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>

|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

 

 

 

|type=pres