Team:Cornell/notebook

|head=February 21th

|text=Today we worked on team structure for the summer; Swati will be the overall team leader, with me and Mark coordinating wetlab, Olya taking meeting minutes, Mac running drylab, and Hannah will be managing human practices. Later on in the summer, we will also develop task groups. For the next week, we've also split into groups to research various aspects of the mycelial materials project.

|text=The drylab team met briefly on June 17 to begin discussing some ideas.  We went over some of the growing methods for Ganoderma lucidum, brainstormed some non-packaging related ideas for a fungal biomaterial, and discussed how we could test some of the physical characteristics of the final product.  We thought it might be a good idea to test the thermal conductivity if it were to be used as a drinking cup or something.  We also determined that people probably do not want to drink out of a fungus cup.   

|head=June 21st

|text=The javascript for toggling technical details took a bit longer than expected, but we finished it up today.

|head=June 22nd

|text=I added a flyout to notebook entries authors with a picture, short description, and link to their bio on the team page.

|head=June 24th

|text=We miniprepped our three constructs. Then we went back to sleep.

|tech=Minipreps of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.

|text=Unfortunately, there were no colonies on the transformation plate for T7+''crtY'', so we redid the digestions for those. We also digested T''trpC'' and ''nptII'' for the eventual cloning of a P''trpC''+''nptII''+T''trpC'' construct (we have to put the parts together in reverse because ''nptII'' contains a PstI site, which we will be trying to remove as well). Gel extractions of digested ''crtI'' and ''crtB'' were also empty (insignificant concentrations), so these digestions were also redone, and all were run on a gel, which looked mostly okay.

|tech=pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (T''trpC'') with EcoRI and XbaI, and pCg13R (''nptII'') with EcoRI and SpeI, and all were separated on a gel; ''crtI'' looked faint, and pAK13D was smeared, but all fragments were cut out for purification tomorrow.

|type=wiki

|text=We finished the hypertexting script and fixed some miscellaneous problems. I think we can call this a wrap. The notebook page is officially done!

|tech=Now we just have to start filling out entries...

|author=Nupur

|type=wet

|text=Yesterday's digestions were gel extracted, ligated, and transformed. Hopefully it worked this time!

|tech=Digested pAK13D was dephosphorylated, then ''crtI'' , ''crtB'', and ''crtY'' were ligated with pAK13D to put them under the T7 promoter, hopefully producing pAK13AB, pAK13AC, and pAK13AD, respectively. pCg13R was also ligated with pC13Q, hopefully producing pCg13U. All four constructs were transformed into DH5α via electroporation.

|author=Rafael

|text=There were no transformants, again :(. On the other hand, we received our tube of ''Ganoderma lucidum'' from Dr. David Hibbett today! Once more with feeling, we redid the four digestion-ligations for ''crtI'', ''crtB'', ''crtY'', and ''nptII''. We also amplified ''hph'', the hygromycin resistance gene, from one of the plasmids we got from Dr. Turgeon's lab, as well as pSB1C3 backbone, and we will use Gibson assembly to put them together (as ''hph'' has both EcoRI and PstI sites)-- the gels looked good for both of these. Finally, we submitted pAb13T (pBARGPE1) for sequencing to determine the sequences of the ''Aspergillus nidulans'' ''gpdA'' promoter and the ''bar'' gene, which confers phosphonothricin resistance.

|tech=:( ;_; T_T D: (tears for the transformations lost). The ''bar'' PCR and pC13F were digested with EcoRI and PstI, and ligated to hopefully form pCp13X. Site-directed mutagenesis was performed both with and without DpnI to digest template DNA. <add something about site-directed mutagenesis design; refer to primers? probably requires Swatinput>

|type=dry

|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

|type=wet

|text=Digest screens were run to see if cloning had been successful,, and Gibson cloning was continued. 

|tech=Minipreps were made from the ''crtY'' plates, from which a digest screen was prepared using ''Not-I'' to see if the ligation worked.  The digestions were run on a gel and some appear to have worked (X/P/S, X/P/A #2/3, and X/P/S/A #⅔ worked).  Also, a glycerol stock of ''nptII BB'' was made and miniprepped, but the concentration was extremely low, so an overnight culture was made from the glycerol stock to be miniprepped tomorrow.  Another transformation was done using the remaining miniprep of ''nptII BB'' from earlier.  Cultures were made for ''Gibson #3'', and a colony PCR perfomed, then run on a gel. Non-GC fragments from Gibson #3 were too small, but GC #⅖ were good (1kb fragment), and #4 had a band at 1kb with another smaller fragment.  Finally, ''nptII BB'' was digested with X/P then column purified, and ''PtrpC'' was digested with S/P then gel purified.  

|author=Rafael

|text=Cloning with ''nptII BB'' and ''PtrpC'' was continued, as well as cloning with the Gibson Assembly method.

|tech=Today ''nptII BB'' and ''PtrpC'' were quantified. Also, ''nptII BB'' transformants were miniprepped, and another glycerol stock of ''nptII BB'' was made.  The DNA for Gibson Assembly was miniprepped, and GC colonies 2,4, and 5 from the Gibson #3 gel yesterday were prepared for sequencing. ''NptII BB'' was digested with the ''PtrpC'' digest from yesterday. More cultures of X/P/S #1/2/3, X/P/A #⅔, and X/P/S/A #⅔ were made.  CYM medium was created, and ''PC13Z'' was transformed from a kit plate.

|text=Today we worked with fungi! We applied different amounts of antibiotic to G. lucidum, to test the sensitivity of our strain of fungi.  In addition, cloning of ''pCg13Y'' was nearly completed.   

|tech=''pCg13Y'' was desalted, transformed, then plated into E. coli.  Antibiotic stocks ''G418'' and ''PPT'' were made (100x).  G. lucidum cultures were inoculated with varying levels of antibiotic to test the strain's sensitivity for future selection purposes.

|text=Our cloning of ''pCg13Y'' was found to be unsuccessful.  ''pC13z'' and ''pAK13AD'' were prepped for cloning.

|tech= Colony PCRs and cultures of ''pCg13Y'' were completed to determine the success of cloning ''nptII BB'' downstream of ''PtrpC''.  Unfortunately, a gel revealed that the dna fragments appeared at 0.6kb instead of 1.4kb.   ''pC13Z'' and ''pAK13AD'' were miniprepped, but had to be thrown out due to contamination of our EB.  New cultures were made to be miniprepped again.  A glycerol stock of ''PC13Z'' was made, too.

|text=This week, drylab split up so members could focus on their strengths.  Rebecca and Sara began designing our feedback controller, with some much appreciated advice from the previous drylab team leader, Dan.  Ritvik, Manny, and Mac began coming up with design considerations for the incubation chamber.  We were most concerned with size; too small and it wouldn't be useful, too big and it would be difficult to control any variables.  We eventually decided that a 1x1x2' chamber would probably be a good compromise.  Rebecca, Sara, Ritvik, and Mac also attended machine shop training this week.  In order to complete our training, we each had to construct a small aluminum lamp using a milling machine and a lathe.  Fortunately, everyone emerged from the training session one lamp richer, and with a full set of fingers on each hand.

|text=Rebecca looked into the components of the temperature feedback control; namely, the temperature sensor, the microcontroller and the heating element.  Put in an order for the temperature sensor, microcontroller (an Arduino Fio) and Xbee ports for wireless communications.

|head=July 15th

|text=Gibson ''hph'' was submitted for sequencing, and cloning of ''crtY'' and ''crtB'' was continued.

|tech=Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays.  Gibson ''hph'' was submitted for sequencing, and ''pAK13AD''s and ''pC13Z''(GFP) were miniprepped.  ''PC13Z'', ''PC13F'', ''PCP13X'', ''PC13M'', and ''PC13E'' were  PCRed with primers 31 and 32, the standard ''pSB1C3'' primers.  A gel was run to check the PCRs-all looked successful except ''pC13M'' and ''pC13E'' which had nonspecific bands around 1kb.  A new technique was used in the following digests, which used a ''restriction cocktail'' of enzymes to cut the dna.    ''PAK13AD'' was digested with E/S/SacI/ClaI so that it could be placed in ''PSB1C3'' using ''SpeI'' instead of ''PstI'' due to the small distance between the ''PstI'' and the ''SacI'' cut sites.  ''PC13F'' was digested with E/S/KpnI so ''PAK13AD'' can be placed in it.  ''PAK13D'' was digested with S/P so ''crtI''/''crtB'' can be placed in it.  All the digests were then column purified rather than gel purified--a move which has improved our ligation efficiencies. Then the purifications were quantified.  Cultures were made for ''PC13P'' and ''PC13K''. Finally, ''PAK13AD'' was dephosphorylated then ligated into ''PC13F'' (''PC13AH''). 

|author=Rafael

|head=July 16th

|text=

|tech=Sequencing results came back positive for the Gibson cloning of hph, so a culture of gibson colony 5 was made. ''PC13AH'' was transformed and plated, and ''pC13K'' and ''pC13K'' were miniprepped.  ''pCp13X'' (S/P), ''PC13F'' (S/P), ''PC13Z'' (S/P) were purified and digested along with ''PC13P'' (X/P) [also dephosphorylated], then all were column purified.  Reran a PCR of ''PC13M'' and ''PC13E'', and one of ''PC13K'' with primers 31/32.  All PCRs were cleaned, then run on a gel along with ''PBARGPE1'' and ''PBARGPE1'' which had been digested with NotI today.  Results: ''PC13E'' had a 3kb fragment (good), ''PC13K'' had a .9kb fragment (should be 1.7-1.8kb), ''PC13M'' had a 1.2-1.5kb fragment (good), ''pBARGPE1'' had a 4kb fragment (super-coiled) and ''pBARGPE1'' with NotI had a 5-6kb fragment (has a NotI site-good).  From these results it can be concluded that everything worked except ''PC13K''.  Ligations of ''PAK13D'' to inserts ''PC13Z'', ''pCp13X'', and ''PC13F'' along with vector ''PC13P'' to inserts ''PC13Z'', ''pCp13X'', and ''PC13F'' were performed.  Also, overnight digestions of ''PC13M'', ''PC13E'', and ''PC13K'' with X/P were set up.  Transformation was attempted for ''PC13AI'' and ''PA13AJ'', but  the cuvettes weren't cleaned properly so it failed.  Finally, our dwindling supplies prompted the ordering of more Spe1 and membrane filters.  

|author=Rafael

|text=The restriction cocktail method of cloning worked! Cloning of pC13AH (''T7'' + ''crtY'') was shown to work using this method.  We are also continuing our cloning of the ''crtI'' and ''crtB'' pathways.   

|tech=All 18 (yes, 18!) of the ''pC13AH'' colonies that had been screened by colony PCR yesterday were miniprepped.  We digested the miniprepped colonies of pC13AH clones (T7 promoter with crtY in pSB1C3) with ''NotI'' to see if the restriction cocktail method of cloning worked.  On the gel that was subsequently run, bands were expected at ~2.1 for pSB1C3 and ~1.2 for ''crtY''+''pT7''.  The restriction cocktail method worked! The bands appeared at the correct lengths on the gel for ''pC13AH''.  There was not enough miniprepped DNA for pC13AH to send for sequencing, so the colonies were cultured again for another miniprep.  None of the ligations into pAK13D (pSB1AK8 + PT7) worked -- pC13Z (''GFP''), pC13M (''crtB''), and pCp13X (''bar'') all failed => try cloning only using restriction cocktail method, instead of attempting PCR with VF2 & VR.  Digested pC13Z, pC13M, pCp13X, pC13K (''crtI'') with XbaI and PstI in addition to ApaLI and SacI for later ligation with pAK13. pAK13D had been previously digested with SpeI and PstI, column purified, and dephosphorylated for insert ligation.  More pAK13D was grown to acquire more DNA to be miniprepped tomorrow.  Nupur performed colony PCR and made cultures of transformants from yesterday. All transformations appeared to have very low efficiency--perhaps an issue with heat shock stocks?  Transformations of ''AI-AL'' (mRFP cds and lox site parts) all yielded colonies, though at really low efficiencies compared to most transformations off of kit plates. These were screened by colony PCR to verify.  Ligations into pC13P all yielded colonies, but at very low efficiencies. These were pC13E (T7 pol), pCp13X (bar), and pC13Z. All were screened by colony PCR as well.  Taq was used to perform 5 colony PCRs, then all colony PCRs were run on a gel.  Also, pCg13s (nptIIBB)was digested with XbaI, PstI, ApaLI, and SacI for later ligation to pC13P, which had been previously digested with SpeI and PstI, column purified, and dephosphorylated. Overnight ligations of pC13Z, pC13M, pCp13X, pC13K, pCg13S with vector pAK13D were set up, along with overnight ligations of pC13K and pCg13S in vector pC13P.   

 

|text= Eric Holmes and Arun Chakravorty presented for a group of scientific researchers (SILS)

|type=wiki

|text=We helped [[Team:Ciencias-UNAM|Ciencias-UNAM]] get set to transfer their HTML website to the wiki. Hopefully we'll get up a tutorial for what to look out for on here soon!

|author=Nupur

 

 

 

 

|text=We started taking measurements of ''Ganoderma'' growth on CYM plates by tracing the central mass of fungus--the differences between the plates are really interesting! Sequencing for pC13AY was successful so we prepared more culture. We did gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG, but only pC13J colonies are positive. We then did digestions for the following vectors: pC13BG, pC13AG, and pC13K. We still do not have any growth on antifungal plates.

 

|tech= We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of Ganoderma growth on CYM plates (plated on September 9) 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.

|text=We tried transformation of ''Cochliobolus'' 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.

|tech=We attempted transforming ''Cochliobolus'' 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 chloromphenicol 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 used less hygromycin for transformation because Cochliobolus concentrations are less than Ganoderma concentrations. We are recording more and more data with regards with fungal resistance! We found a new contaminant on one plate but we will keep an eye out for it and make sure it does not come into contact with Ganoderma. We ran the gel extracted ''PpelA'' and pC13F PCRs on a gel but there were no bands. However, they may still be successful as the concentration were fairly low and may not have appeared on the gel. We ran the pC13CO digests on the gel and looked good. We ran a PCR of pC13AI to amplify ''lox'' sites for Gibson with harsh conditions to remove the nonspecific amplification.   

|text= Something may be wrong with the pC13AI plasmid based on gel screens. We ligated the constructs from Tuesday--''crtB'' with ''crtI'' and ''crtI'' with ''PtrpC''.  

|tech= We quantified the holin minipreps. Both were around 48ng/uL, which is low to send to sequencing. We will reculture and miniprep when LB is ready. We ran a PCR of pC13AI with primers 45/52 and 48/51. We ran the PCR products on a 1% gel and found bands at 1.5kb and 1.2kb for ''lox1'' and 1.5kb and 1kb for ''lox2''. There may be something wrong with the pC13AI plasmid. We also ran a 1 step PCR of ''lox1'' and ''lox2'' with a 59C annealing temperature. We submitted pC13J for sequencing with Primer 31. We also ligated the constructs from Tue--in short, ''crtB'' + ''crtI'', ''crtI'' + ''PtrpC''. We ran a PCR with Q5 polymerase for inserts in pC13M, pC13J and pAK13CR and ran a gel for them--pC13J was good, pC13M had a faint band but nothing for pAK13CR.

|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.

|text= We set up the PCR to append ''KpnI'' and ''BamHI'' cut sites to pC13F again with different annealing temperatures and ran a gel to test the PCRs. Ran Gibson for pC13AI. A large Q5 PCR was done on fluorescent constructs pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. We ran the gel for pAK13CR from yesterday. We ligated the following: ''rbs'' + ''crtE'' to make pAK13CQ, ''rbs'' + ''crtB'' to make pC13CV, ''ctrB'' + pC13BG to make pC13DP, ''rbs'' + ''crtI'' to make pAK13CS.  

 

|tech= We ran a Gibson of the DpnI digest of pC13AI PCRs. In terms of characterization, we ran a huge Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F using standard biobrick forward and reverse primers 23 and 24. We set up the PCR to append KpnI and BamHI cut sites to pC13F again with different annealing temperatures and ran a gel to test the PCRs. We ran a gel for the PCR products--insert of pAK13CR--from yesterday, and then we did a PCR clean up of the inserts of pAK13CR and pC13I. We then did electroporation for pC13DJ and pC13BD. The latter arced... We digested pSB1C3 with EcoRI-HF and PstI, pSB1C3 with XbaI and PstI, pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, pSB1AK8 with SpeI and PstI, pC13J with XbaI and PstI, pC13M with XbaI and PstI. We then dephosphorylated digested pSB1C3 and pSB1AK8. Ligation was performed for the following: ''rbs'' + ''crtE'' (from pC13J) into pSB1AK8 to create pAK13CQ, ''rbs'' + ''crtB'' (from pAK13CR) into pSB1C3 to create pC13CV, ''ctrB'' (from pC13M) into pC13BG to create pC13DP, ''rbs'' + ''crtI'' (from pC13L) into pSB1AK8 to create pAK13CS.  

|text= We plated pC13AI. We are continuing the <i>Aspergillus</i> growth assay.

|tech= We did a PCR cleanup of the pC13AI Gibson pieces, electroporated (time constant was 2.88 - it arced), and plated the cells, We continued taking masses for <i>Aspergillus</i> with ''afp1'' growth assay. We began growth assay with inoculating discs.  

|text= Today we ran a gel to verify insert lengths from Q5 PCR products of fluorescent constructs. We also transformed our T7 + hygromycin resistance construct into <i>E. coli</i> for characterization, and set up a zone of inhibition experiment to test the efficacy of antibiotics and antifungals on <i>G. lucidum.</i>

 

|tech= Two gels, each with 20 wells, were run simultaneously. 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 re-cultured (with IPTG) pC13BA. The pC13BN transformations yielded no colonies.  

|author=Danielle

|text= The gel of pC13AI showed nothing - the EtBr (for gel staining) needs to be replaced.

|tech= We miniprepped the pC13AI cultures. We amplified with 45/52 and 48/51 and ran a gel, and got products that are 1.5kb. Nothing appeared on the gel, not even the ladder. The EtBr needs to be replaced.

|author=Danielle

|text= Sequencing of pC13CO did not turn out to be positive. Still trying to get data from aspergillus and growth assay.

|tech= The sequencing of pC13CO came back as ''PtrpC'' + ''GFP''. We are not sure why or how. We either miniprepped the wrong thing or put the wrong thing into sequencing. We continue to take measurements for growth assay. We put a lot of aspergillus around the inoculating discs for gathering more data.

|author=Danielle