafael

|author=Rafael

|type=wet

|type=wet

|head=August 20th

|head=August 20th

|tech= We transformed and plated the pA13AW ligation from yesterday. We also retried the PCR to append KpnI and BamHI cut sites to pC13F again, using the newly received Q5 mastermix and higher annealing temperatures. We ran a massive gel screen and found that the cut site PCR and Gibson assembly may have been successful, but that the construction pC13AY most likely failed. We then digested pC13F with cut sites appended with BamHI and KpnI  for ligation with pC13BE to make pC13BF. We digested pA13CZ with SpeI and PstI, which is to be ligated with ''GFP'' from pC13Z and ''mRFP'' from pC13AK. We ligated the Xpa1, Pst1, Spe1, Apa digested pC13AK and Spe1 and Pst1 digested and dephosphorylated pC13BG to make pC13CG, which we transformed into BL21-A1 cells to check transformation efficiency. We also tranformed into DH5α and plated both for comparison. The sequencing results of pC13BJ 7, pC13BK 6, pC13BL 7, and pC13BM 6 confirmed that we were successful, so we made new cultures to create glycerol stocks from, but the initial cultures were thrown out and the plates were difficult to re-pick from, so we hope that cells made it into the culture and have the correct plasmid. We did a PCR clean-up for pAK13AB, pAK13AC, pC13A, pC13A, pC13N, pC13L. We digested pC13I, pC13J, pC13N, pC13L with XbaI & PstI, pAK13AB & pAK13AC with EcoRI & PstI. We also digested pC13F with XbaI and PstI, also with EcoRI and PstI, as well as pAK13D with XbaI and PstI. We transformed ligations of pC13BQ, pC13BR, pC13CC, and pC13CD into <i>E. coli</i>. We also interpreted sequencing results of pC13AS, pC13W, and pC13Y - the latter two look good, and pC13AS may have an overlap, suggesting a mixed population of plasmid.

|tech= We transformed and plated the pA13AW ligation from yesterday. We also retried the PCR to append KpnI and BamHI cut sites to pC13F again, using the newly received Q5 mastermix and higher annealing temperatures. We ran a massive gel screen and found that the cut site PCR and Gibson assembly may have been successful, but that the construction pC13AY most likely failed. We then digested pC13F with cut sites appended with BamHI and KpnI  for ligation with pC13BE to make pC13BF. We digested pA13CZ with SpeI and PstI, which is to be ligated with ''GFP'' from pC13Z and ''mRFP'' from pC13AK. We ligated the Xpa1, Pst1, Spe1, Apa digested pC13AK and Spe1 and Pst1 digested and dephosphorylated pC13BG to make pC13CG, which we transformed into BL21-A1 cells to check transformation efficiency. We also tranformed into DH5α and plated both for comparison. The sequencing results of pC13BJ 7, pC13BK 6, pC13BL 7, and pC13BM 6 confirmed that we were successful, so we made new cultures to create glycerol stocks from, but the initial cultures were thrown out and the plates were difficult to re-pick from, so we hope that cells made it into the culture and have the correct plasmid. We did a PCR clean-up for pAK13AB, pAK13AC, pC13A, pC13A, pC13N, pC13L. We digested pC13I, pC13J, pC13N, pC13L with XbaI & PstI, pAK13AB & pAK13AC with EcoRI & PstI. We also digested pC13F with XbaI and PstI, also with EcoRI and PstI, as well as pAK13D with XbaI and PstI. We transformed ligations of pC13BQ, pC13BR, pC13CC, and pC13CD into <i>E. coli</i>. We also interpreted sequencing results of pC13AS, pC13W, and pC13Y - the latter two look good, and pC13AS may have an overlap, suggesting a mixed population of plasmid.

|author=Rafael

|author=Rafael

|type=wet

|type=wet

|head=August 21st

|head=August 21st

|author=Rafael

|author=Rafael

}}

}}

|head=August 22nd

|head=August 22nd

|text= We did a bunch of ligations and are continuing cloning of ''PgpdA'', ''Anderson promoter'' with ''T7 Polymerase'', ''GFP'', geneticin resistance, and a strong RBS. We ligated pC13I and pC13AF.  

|text= We did a bunch of ligations and are continuing cloning of ''PgpdA'', ''Anderson promoter'' with ''T7 Polymerase'', ''GFP'', geneticin resistance, and a strong RBS. We ligated pC13I and pC13AF.  

|author=Rafael

|author=Rafael

}}

}}

|type=wet

|type=wet

|head=August 23rd

|head=August 23rd

|tech= Today we made liquid cultures of and ran colony PCRs of pA13DC, pA13DB, pC13AU, pC13AT, and pC13CB. pC13CF did not grow any colonies. We then ran a gel of the PCR products. pC13CB colony 5, pC13AT colonies 1-3 and 5, pC13DC colonies 2-5, pC13DB colonies 1-5, and pC13AU colonies 1 and 5 are looked like they had the proper insert lengths from the gel image. Sequencing results came back confirming that pC13CG 3 worked We digested our site directed mutagenesis attempt from yesterday with DpnI and stored it in the fridge. We ran a gel combined with other DNA from other team members, which resulted in a smeared band that was in the correct place. We believe this will be successful, and we will transform our site directed mutagenesis product as soon as desalting membranes arrive. We mini-prepped and plated pC13AG, pAK13CR, pAK13CS. We miniprepped pC13BQ and pC13BR, and submitted pC13CC, pC13CD, pC13BQ, and pC13BR for sequencing. We ran PCRs of the inserts in pAK13BB and pAK13BU to prepare for further cloning.

|tech= Today we made liquid cultures of and ran colony PCRs of pA13DC, pA13DB, pC13AU, pC13AT, and pC13CB. pC13CF did not grow any colonies. We then ran a gel of the PCR products. pC13CB colony 5, pC13AT colonies 1-3 and 5, pC13DC colonies 2-5, pC13DB colonies 1-5, and pC13AU colonies 1 and 5 are looked like they had the proper insert lengths from the gel image. Sequencing results came back confirming that pC13CG 3 worked We digested our site directed mutagenesis attempt from yesterday with DpnI and stored it in the fridge. We ran a gel combined with other DNA from other team members, which resulted in a smeared band that was in the correct place. We believe this will be successful, and we will transform our site directed mutagenesis product as soon as desalting membranes arrive. We mini-prepped and plated pC13AG, pAK13CR, pAK13CS. We miniprepped pC13BQ and pC13BR, and submitted pC13CC, pC13CD, pC13BQ, and pC13BR for sequencing. We ran PCRs of the inserts in pAK13BB and pAK13BU to prepare for further cloning.

|author=Rafael

|author=Rafael

|head=August 24th

|head=August 24th

|text= Running into some problems with ''PgpdA'' cloning. PCR confirmed that pC13AG and pAK13CR worked! We picked more colonies of pAK13CS for screening.  

|text= Running into some problems with ''PgpdA'' cloning. PCR confirmed that pC13AG and pAK13CR worked! We picked more colonies of pAK13CS for screening.  

|tech= We cultured transformants for our Gibson assembly of pC13BH using 1:2 vector to insert ratios, but got no colonies for the 1:6 ratio Gibson and the pC13BF ligation. We also digested our pA13AW minipreps for gel screening. Our PCR confirmed that pC13AG and pAK13CR worked. We picked more colonies of pAK13CS from electroporation plate to screen more colonies.  

|tech= We cultured transformants for our Gibson assembly of pC13BH using 1:2 vector to insert ratios, but got no colonies for the 1:6 ratio Gibson and the pC13BF ligation. We also digested our pA13AW minipreps for gel screening. Our PCR confirmed that pC13AG and pAK13CR worked. We picked more colonies of pAK13CS from electroporation plate to screen more colonies.  

|author=Rafael

|author=Rafael

}}

}}

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|text=Christine's back! She started looking at the components for a humidity sensor and placed an order for a mistmaker which would be able to control humidity levels. In the meantime, Sara studied the details of the heating circuit which utilizes a transistor to amplify signals and switches it such that the resistor could be used as the heating element.   

|text=Christine's back! She started looking at the components for a humidity sensor and placed an order for a mistmaker which would be able to control humidity levels. In the meantime, Sara studied the details of the heating circuit which utilizes a transistor to amplify signals and switches it such that the resistor could be used as the heating element.   

|author=Rebecca

|author=Rebecca

}}

}}

<html>

<html>

<h5 class="centered">(08/26 - 09/01)</h5>

<h5 class="centered">(08/26 - 09/01)</h5>

</html>

</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|text=Our materials are here and everyone in the fabrication group has returned to Ithaca!  This week, Manny and Mac focused their efforts on getting the plastic cut to the proper dimensions.  Unfortunately, this proved more to be more difficult than anyone could have imagined.  After transporting a rather hefty 48"x48" piece of plastic across campus from Weill Hall to the Emerson Lab machine shop, we were told that we would have to take it elsewhere to have it cut (plastic is bad for blades).  Disappointed, but never defeated, we decided to try our luck with another machine shop.  Also, carrying plastic around campus isn't much fun, so we held it on the roof of a car instead (not recommended).  Fortunately, no traffic violations were received, and the Clark hall machine shop was more than happy to cut up our plastic for us.   

|text=Our materials are here and everyone in the fabrication group has returned to Ithaca!  This week, Manny and Mac focused their efforts on getting the plastic cut to the proper dimensions.  Unfortunately, this proved more to be more difficult than anyone could have imagined.  After transporting a rather hefty 48"x48" piece of plastic across campus from Weill Hall to the Emerson Lab machine shop, we were told that we would have to take it elsewhere to have it cut (plastic is bad for blades).  Disappointed, but never defeated, we decided to try our luck with another machine shop.  Also, carrying plastic around campus isn't much fun, so we held it on the roof of a car instead (not recommended).  Fortunately, no traffic violations were received, and the Clark hall machine shop was more than happy to cut up our plastic for us.   

|author=Mac  

|author=Mac  

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

<h5 class="centered">(09/02 - 09/08)</h5>

<h5 class="centered">(09/02 - 09/08)</h5>

</html>

</html>

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=dry

|type=dry

|head=September 4th

|head=September 4th

|text= Transformed the ''PpelA'' and ''Anderson promoter'' + ''T7 polymerase'' into pSB1C3 as well as digest and ligate other promoters. The pC13BN construct was incorrect, so we will have to redo all of the Gibson transformations for the ''lox'' constructs. On a brighter note, sequence confirmation of pA13DB and pA13DC came back today!

|text= Transformed the ''PpelA'' and ''Anderson promoter'' + ''T7 polymerase'' into pSB1C3 as well as digest and ligate other promoters. The pC13BN construct was incorrect, so we will have to redo all of the Gibson transformations for the ''lox'' constructs. On a brighter note, sequence confirmation of pA13DB and pA13DC came back today!

|tech= We got transformants for all ''lox'' constructs. We got sequencing back for pC13BN and it was incorrect, so we will redo the Gibson transformation and all the subsequent ''lox'' constructs. We also electroporated pC13BF and pC13AZ and digested pC13F and pA13AW with XbaI and PstI for ligation to make pC13AZ. pC13F and pC13BE KpnI and BamHI digests were then ligated to make pC13BH. pA13DB colony 1 and pA13DC colony 4 were sequence confirmed today. Lastly, we made cultures from restreaked pC13BM and pC13BK plates.

|tech= We got transformants for all ''lox'' constructs. We got sequencing back for pC13BN and it was incorrect, so we will redo the Gibson transformation and all the subsequent ''lox'' constructs. We also electroporated pC13BF and pC13AZ and digested pC13F and pA13AW with XbaI and PstI for ligation to make pC13AZ. pC13F and pC13BE KpnI and BamHI digests were then ligated to make pC13BH. pA13DB colony 1 and pA13DC colony 4 were sequence confirmed today. Lastly, we made cultures from restreaked pC13BM and pC13BK plates.

|author=Rafael

|author=Rafael

|head=September 5th

|head=September 5th

|text= Today in lab we gel screened a bunch of old minipreps to see if they were still viable and made cultures of the ''Anderson promoter'' + ''T7 polymerase'' in pSB1C3. We digested the ''lox'' site constructs in order to ligate with pC13BN. In a meeting with Professor Turgeon we decided that we would leave <i>G. lucidum</i> behind for now and work with <i>Cochliobolus</i>, which her lab transforms regularly and has more expertise with.

|text= Today in lab we gel screened a bunch of old minipreps to see if they were still viable and made cultures of the ''Anderson promoter'' + ''T7 polymerase'' in pSB1C3. We digested the ''lox'' site constructs in order to ligate with pC13BN. In a meeting with Professor Turgeon we decided that we would leave <i>G. lucidum</i> behind for now and work with <i>Cochliobolus</i>, which her lab transforms regularly and has more expertise with.

|tech= The Gibson was digested with DpnI and electroporated into DH5a. We are growing DH5a for electrocompetent stocks. We did a PCR of pSB1C3 to append cut sites in order to ligate with ''holin''. We digested pC13Y, pC13BQ, pC13BR, and pC13CN for ligation with pC13BN and made cultures of three pC13AZ colonies. Gel screen of pC13F and pA13AW appeared successful and were then column purified. We also gel screened pC13P miniprep, SpeI and PstI digested pC13P, PCR of ''PgpdA'' for homologous regions, PCR of pC13F for homologous regions, and BamHI/KpnI digested pA13BE to verify the viability of old minipreps. We then amplified ''T7 polymerase'' from pC13E. We did a Q5 PCR for pC13CV, pC13I and pC13L. Our plates from transformation on Saturday showed conidia growth on the controls with no hygromycin. None of the plates with hygromycin showed any growth. We decided that we will now use <i>Cochliobolus</i> to protoplast and transform after meeting with Professor Turgeon again. To determine if the DNA is in the fungal genome and stable, we will plate on non-antibiotic and antibiotic plates, then replate transformants from the antibiotic plate onto non-antibiotic plates and back onto antibiotic plates. Since protoplasting is failing, we will turn to agrobacterium for <i>G. lucidum</i> transformation in the future. We will also check for homology between the promoter gpd in <i>G. lucidum</i> and <i>Cochliobolus</i> to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after <i>Cochliobolus</i> gpd by cutting into that region in the genomic promoter to transform. We also created glycerol stocks of pC13BM and pC13BK cultures and miniprepped them to submit for sequencing. We set up new cultures of colonies from transformation of pC13BC, pC13BV, and pC13CE, and ran colony PCRs of them (using our brand new tube of Q5 polymerase!) to check for inserts.

|tech= The Gibson was digested with DpnI and electroporated into DH5a. We are growing DH5a for electrocompetent stocks. We did a PCR of pSB1C3 to append cut sites in order to ligate with ''holin''. We digested pC13Y, pC13BQ, pC13BR, and pC13CN for ligation with pC13BN and made cultures of three pC13AZ colonies. Gel screen of pC13F and pA13AW appeared successful and were then column purified. We also gel screened pC13P miniprep, SpeI and PstI digested pC13P, PCR of ''PgpdA'' for homologous regions, PCR of pC13F for homologous regions, and BamHI/KpnI digested pA13BE to verify the viability of old minipreps. We then amplified ''T7 polymerase'' from pC13E. We did a Q5 PCR for pC13CV, pC13I and pC13L. Our plates from transformation on Saturday showed conidia growth on the controls with no hygromycin. None of the plates with hygromycin showed any growth. We decided that we will now use <i>Cochliobolus</i> to protoplast and transform after meeting with Professor Turgeon again. To determine if the DNA is in the fungal genome and stable, we will plate on non-antibiotic and antibiotic plates, then replate transformants from the antibiotic plate onto non-antibiotic plates and back onto antibiotic plates. Since protoplasting is failing, we will turn to agrobacterium for <i>G. lucidum</i> transformation in the future. We will also check for homology between the promoter gpd in <i>G. lucidum</i> and <i>Cochliobolus</i> to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after <i>Cochliobolus</i> gpd by cutting into that region in the genomic promoter to transform. We also created glycerol stocks of pC13BM and pC13BK cultures and miniprepped them to submit for sequencing. We set up new cultures of colonies from transformation of pC13BC, pC13BV, and pC13CE, and ran colony PCRs of them (using our brand new tube of Q5 polymerase!) to check for inserts.

|author=Rafael

|author=Rafael

|head=September 6th

|head=September 6th

|text= Most of old minipreps were no good and the ''PpelA'' transformation failed. The ''Anderson promoter+T7 polymerase'' gel results looked good and were submitted for sequencing. Oh no, the PCR of pC13CV, pC13I and pC13L failed again--we suspect something is wrong with our reagents. Use the new dNTPs from now on!

|text= Most of old minipreps were no good and the ''PpelA'' transformation failed. The ''Anderson promoter+T7 polymerase'' gel results looked good and were submitted for sequencing. Oh no, the PCR of pC13CV, pC13I and pC13L failed again--we suspect something is wrong with our reagents. Use the new dNTPs from now on!

|tech= We digested, ligated, and transformed to create pC13DD. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin''. Gel results indicate that pC13P miniprep, pC13P SpeI/PstI digests, ''PgpdA'' PCR for Gibson and the pA13BE BamHI/KpnI/SacI digest are not viable, but the pC13F PCR for Gibson might be okay. pC13BF transformation failed as well. The pC13AZ cultures were digest screened; results were good, so they were sent for sequencing. pC13P was plated and pC13E was cultured. We submitted pC13AF (colony #2) for sequencing. The Q5 PCR from yesterday--pC13CV, pC13I and pC13L--was a failure. We suspected that something is wrong with our reagents. Use the new dNTPs from now on! We ran PCRs to prepare the necessary parts for our Gibson assembly to produce parts with flanking <i>G. lucidum</i> homologous regions. We spun down the cultures of pC13BC, pC13BV, and pC13CE and stored them in the fridge.

|tech= We digested, ligated, and transformed to create pC13DD. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin''. Gel results indicate that pC13P miniprep, pC13P SpeI/PstI digests, ''PgpdA'' PCR for Gibson and the pA13BE BamHI/KpnI/SacI digest are not viable, but the pC13F PCR for Gibson might be okay. pC13BF transformation failed as well. The pC13AZ cultures were digest screened; results were good, so they were sent for sequencing. pC13P was plated and pC13E was cultured. We submitted pC13AF (colony #2) for sequencing. The Q5 PCR from yesterday--pC13CV, pC13I and pC13L--was a failure. We suspected that something is wrong with our reagents. Use the new dNTPs from now on! We ran PCRs to prepare the necessary parts for our Gibson assembly to produce parts with flanking <i>G. lucidum</i> homologous regions. We spun down the cultures of pC13BC, pC13BV, and pC13CE and stored them in the fridge.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type= anim

|type= anim

|head=September 7th

|head=September 7th

|text= Digested and ligated the ''Anderson promoter+T7 polymerase'' into pSB1C3 and pSB2K3 as well as start cultures from glycerol stocks and transformation and gel screened the ''Anderson promoter'' + ''T7 polymerase''. We transformed ''afp1'' and ''Cht1_2''. For fluorescence, we electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ to obtain more plasmid DNA. We redid a Q5 PCR for pC13CV, pC13I, pC13AF and pC13L with the new dNTPs--PCR products were all bad except for pC13L.

|text= Digested and ligated the ''Anderson promoter+T7 polymerase'' into pSB1C3 and pSB2K3 as well as start cultures from glycerol stocks and transformation and gel screened the ''Anderson promoter'' + ''T7 polymerase''. We transformed ''afp1'' and ''Cht1_2''. For fluorescence, we electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ to obtain more plasmid DNA. We redid a Q5 PCR for pC13CV, pC13I, pC13AF and pC13L with the new dNTPs--PCR products were all bad except for pC13L.

|tech= We did Q5 PCR to amplify pSB1C3 for Gibson with ''afp1'' and ''Cht1_2''. 3uL of PCR product was run on the gel, found to correct, and was cleaned. We ran a Gibson of pSB1C3 with both pC13CL and pC13BA. Both of these were transformed, ''Cht1_2'' into DH5a (time constant 4.82) and ''afp1'' into BL21 (time constant 4.84). We made glycerol stock of pC13BN. We ran a gel of pSB1C3 PCR from yesterday, which was successful, and performed a PCR clean up. We digested pSB1C3 and pC13CN with HindIII and BglII. We ligated these together to create pC13CO and transformed it into DH5a (time constant 4.82). We miniprepped pC13BN culture and did a double digest on pC13BN culture with KpnI and HindIII to see if internal cut sites were present. There was some digestion that occurred when it was run on a gel, so we are confident the sequence is correct. We double digested pC13BN again with KpnI and HindIII. We ligated it with pC13Y (time constant 4.86), pC13BR (4.86), pC13BQ (4.90), pC13CD (4.80). We got transformants for pC13DD. A colony PCR was run and pC13DD colony 3 looked good. This colony was grown in liquid culture for miniprepping and sequencing. We grew cultures of pC13BX, pC13CK, and pC13BN for submission (pC13BX submitted directly; pC13CK and pC13BN need sequence confirmation first). pC13BF transformants were cultured, pC13AZ cultures were digested with EcoRI/PstI and gel screened; results were good, so they were submitted for sequencing. Digested pK13AL and pA13AW with XbaI/PstI for ligation to make pK13AX. pC13P and pC13E were digested with EcoRI/PstI and ligated to make pC13AY. Cultures of pC13Q and pC13BE were made from glycerol stocks. We electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ. We also ran a Q5 PCR of pC13CB and pAK13AO using standard BioBrick primers 23 and 24 in order to confirm lengths of inserts. We redid the Q5 PCR with the new dNTPs for pC13CV, pC13I, pC13AF  and pC13L--pC13CV, pC13I and pC13AF were bad, but pC13L was good! We suspect that electroporation did not go well to begin with. We reran the 2-step Q5 PCR for pC13A. We ran a gel of our PCR to create Gibson assembly parts, and it appeared unsuccessful. We ran the colony PCRs of pC13BC, pC13BV, and pC13CE on a gel, and only observed bands for a few pC13BC colonies.

|tech= We did Q5 PCR to amplify pSB1C3 for Gibson with ''afp1'' and ''Cht1_2''. 3uL of PCR product was run on the gel, found to correct, and was cleaned. We ran a Gibson of pSB1C3 with both pC13CL and pC13BA. Both of these were transformed, ''Cht1_2'' into DH5a (time constant 4.82) and ''afp1'' into BL21 (time constant 4.84). We made glycerol stock of pC13BN. We ran a gel of pSB1C3 PCR from yesterday, which was successful, and performed a PCR clean up. We digested pSB1C3 and pC13CN with HindIII and BglII. We ligated these together to create pC13CO and transformed it into DH5a (time constant 4.82). We miniprepped pC13BN culture and did a double digest on pC13BN culture with KpnI and HindIII to see if internal cut sites were present. There was some digestion that occurred when it was run on a gel, so we are confident the sequence is correct. We double digested pC13BN again with KpnI and HindIII. We ligated it with pC13Y (time constant 4.86), pC13BR (4.86), pC13BQ (4.90), pC13CD (4.80). We got transformants for pC13DD. A colony PCR was run and pC13DD colony 3 looked good. This colony was grown in liquid culture for miniprepping and sequencing. We grew cultures of pC13BX, pC13CK, and pC13BN for submission (pC13BX submitted directly; pC13CK and pC13BN need sequence confirmation first). pC13BF transformants were cultured, pC13AZ cultures were digested with EcoRI/PstI and gel screened; results were good, so they were submitted for sequencing. Digested pK13AL and pA13AW with XbaI/PstI for ligation to make pK13AX. pC13P and pC13E were digested with EcoRI/PstI and ligated to make pC13AY. Cultures of pC13Q and pC13BE were made from glycerol stocks. We electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ. We also ran a Q5 PCR of pC13CB and pAK13AO using standard BioBrick primers 23 and 24 in order to confirm lengths of inserts. We redid the Q5 PCR with the new dNTPs for pC13CV, pC13I, pC13AF  and pC13L--pC13CV, pC13I and pC13AF were bad, but pC13L was good! We suspect that electroporation did not go well to begin with. We reran the 2-step Q5 PCR for pC13A. We ran a gel of our PCR to create Gibson assembly parts, and it appeared unsuccessful. We ran the colony PCRs of pC13BC, pC13BV, and pC13CE on a gel, and only observed bands for a few pC13BC colonies.

|author=Rafael

|author=Rafael

}}

}}

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|type=hprac

|type=hprac

|tech=  

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|author=Arun

|author=Arun

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{{:Team:Cornell/templates/nbentry

|author=Eric, Arun  

|author=Eric, Arun  

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<h5 class="centered">(09/09 - 09/15)</h5>

<h5 class="centered">(09/09 - 09/15)</h5>

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{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=wet

|type=wet

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=anim

|type=anim

|head=September 10th

|head=September 10th

|text= ''Anderson promoter'' + ''T7 polymerase'', in pSB1C3 and BBa_J61002 were miniprepped, glycerol stocked, and prepared for biobrick submission. For fluorescent constructs, a Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. The pC13BN construct was incorrect. An overnight culture was prepared for ''rbs'' + ''crtI'' (from pC13L) at 4C. We made our first attempt at protoplasting and transforming <i>Cochliobolus</i> with the help of the Turgeon Lab. We set up cultures of pC13X and pC13AV in preparation for part submission. We started the growth experiment for testing <i>G. lucidum</i>’s antibiotic sensitivity.

|text= ''Anderson promoter'' + ''T7 polymerase'', in pSB1C3 and BBa_J61002 were miniprepped, glycerol stocked, and prepared for biobrick submission. For fluorescent constructs, a Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. The pC13BN construct was incorrect. An overnight culture was prepared for ''rbs'' + ''crtI'' (from pC13L) at 4C. We made our first attempt at protoplasting and transforming <i>Cochliobolus</i> with the help of the Turgeon Lab. We set up cultures of pC13X and pC13AV in preparation for part submission. We started the growth experiment for testing <i>G. lucidum</i>’s antibiotic sensitivity.

|tech= We prepared pC13CO, pC13CL, pC13BA for sequencing. We made glycerol stocks of pC13CL and pC13BA. We got a sequence confirmation  for pC13CK. We got sequencing for pC13BN and had a portion of the lac operon inside the prefix and suffix...a weird recombination must have occurred. pC13AZ sequencing results were good! That and pA13AM and pA13AW were miniprepped for glycerol stocks and biobrick submission. A PCR to add homologous regions to pC13F for Gibson assembly was done. The gel of Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. Thus, we miniprepped the colonies with correct plasmid insert lengths and submitted them for sequencing. We ran a PCR and electroporation for pC13CV  and pC13I, which turned out to be bad. We made overnight E. Coli culture for pC13AF (colony #2). An overnight ligation was done for ''rbs'' + ''crtI'' (from pC13L) at 4C. We ran a PCR to check the inserts in pC13A, pC13I and pC13J. We protoplasted and transformed <i>Cochliobolus</i> with Dongliang, a post-doctoral research associate in the Turgeon Lab. We used our constructs of pAh13G / pC13BR linearized with SphI, as well as the highly efficient <i>Cochliobolus</i> vector PNG from the Turgeon lab as a "positive" control. We submitted pC13BM 1 and pC13BK 1 for sequencing. We homogenized a dilute culture of <i>G. lucidum</i> and inoculated each of the plates for the antibiotic sensitivity test with 20 μL aliquots, without spreading.

|tech= We prepared pC13CO, pC13CL, pC13BA for sequencing. We made glycerol stocks of pC13CL and pC13BA. We got a sequence confirmation  for pC13CK. We got sequencing for pC13BN and had a portion of the lac operon inside the prefix and suffix...a weird recombination must have occurred. pC13AZ sequencing results were good! That and pA13AM and pA13AW were miniprepped for glycerol stocks and biobrick submission. A PCR to add homologous regions to pC13F for Gibson assembly was done. The gel of Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. Thus, we miniprepped the colonies with correct plasmid insert lengths and submitted them for sequencing. We ran a PCR and electroporation for pC13CV  and pC13I, which turned out to be bad. We made overnight E. Coli culture for pC13AF (colony #2). An overnight ligation was done for ''rbs'' + ''crtI'' (from pC13L) at 4C. We ran a PCR to check the inserts in pC13A, pC13I and pC13J. We protoplasted and transformed <i>Cochliobolus</i> with Dongliang, a post-doctoral research associate in the Turgeon Lab. We used our constructs of pAh13G / pC13BR linearized with SphI, as well as the highly efficient <i>Cochliobolus</i> vector PNG from the Turgeon lab as a "positive" control. We submitted pC13BM 1 and pC13BK 1 for sequencing. We homogenized a dilute culture of <i>G. lucidum</i> and inoculated each of the plates for the antibiotic sensitivity test with 20 μL aliquots, without spreading.

|author=Rafael

|author=Rafael

}}

}}

{{:Team:Cornell/templates/nbentry

{{:Team:Cornell/templates/nbentry

|type=anim

|type=anim

|head=September 11th

|head=September 11th

|text= Redid transformations of ''PpelA'' and the ''Anderson promoter+T7 polymerase''. We sent the ''Anderson promoter'', pC13CO, pC13CL, and pC13BA to sequencing. Gel electroporation for pC13I showed that it was good, but nothing for pC13J. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists in iGEM! We continued with protoplasting <i>Cochliobolus</i> in the Turgeon Lab, and we ran a Gibson assembly of homology regions in <i>G. lucidum</i>.

|text= Redid transformations of ''PpelA'' and the ''Anderson promoter+T7 polymerase''. We sent the ''Anderson promoter'', pC13CO, pC13CL, and pC13BA to sequencing. Gel electroporation for pC13I showed that it was good, but nothing for pC13J. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists in iGEM! We continued with protoplasting <i>Cochliobolus</i> in the Turgeon Lab, and we ran a Gibson assembly of homology regions in <i>G. lucidum</i>.

|tech= We submitted pC13CO, pC13CL, and pC13BA for sequencing. After, we miniprepped and made stocks of pC13AY and pC13AZ. Also, we submitted pA13AW for sequencing. Transformation of pK13AX was unsuccessful because the wrong resistance plate was used. pC13BF and pK13AX were transformed today. We miniprepped the overnight culture of pC13AF. Gel electroporation showed that pC13I samples are good, but none of the pC13J samples are positive. We ran a PCR for the inserts in pAK13CR. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists! We made 1000x hygromycin and 100x geniticin. We checked plates from our protoplasting and transforming attempt under the microscope. It appeared unsuccessful so next time we will use all the protoplasts, at least 300 µL. We plated a layer of cym agar and the respective antibiotic on top of the regeneration agar gel as well as inoculating 4 new plates of <i>Cochliobolus</i>. We also ran our Gibson assembly of homology regions in <i>G. lucidum</i>. We miniprepped pC13X and pC13AV in anticipation of part submission, and ran a PCR (to check for inserts) from minipreps of pC13BC, pC13BV, and pC13CE, as well as pC13X and pC13AV (just in case).

|tech= We submitted pC13CO, pC13CL, and pC13BA for sequencing. After, we miniprepped and made stocks of pC13AY and pC13AZ. Also, we submitted pA13AW for sequencing. Transformation of pK13AX was unsuccessful because the wrong resistance plate was used. pC13BF and pK13AX were transformed today. We miniprepped the overnight culture of pC13AF. Gel electroporation showed that pC13I samples are good, but none of the pC13J samples are positive. We ran a PCR for the inserts in pAK13CR. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists! We made 1000x hygromycin and 100x geniticin. We checked plates from our protoplasting and transforming attempt under the microscope. It appeared unsuccessful so next time we will use all the protoplasts, at least 300 µL. We plated a layer of cym agar and the respective antibiotic on top of the regeneration agar gel as well as inoculating 4 new plates of <i>Cochliobolus</i>. We also ran our Gibson assembly of homology regions in <i>G. lucidum</i>. We miniprepped pC13X and pC13AV in anticipation of part submission, and ran a PCR (to check for inserts) from minipreps of pC13BC, pC13BV, and pC13CE, as well as pC13X and pC13AV (just in case).

|author=Rafael

|author=Rafael

|head=September 12th

|head=September 12th

|text= Ran Gibson assembly for the <i>G. lucidum</i> promoter into pSB1C3. pC13BA is now a BioBrick! We also began cloning a construct with both fluorescence and resistance genes by ligating pC13BR and pC13AT. We successfully electroporated ''E.Coli'' with pC13J! We digested pAK13CR, and then we ligated ''rbs'' + ''crtB'' into pSB1C3, ''crtE'' into pSB1C3. After finding our Gibson assembly from last night left in the water bath, we attempted it again as well as preparing some of our Biobricks for submission. We checked pC13BC, pC13BV, and pC13CE and prepared the successful constructs for sequencing.

|text= Ran Gibson assembly for the <i>G. lucidum</i> promoter into pSB1C3. pC13BA is now a BioBrick! We also began cloning a construct with both fluorescence and resistance genes by ligating pC13BR and pC13AT. We successfully electroporated ''E.Coli'' with pC13J! We digested pAK13CR, and then we ligated ''rbs'' + ''crtB'' into pSB1C3, ''crtE'' into pSB1C3. After finding our Gibson assembly from last night left in the water bath, we attempted it again as well as preparing some of our Biobricks for submission. We checked pC13BC, pC13BV, and pC13CE and prepared the successful constructs for sequencing.

|tech= We prepped pC13BX and pC13BA for BioBrick submission and got sequencing back. pC13BA was correct, and pC13CO and pC13CL were incorrect. We redid the Gibson for pC13CL and transformed it. We redid pC13CO digestions, ligation, and transformations. We transformed the redone ''lox'' Gibson from yesterday. We streaked out CYM plates with <i>Aspergillus Niger</i>, ran the Gibson assembly for ''PgpdA'' in pC13F, and transformed. We also digested pC13BR with SpeI and PstI and digested pC13AT with Xba and PstI, ran a gel of digestions to verify lengths, and column purified the inserts from digestion. We did an electroporation with pC13J and it was successful! We ran a gel for pAK13CR for confirmation and then we did a PCR clean up. We digested pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, and then we ligated ''rbs'' + ''crtB'' (from pAK13CR) into pSB1C3, ''crtE'' (from pC13I) into pSB1C3. We prepared pC13BJ pC13BK pC13BL pC13BM and pC13BR for Biobrick submission. It seems our Gibson assemblies from yesterday were left in the water bath all night, so we reran them (pC13BK + pC13BM + pC13CD, pC13BQ, or pC13Y) before we transformed and plated the new assembled plasmids. We made a culture of pC13CG from glycerol stock. We ran a gel of the PCRs of pC13BC, pC13BV, pC13CE, pC13X, and pC13AV - there were correct bands for at least two of everything, and successful pC13BC, pC13BV, and pC13CE constructs were prepared for sequencing. We also set up a culture of pC13BQ in anticipation of part submission.

|tech= We prepped pC13BX and pC13BA for BioBrick submission and got sequencing back. pC13BA was correct, and pC13CO and pC13CL were incorrect. We redid the Gibson for pC13CL and transformed it. We redid pC13CO digestions, ligation, and transformations. We transformed the redone ''lox'' Gibson from yesterday. We streaked out CYM plates with <i>Aspergillus Niger</i>, ran the Gibson assembly for ''PgpdA'' in pC13F, and transformed. We also digested pC13BR with SpeI and PstI and digested pC13AT with Xba and PstI, ran a gel of digestions to verify lengths, and column purified the inserts from digestion. We did an electroporation with pC13J and it was successful! We ran a gel for pAK13CR for confirmation and then we did a PCR clean up. We digested pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, and then we ligated ''rbs'' + ''crtB'' (from pAK13CR) into pSB1C3, ''crtE'' (from pC13I) into pSB1C3. We prepared pC13BJ pC13BK pC13BL pC13BM and pC13BR for Biobrick submission. It seems our Gibson assemblies from yesterday were left in the water bath all night, so we reran them (pC13BK + pC13BM + pC13CD, pC13BQ, or pC13Y) before we transformed and plated the new assembled plasmids. We made a culture of pC13CG from glycerol stock. We ran a gel of the PCRs of pC13BC, pC13BV, pC13CE, pC13X, and pC13AV - there were correct bands for at least two of everything, and successful pC13BC, pC13BV, and pC13CE constructs were prepared for sequencing. We also set up a culture of pC13BQ in anticipation of part submission.

|author=Rafael

|author=Rafael

}}

}}

|head=September 13th

|head=September 13th

|text= Gibson assembly for the <i>G. lucidum</i> promoter yielded no colonies. Numerous ligations and electroporations were done for fluorescent constructs. We are starting the ''afp1'' <i>Aspergillus</i> growth assay. We did electroporation for pC13I, pC13CV and pAK13CS but pAK13CS arced. We prepared numerous Biobricks for submission to iGEM HQ.

|text= Gibson assembly for the <i>G. lucidum</i> promoter yielded no colonies. Numerous ligations and electroporations were done for fluorescent constructs. We are starting the ''afp1'' <i>Aspergillus</i> growth assay. We did electroporation for pC13I, pC13CV and pAK13CS but pAK13CS arced. We prepared numerous Biobricks for submission to iGEM HQ.

|tech= We submitted pC13BC, pC13BV, and pC13CE for sequencing. We reinoculated cultures of pC13BN, pC13CL, pC13CO. Then, we conducted a preliminary test on activity of ''afp1''. We cut circles of various sizes from dialyzing membranes and dipped in grown ''afp1'' in BL21 culture. The membrane pieces were then placed on the fully grown <i>Aspergillus</i> plate. This preliminary test will see whether or not there is activity of ''afp1'' on the <i>Aspergillus</i>.  We are hoping for a ring of death on the <i>Aspergillus</i> plate. Unfortunately, there were no colonies for the ''PgpdA'' assembly. We then ran a gel for some of the remaining assembled DNA and no bands appeared. We also digested pC13AU with Xba and PstI to ligate downstream of pC13BR and pC13BQ. We then column purified pC13BQ, pC13BR, pC13AT, pAK13AO, pC13AU, pC13F. After, we dephosphorylated pC13BQ, pC13BR, pC13F, and ligated pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, and pAK13AO with pC13F. Next, we electroporated all ligations into both BL21 and DH5α cells. Hopefully, a fluorescence trial can be done tomorrow with these colonies! We made overnight culture for pC13J. We did electroporation for pC13I, pC13CV and pAK13CS. The last one unfortunately arced. Our Gibson-transformed plates had too many colonies so we restreaked them. We miniprepped pC13CG and prepared it for Biobrick submission. We also checked our plates in the Turgeon lab. The control plate is growing well, and the others appear to have something very small starting to grow. We will give them more time before checking again. We also received our sequencing results from pC13BM 1, pC13BK 1, pC13BC, pC13BV, and pC13CE, which all appear to have been successful. We set up a culture of pC13CD from its glycerol stock to re-miniprep and submit as a biobrick.

|tech= We submitted pC13BC, pC13BV, and pC13CE for sequencing. We reinoculated cultures of pC13BN, pC13CL, pC13CO. Then, we conducted a preliminary test on activity of ''afp1''. We cut circles of various sizes from dialyzing membranes and dipped in grown ''afp1'' in BL21 culture. The membrane pieces were then placed on the fully grown <i>Aspergillus</i> plate. This preliminary test will see whether or not there is activity of ''afp1'' on the <i>Aspergillus</i>.  We are hoping for a ring of death on the <i>Aspergillus</i> plate. Unfortunately, there were no colonies for the ''PgpdA'' assembly. We then ran a gel for some of the remaining assembled DNA and no bands appeared. We also digested pC13AU with Xba and PstI to ligate downstream of pC13BR and pC13BQ. We then column purified pC13BQ, pC13BR, pC13AT, pAK13AO, pC13AU, pC13F. After, we dephosphorylated pC13BQ, pC13BR, pC13F, and ligated pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, and pAK13AO with pC13F. Next, we electroporated all ligations into both BL21 and DH5α cells. Hopefully, a fluorescence trial can be done tomorrow with these colonies! We made overnight culture for pC13J. We did electroporation for pC13I, pC13CV and pAK13CS. The last one unfortunately arced. Our Gibson-transformed plates had too many colonies so we restreaked them. We miniprepped pC13CG and prepared it for Biobrick submission. We also checked our plates in the Turgeon lab. The control plate is growing well, and the others appear to have something very small starting to grow. We will give them more time before checking again. We also received our sequencing results from pC13BM 1, pC13BK 1, pC13BC, pC13BV, and pC13CE, which all appear to have been successful. We set up a culture of pC13CD from its glycerol stock to re-miniprep and submit as a biobrick.

|author=Rafael

|author=Rafael

}}

}}

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|type=dry

|type=dry

|head=September 14th

|head=September 14th

|text= The ''Anderson promoter'' + ''T7 polymerase'' were electroporated, but both ligations arced, so transformations are very likely to not succeed. We are unsuccessful with pC13CL and pC13BN. We miniprepped pC13J cultures.  

|text= The ''Anderson promoter'' + ''T7 polymerase'' were electroporated, but both ligations arced, so transformations are very likely to not succeed. We are unsuccessful with pC13CL and pC13BN. We miniprepped pC13J cultures.  

|tech= Electroporated pK13AX, but both arced, so outlook is grim. We also picked 12 colonies from potential pAK13CY colonies and grew corresponding colonies in liquid culture. We then prepped pC13AQ, pC13AT, and pC13AU for biobrick submission. Later that day, we picked colonies for pC13BN and pC13CL from plates from Thursday, did a colony PCR, and grew cultures for each. We re-electroporated the pC13CO construct, and plated four different versions of it. We ran a gel of all 14 colonies of pC13BN and pC13CL. The pC13CL one came out blank and the pC13BN had bands, but not where they were expected. We miniprepped pC13J and made glycerol stock. We ran PCR to check the following inserts: pC13AF, pC13P, pC13CV, pC13I and pC13BG. We ran colony PCRs on the restreaked plates containing our Gibson assembly products of homology regions. We miniprepped pC13BQ and pC13CD for part submission.

|tech= Electroporated pK13AX, but both arced, so outlook is grim. We also picked 12 colonies from potential pAK13CY colonies and grew corresponding colonies in liquid culture. We then prepped pC13AQ, pC13AT, and pC13AU for biobrick submission. Later that day, we picked colonies for pC13BN and pC13CL from plates from Thursday, did a colony PCR, and grew cultures for each. We re-electroporated the pC13CO construct, and plated four different versions of it. We ran a gel of all 14 colonies of pC13BN and pC13CL. The pC13CL one came out blank and the pC13BN had bands, but not where they were expected. We miniprepped pC13J and made glycerol stock. We ran PCR to check the following inserts: pC13AF, pC13P, pC13CV, pC13I and pC13BG. We ran colony PCRs on the restreaked plates containing our Gibson assembly products of homology regions. We miniprepped pC13BQ and pC13CD for part submission.

|author=Rafael

|author=Rafael

}}

}}

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|author=Arun, Eric

|author=Arun, Eric

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<h5 class="centered">(09/16 - 09/22)</h5>

<h5 class="centered">(09/16 - 09/22)</h5>

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{{:Team:Cornell/templates/nbentry

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|type=wet

|type=wet

|tech= We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of <i>G. lucidum</i> growth on CYM plates (plated on September 10) through tracing the central mass of fungus--the differences between the plates are really interesting, but there’s a small contamination on one plate. We transplanted mycelia from the G418 plates. We are slightly concerned that there’s still no growth on the antifungal plates. We submitted pC13CL and pC13BN for sequencing. We ran the gel for the colony PCR of pC13CO but they all failed. We will repick the white colonies from pC13CO plates. We had successful sequencing for pC13AY and we made cultures for glycerol stocks. We also retired the PCR to append cut sites to pC13F using a higher concentration of dNTPs and began a digestion of pA13BE in order to gel extract ''PpelA''. We electroporated ''E. Coli'' with pAK13CS, which unfortunately arced again… We performed gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG. Only pC13J colonies are positive. We also did digestions for the following vectors: pC13BG with SpeI and PstI, pC13AG with EcoRI-HF and XbaI, and pC13K with EcoRI-HF and XbaI.

|tech= We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of <i>G. lucidum</i> growth on CYM plates (plated on September 10) through tracing the central mass of fungus--the differences between the plates are really interesting, but there’s a small contamination on one plate. We transplanted mycelia from the G418 plates. We are slightly concerned that there’s still no growth on the antifungal plates. We submitted pC13CL and pC13BN for sequencing. We ran the gel for the colony PCR of pC13CO but they all failed. We will repick the white colonies from pC13CO plates. We had successful sequencing for pC13AY and we made cultures for glycerol stocks. We also retired the PCR to append cut sites to pC13F using a higher concentration of dNTPs and began a digestion of pA13BE in order to gel extract ''PpelA''. We electroporated ''E. Coli'' with pAK13CS, which unfortunately arced again… We performed gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG. Only pC13J colonies are positive. We also did digestions for the following vectors: pC13BG with SpeI and PstI, pC13AG with EcoRI-HF and XbaI, and pC13K with EcoRI-HF and XbaI.

|author=Rafael

|author=Rafael

}}

}}

|head=September 17th

|head=September 17th

|text=We tried transformation of <i>Cochliobolus</i> again. We ran a PCR to append cut sites to pC13F but no band was shown on a gel. We redid the Gibson of ''Cht1_2'' and pSB1C3. When we ran a PCR fo pC13AI, weird band patterning was observed. We purified the vectors we digested yesterday: pC13BG, pC13AG and pC13K. In addition, we purified inserts of pC13AF and pC13P. We continued recording measurements of <i>G. lucidum</i>’s growth in the presence of antibiotics.

|text=We tried transformation of <i>Cochliobolus</i> again. We ran a PCR to append cut sites to pC13F but no band was shown on a gel. We redid the Gibson of ''Cht1_2'' and pSB1C3. When we ran a PCR fo pC13AI, weird band patterning was observed. We purified the vectors we digested yesterday: pC13BG, pC13AG and pC13K. In addition, we purified inserts of pC13AF and pC13P. We continued recording measurements of <i>G. lucidum</i>’s growth in the presence of antibiotics.

|tech=We attempted transforming <i>Cochliobolus</i> with pC13BR, pC13CD, pUCATPH and pC13AY. We ran a gel of the pA13BE digest and extracted ''PpelA''. We also reran a PCR to append cut sites to pC13F again using a different tube of dNTPs and re-diluted primers. We ran a colony PCR and screened it on a gel, but no bands appeared. Some of the cultures were miniprepped anyways, and they will be screened for presence of the insert (~340bp). We detected some growth on the afp1 assay plates. We redid the Gibson of ''Cht1_2'' and pSB1C3 PCR. We electroporated this Gibson into DH5a cells and plated it on chloramphenicol LB plates. We reran the PCR of pC13AI with primers 45/52 and 48/51, annealing temperature of 61.5C and extended for 30s. We did a double digest of pC13CO with EcoRI-HF & PstI. We ran a 1% TAE gel of pC13AI PCR products for 30 minutes at 100V. The bands for pC13AI with 45/53 were seen at 1.5kb and 1.1kb; bands for pC13AI with 48/51 were seen at 1.5kb and 1kb. We still have a weird banding pattern. We may need to do a gradient PCR to find a better temperature for the primers and to test the PCR without the GC buffer. We purified and dephosphorylated the vectors we digested yesterday--pC13BG, pC13AG and pC13K. We also purified inserts of pC13AF with EcoRI-HF and SpeI, and pC13P with EcoRI-HF and SpeI.  

|tech=We attempted transforming <i>Cochliobolus</i> with pC13BR, pC13CD, pUCATPH and pC13AY. We ran a gel of the pA13BE digest and extracted ''PpelA''. We also reran a PCR to append cut sites to pC13F again using a different tube of dNTPs and re-diluted primers. We ran a colony PCR and screened it on a gel, but no bands appeared. Some of the cultures were miniprepped anyways, and they will be screened for presence of the insert (~340bp). We detected some growth on the afp1 assay plates. We redid the Gibson of ''Cht1_2'' and pSB1C3 PCR. We electroporated this Gibson into DH5a cells and plated it on chloramphenicol LB plates. We reran the PCR of pC13AI with primers 45/52 and 48/51, annealing temperature of 61.5C and extended for 30s. We did a double digest of pC13CO with EcoRI-HF & PstI. We ran a 1% TAE gel of pC13AI PCR products for 30 minutes at 100V. The bands for pC13AI with 45/53 were seen at 1.5kb and 1.1kb; bands for pC13AI with 48/51 were seen at 1.5kb and 1kb. We still have a weird banding pattern. We may need to do a gradient PCR to find a better temperature for the primers and to test the PCR without the GC buffer. We purified and dephosphorylated the vectors we digested yesterday--pC13BG, pC13AG and pC13K. We also purified inserts of pC13AF with EcoRI-HF and SpeI, and pC13P with EcoRI-HF and SpeI.  

|author=Rafael

|author=Rafael

}}

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|type=anim

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|type=wet

|type=wet

|head=September 19th

|head=September 19th

|author=Rafael

|author=Rafael

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|tech= We began another growth assay because the previous ones showed no results. The cells may bave been dead by the time the cells reached them. This method of spreading the ''afp1'' culture like an antibiotic and plating mycelium smoothie on the top should give more consistent results. We retried electroporation with pAK13CS and it sadly arced again… We also ran a Q5 PCR for the insert in pAK13CR. We prepared and autoclaved CYM + agar, for making plates.

|tech= We began another growth assay because the previous ones showed no results. The cells may bave been dead by the time the cells reached them. This method of spreading the ''afp1'' culture like an antibiotic and plating mycelium smoothie on the top should give more consistent results. We retried electroporation with pAK13CS and it sadly arced again… We also ran a Q5 PCR for the insert in pAK13CR. We prepared and autoclaved CYM + agar, for making plates.

|author=Rafael

|author=Rafael

}}

}}

|author=Rafael

|author=Rafael

}}

}}

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|type=anim

|type=anim

|author=Rafael

|author=Rafael

}}

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<h5 class="centered">(09/23 - 09/29)</h5>

<h5 class="centered">(09/23 - 09/29)</h5>

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|type=wet

|type=wet

|tech= Two gels, each with 20 wells, were run simultaneously for fluorescence. The DNA samples loaded were from a Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. pC13BV was transformed into electrocompetent BL21 <i>E. coli</i>. A culture of <i>G. lucidum</i> was diluted and homogenized, and 150 μL aliquots were spread on plates and let dry. Paper discs were soaked in 100X solutions of geniticin, hygromycin, ampicillin, kanamycin, and chloramphenicol and placed in the center of each plate. We miniprepped ''holin'' 8 & 9. The results from the growth assay do not look good. However, we found that the T7 polymerase in BL21 is controlled with an IPTG inducible promoter, so more pC13BA was grown with IPTG and plates were all re-inoculated with the corresponding amounts. An appropriate volume of LB was also added to some plates to account for mass changes. The zone of inhibition tests were also restarted with recultured (with IPTG) pC13BA. The pC13BN transformations yielded no colonies.

|tech= Two gels, each with 20 wells, were run simultaneously for fluorescence. The DNA samples loaded were from a Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. pC13BV was transformed into electrocompetent BL21 <i>E. coli</i>. A culture of <i>G. lucidum</i> was diluted and homogenized, and 150 μL aliquots were spread on plates and let dry. Paper discs were soaked in 100X solutions of geniticin, hygromycin, ampicillin, kanamycin, and chloramphenicol and placed in the center of each plate. We miniprepped ''holin'' 8 & 9. The results from the growth assay do not look good. However, we found that the T7 polymerase in BL21 is controlled with an IPTG inducible promoter, so more pC13BA was grown with IPTG and plates were all re-inoculated with the corresponding amounts. An appropriate volume of LB was also added to some plates to account for mass changes. The zone of inhibition tests were also restarted with recultured (with IPTG) pC13BA. The pC13BN transformations yielded no colonies.

|author=Rafael

|author=Rafael

}}

}}

|head=September 24th

|head=September 24th

|text=The gel of pC13AI showed nothing - the EtBr (for gel staining) needs to be replaced. We also continued putting our T7+resistances constructs into <i>E. coli</i> for part characterization.

|text=The gel of pC13AI showed nothing - the EtBr (for gel staining) needs to be replaced. We also continued putting our T7+resistances constructs into <i>E. coli</i> for part characterization.

|tech=We miniprepped the pC13AI cultures. We amplified with primers 45-52 and primers 48-51 and ran a gel (expected size at 1.5kb). Nothing appeared on the gel, not even the ladder. The EtBr should be replaced. There were too many transformants for pC13BV in the BL21 strain, so those cells were restreaked onto a new plate. Additionally, we transformed pC13BC into BL21 as well, and characterize that part as well.

|tech=We miniprepped the pC13AI cultures. We amplified with primers 45-52 and primers 48-51 and ran a gel (expected size at 1.5kb). Nothing appeared on the gel, not even the ladder. The EtBr should be replaced. There were too many transformants for pC13BV in the BL21 strain, so those cells were restreaked onto a new plate. Additionally, we transformed pC13BC into BL21 as well, and characterize that part as well.

|author=Rafael

|author=Rafael

|author=Rafael

|author=Rafael

}}

}}

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|head=September 27th

|head=September 27th

|text=Today we met again with Oat to make some changes to our images.  Arun moved onto his next color scheme: the same deep maroon with elephant grey banners.  In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive.  Arun also experimented with backgrounds of clouds, dirt, and toolkit to make the introduction slides very sharp. It look like we now have a solid design for our presentation.

|text=Today we met again with Oat to make some changes to our images.  Arun moved onto his next color scheme: the same deep maroon with elephant grey banners.  In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive.  Arun also experimented with backgrounds of clouds, dirt, and toolkit to make the introduction slides very sharp. It look like we now have a solid design for our presentation.

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<h5 class="centered">(09/30 - 10/06)</h5>

<h5 class="centered">(09/30 - 10/06)</h5>

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<h5 class="centered">(10/07 - 10/13)</h5>

<h5 class="centered">(10/07 - 10/13)</h5>

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<h5 class="centered">(10/14 - 10/20)</h5>

<h5 class="centered">(10/14 - 10/20)</h5>

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<h5 class="centered">(10/21 - 10/28)</h5>

<h5 class="centered">(10/21 - 10/28)</h5>

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