Team:Cornell/notebook

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Speed-taboodunit is a mashup of speed-dating, taboo, and whodunit; the idea is that each person writes on a piece of paper something unusual about themselves, underlining the key words, then these pieces of paper are randomly redistributed. Everyone organizes in a speed-dating format, with two lines of people, and you have a minute to talk to them and try to find out if they're the person described on their piece of paper-- with the catch that they can't use any of the underlined words. People who successfully find their person leave the group, but otherwise after the minute everyone moves down the line. As for why we came up with this specific icebreaker, we wanted to hit two key requirements of icebreakers: they have to be fun, and they have to make specific accommodations for being shy or quiet. Because each person has the specific objective of finding their person, this helps them overcome the shyness, and as for the fun, everyone loves taboo!

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A lot of iGEM teams we've looked at don't have much beyond the biology, so we really try hard to do interdisciplinary projects, as they provide a great opportunity for learning to work with different kinds of people.

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We transformed parts containing carotenoid pathway genes crtE, crtI, crtB, and crtY (BBa_K523022, BBa_K539119, BBa_K145001), as well as the T7 promoter (both in pIVEX2.3d as well as BBa_I712074), the T7 polymerase gene (BBa_K145001), and the transformation efficiency kit part (BBa_J04450).

From the plasmid pUCATPH (pAh13G), we amplified the trpC promoter (PtrpC, a constitutive fungal promoter) and the trpC terminator (TtrpC). From pNG (pAg13H), we amplified nptII, the geneticin (a plant/fungal antibiotic) resistance gene.

We miniprepped the crtEIB (BBa_K523022), crtY (BBa_K539119), and T7 (BBa_I712074) biobricks. The gels confirmed our PCRs for PtrpC, TtrpC, and nptII, which we then digested to insert into pSB1C3.

PtrpC, TtrpC, and nptII were column-purified, digested pSB1C3 backbone was gel-purified and dephosphorylated, and each was ligated into the pSB1C3 backbone.

We transformed TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R) constructs into DH5α via electroporation.

We used Q5 PCRs with the standard biobrick primers VF2 and VR to attempt to confirm the presence of TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R).

Minipreps of TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.

The Q5 PCRs of TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R) showed bands of the correct size, so sequencing with VF2 and VR was submitted to make sure nothing strange had happened.

pC13B (crtY) was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). However, the gel showed only one band for each (what one would expect for pAK13D, but pC13B should have had two bands).

pC13B (crtY) was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). The gel this time had two bands at the correct locations for pC13B, so we're hoping it's a success this time. We also started overnight LB cultures of pAb13T (pBARGPE1).

If successful, the ligation will form pAK13AD. pC13K (crtI, BBa_K118003) and pC13M (crtB, BBa_K118002) were digested with XbaI and PstI for insertion into pAK13D, and separated on a gel. pAb13T (pBARGPE1) was miniprepped and quantified by nanodrop.

pC13K (crtI, BBa_K118003) and pC13M (crtB, BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (TtrpC) 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.

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.

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.

:( ;_; 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>

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.

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.

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.

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.

Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. Gibson hph was submitted for sequencing, and pAK13ADs 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).

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.

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.

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.

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 G. lucidum 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.

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.

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 G. lucidum protoplasting will remain sterile. We made another stock of G. lucidum 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.

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.

We did a fast ligation (30 minutes, room temperature) of Cre with pC13F and electroporated. The time constant was low, so the transformation may not have worked. We continued the ligation overnight. We were still waiting on a miniprep kit so we pelleted our pA13AM and pC13BG cultures to prevent further growth. We transformed with our new pC13AY ligation to hopefully get some colonies this time. We also ran a PCR of pC13F to append homologous regions to G. lucidum PgpdA for Gibson assembly to create pC13BH and another PCR to retry appending BamHI and KpnI cut sites to pC13F using half to reaction volume to see if efficiency would be improved. We also electroporated the 4 ligations for fluorescence from yesterday (pAK13D with GFP, pAK13D with mRFP, pC13P with GFP and pC13P with mRFP) along with a control of pC13F to make sure that the electrocompetent cell stocks made yesterday are good. We plated the electroporated cells onto selective antibiotic resistance plates and incubated in 37 degree incubator overnight. We made our first attempt at protoplasting G. lucidum today. However we were having difficulty forming pellets of protoplasts, so we viewed the samples under a microscope. It appeared that the protoplasting attempt was unsuccessful. pC13M and pC13K plates were lawns on their plates. We set up overnight cultures of pC13AS, pC13W, and pC13Y, planning to miniprep and make glycerol stocks.

The electroporation from the 10th did not work (there were no colonies). Electroporation was redone on the overnight ligation of Cre and pC13F and a better time constant was observed (even with poor cuvette drying). We transformed limonene synthase from 2013 kit plate 4. We discovered that we need spectinomycin to use the holin parts registry plates. We got colonies from the pC13AY transformation and cultured them for gel screening. For fluorescence, no colonies were found on pAK13AN and pAK13AO plates. We centrifuged potential pC13AQ cultures for 10 min at 5000g and poured off supernatant. We kept the cell pellets in the fridge for mini prepping tomorrow. We also found two colonies each on the PtrpC and GFP plate and PtrpC and mRFP plate. We picked off all the colonies and grew each up in 5 ml liquid LB with CM antibiotic. pC13M and pC13K plates are now ready for miniprepping. We could not miniprep the cultures of pC13AS, pC13W, and pC13Y due to running out of our miniprepping supplies.

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 G. lucidum 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.

We spun down the pC13BX and pC13CJ cultures and put them in the fridge to be miniprepped. We also ligated T7 with mRFP, PtrpC with GFP, and PtrpC with mRFP since restriction cocktail digests looked good from the gel yesterday. We then electroporated the ligations and plated them. The plates were left overnight in the incubator. We created another culture of G. lucidum this time in an effort to obtain solid pieces at the top of the media instead of mycelium floating throughout.

We ran a PCR to append homologous regions to G. lucidum PgpdA for Gibson assembly with pC13F that succeeded as verified by a gel screen, but we accidentally used a RNA binding tube during purification and lost all the DNA. Needless to say, that mistake won’t happen again. Luckily, when we ran the same PCR again it was successful. We ran a colony PCR from the grown cultures of pC13BX and pC13CJ to visualize the insert using Q5. We got a positive insert for pC13BX. So this will be miniprepped and sent for sequencing once the miniprep kit is in. Unfortunately, no colonies appeared on the potential pAK13AO plate. We also decided to transform RBS (pA13CZ) off of a kit plate for subsequent miniprepping/digestion/ligation with GFP and mRFP. The T7 promoter needs a ribosomal binding site in order to express in E. coli. We then retried the ligation of pAK13D with mRFP using a 6:1 insert to vector ratio. We attempted to protoplast a huge chunk of mycelium, but had difficulty forming a pellet again. At this point, we decided to enlist the help of Professor Gillian Turgeon, who performs fungal transformations in her lab. We made some hygromycin plates to test transformants, and digested pAh13G with Sph1 to linearize, but this time we column purified the product.

We ran a Gibson assembly of pC13F and G. lucidum 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.

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 G. lucidum 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.

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.

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.

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.

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 E. coli. 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.

We made cultures of the successfully transformed pA13AW and ran a gel screen of the digests of pC13F and pC13BE and found that they did not have enough DNA to be visible. The G. lucidum PgpdA and pC13F parts with homologous regions needed for Gibson assembly and found them successful. We then ligated and transformed pC13BF although it sparked during electroporation, so we will most likely have to retransform. A sequence confirmation came back for pC13AQ! pC13AT turned out to be PtrpC and mRFP.Thus, we redigested PtrpC in pC13P and pCg13Y with SpeI and Pst1-HF. pCg13Y will be used to create pC13CB. We also redigested pC13Z, pC13AK, and pC13AQ with Xba, Pst1-HF, ApaI and Sac1-HF in order to get necessary inserts. We threw away the cultures of pC13BJ" 7, pC13BK" 6, pC13BL" 7, and pC13BM" 6 from yesterday and didn’t make glycerol stocks because we were not sure if we got the correct colonies. We will transform the sequence confirmed minipreps when more DNA is needed. We attempted site directed mutagenesis of ptrpC + hph + ttrpC in pAh13G. The extension time was a little short for pAh13G so it might not work. We ran gel a gel, which had a band at 100 base pairs, meaning it didn’t work. We will attempt this again tomorrow with a longer extension time. Colonies of BL21-A1 and DH5α with pC13CG grew. BL21-A1 seems to have a higher transformation efficiency.We started cultures and performed a colony PCR from the DH5α plate. We ran a gel of the colony PCR and pC13CG 1, 3, and 4 had the correct band at about 1.8 kb. pC13A (rbs + crtE) and pC13F (XbaI,PstI) did not have enough DNA. As a result, ligations for creating pC13I and pC13J are on hold. We ligated and electroporated pC13AF, pC13AG, pAK13CR, pAK13CS from other digestions, and plated them overnight. We found the the transformations of pC13BR and pC13BQ were too efficient and had to restreak the plates in order to isolate single colonies. We ran colony PCRs of pC13BU, pC13CC, and pC13CD, and at least two copies of each construct seem to be successful!

We redid the Gibson assembly of pC13BH trying 1:2 and 1:6 vector to insert ratios and transformed it along with pC13BF as it sparked when we tried yesterday. We also miniprepped our pA13AW cultures. After, we vector dephosphorylated pA13CZ, pC13P, and pCg13Y for fluorescent characterization constructs. We then ligated PgpdA with GFP, pC13AQ with pC13Y, pC13P and mRFP, pC13P with GFP, pA13CZ with GFP, and pA13CZ with mRFP. All ligations were then electroporated and incubated overnight. We sent pC13BX and pC13CJ to sequencing. We digested and ligated pC13CJ and pAK13D and electroporated into DH5a cells. We miniprepped and made glycerol stocks of pC13CG 1,3,4 (labeled TEMP). We will make sure to go back and throw out extras and failed attempts after sequencing results return. We sent pC13CG 3 in for sequencing, using the last of our DNA, meaning if we need more it will have to come from the glycerol stock. We performed our second attempt at site directed mutagenesis of pAh13G with a longer extension time. We recreated and digested inserts pC13A (rbs + crtE) and pC13F (XbaI, PstI). We ligated pC13I and pC13J. pC13AF electroporation did not yield any colonies. We made glycerol stocks of pC13AG, pAK13CR, pAK13CS, pAK13BU, pC13CC, and pC13CD and put them at -80 degrees C. We miniprepped the successful pAK13BU, pC13CC, and pC13CD and prepared them for sequencing. We also ran colony PCRs of pC13BR and pC13BQ, and set up overnight cultures of the successful constructs.

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.

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.

The extra colonies for pAK13CS that we made yesterday all failed. We will need to restart pAK13CS. We are recreating insert from pAK13AB for recreating pC13AF.

The transformed colonies of pC13BH had the PgpdA insert amplified via colony PCR and gel screened to check for the correct length. The gel however was blurry, so the cultures were miniprepped for digest screening. Gel screen pA13AW digested with XbaI and PstI. Gel results indicate that the cloning was successful! We then amplified the T7 polymerase gene for more insert DNA. We ran a PCR and digested pC13L to prepare insert for recreating pAK13CS. We recreated ligation of pC13AF. We looked at sequencing results for pC13BQ, pC13BR, pC13CC, and pC13CD - all looked good, other than some minor differences between our copies of the trpC promoter and terminators and the sequences in the online database.

We made cultures of pC13BX colony 1 and pC13CJ colony 4 to make glycerol stocks. We ran PCR of lox sites to amplify by Gibson. We ligated pAK13D with limonene. We preppred cre for reverse sequencing. We transformed holin off of kit plate and plated it. Digested pC13BH preps using EcoRI and PstI and screened alongside T7 polymerase. Results indicate that only the T7 PCR worked. The backbone pC13F and pA13AM were digested with XbaI and PstI, retrieving the T7 polymerase gene from the latter. Gel results for the digestions revealed correct banding patterns only for pC13F. We also miniprepped and prepared for sequencing pC13AU, pC13AT, pC13CB, pA13DC, and pA13DB. We prepared insert from pAK13CR for creating pC13CV. We submitted pC13AG for sequencing. We ran PCRs of pAK13BB and pAK13BU, and digested the inserts with EcoRI and PstI. We also digested pC13CD with SpeI and PstI.

One colony of pC13BH had a good banding pattern and was submitted for sequencing. Digested T7 polymerase PCR with XbaI and PstI and pC13F with BamHI and KpnI (sites were appended via PCR). pC13F that was digested with XbaI and PstI as well as with BamHI and KpnI were dephosphorylated for ligation. After confirming T7 polymerase PCR on a gel, it was ligated into pA13AM and pC13P. Additionally, Pc + T7 polymerase was ligated into pC13F and PpelA was ligated into pC13F. We ran those PCR's for the Gibson of pC13BN on a gel. lox 2 worked, but lox 1 did not, so I purified lox 2 and reran the PCR of lox 1 (it is in the small fridge labeled lox 1). I also made glycerol stocks of pC13BX and pC13CJ so that we have those in case we need to do more minipreps on them. We also had transformants for pC13CN so I made cultures with spectinomycin for all of them and ran a colony PCR on them. The colony PCR did not work due to incorrect protocol. We do not have the correct primers to do a PCR on this plasmid because its in Bbb format, so we just have to assume the plasmid is present because the cultures are growing in Spec. Tomorrow we need to miniprep those cultures that are in the incubator. We blended G. lucidum and made two 100mL cultures with 75mL cells and 25 mL cells. We will now let these grow for 2 days before attempting protoplasting once more. We created large batches of vectors (pC13F (XbaI,PstI) and (EcoRI-HF,PstI), pC13D (XbaI,PstI)). pC13AG sequence is sequence confirmed! We tried to dephosphorylate, but mistakenly used DNA Ligase on pC13F and pC13Q instead, so we’ll have to redo that tomorrow.

Today we electroporated Pc + T7 polymerase in pC13F, PpelA in pC13F, and T7 polymerase in pA13AM and pC13P. We prepared pAK13AB and pC13AG for glycerol stock. We ligated pAK13CS and pC13CV. We electroporated and plated pC13I, pC13J, pAK13CS, pC13CV. pC13J arced, so we ran a new Q5 PCR to begin preparation of insert from pC13A. We re-digested pC13Q with XbaI and PstI and pC13F with EcoRI and PstI, and subsequently dephosphorylated pC13CD and pC13F. We set up ligations to create pC13BC, pC13BV, and pC13CE.

Set up cultures for Pc + T7 polymerase in pC13F, PpelA in pC13F, and T7 polymerase in pA13AM and pC13P and ran a colony PCR for each. Each PCR was visualized on a gel; only PpelA in pC13F appeared to work. We quantified DNA: lox 1: 72ng/uL, lox 2: 34ng/uL, pC13CN 640.9 & 820.9ng/uL. We ran Gibson on the lox PCRs, transformed them into DH5a cells. We made glycerol stocks of pC13CN. We have pC13CK transformants, ran colony PCR and made cultures. We got reverse sequencing on cre and it looks good. We inoculated a culture of DH5a for electrocompetent stocks. pC13AU and pC13AT was sequence confirmed today. However, for pC13CB, PtrpC and nptII BB re-ligated back to itself pA13DB colony 1 and pA13DB colony 4, pA13DC colony 3 and pA13DC colony 4 all failed to sequence. We discarded all of our pC13CG glycerol stocks except for the successful pC13CG 3, and met with members of Professor Turgeon's lab. We also blended our 25 mL culture from 2 days prior, and made new 100ml cultures with (75 mL and 25 mL, again.) We prepared pAK13AB and pC13AG for glycerol stock. We made glycerol stock of pAK13AB and pC13AG. We redid a PCR with Phusion polymerase in an attempt to amplify rbs + crtE (pC13J). We then made overnight E. Coli cultures of pC13I, pC13AF and pC13CV. We transformed the ligations of pC13BC, pC13BV, and pC13CE into E. coli, but both attempts for pC13CE arced, so we started another ligation for pC13CE.

We ran colony PCR of pAK13CK on gel and nothing appeared. This could be because we used a janky Vent protocol. We miniprepped liquid cultures of pC13CK. A digest screen was run on the minipreps, but they did not look good. We will redo cloning for the construct. In other news, we got pC13BN transformants! We ran a colony PCR and grew up liquid cultures of pC13BN. The colony PCR looks good, and we will miniprep liquid cultures of pC13BN tomorrow. We made electrocompetent DH5a stocks, which are marked with blue circles with a line through it. Lastly, we double digested pC13Z and pC13AK with Xba, PstI, Sac, and ApaLI since we were running low on insert DNA. The gel for we ran for the double digestion looked good since we got 4 distinct bands for GFP and mRFP each. Next, we column purified pC13Z and pC13AK double digests and stored them in the freezer for later use. Today we attempted the first day of protoplasting and transforming G. lucidum in the Turgeon Lab. We used both our 3 day old and 1 day old cultures of G. lucidum in this attempt. Our initial pellet of both cultures appeared about the same size, though the younger one may have been slightly larger. We observed that the cells didn’t look like protoplasts, as they weren’t round and were smaller/very few in number. We looked at the solution after filtering and were able to see protoplasts, so it appears that something didn’t work with buffer/centrifuging. We miniprepped and made glycerol stock of the overnight cultures from yesterday--pC13I, pC13AF and pC13CV. We then digested these products. We electroporated for pAK13CS and plated the culture on Ampicillin plates. In addition, the PCR of pC13J worked! The transformations for pC13BC and pC13BV were too efficient, so we had to restreak them out to single colonies.

We ran a PCR for amplification of pC13Y, pC13BQ, pC13BR, and pC13CD to put between the lox sites. The PCR products were then cleaned and quantified. We redid the ligation of pC13D with pC13CJ to form pC13CK. We electroporated the ligation into DH5a. We miniprepped the pC13BN 2 cultures. We then ran a Vent colony PCR of pC13CB colonies 1-5. We also grew up 5ml cultures of pC13CB colonies 1-5 and left them in the incubator overnight. We finished our attempt at protoplasting G. lucidum with the help of members of the Turgeon lab, with hygromycin as our selection marker, and we left the plates in the Turgeon lab incubator. We then combined our three blended cultures and blended again to make a new culture for tomorrow. We planned out a protoplasting schedule for this week to try running the protocol under different conditions since it takes a long time to get results. We believe there might be bacteria growing in the fungal cultures we are using for protoplasting since phosphinothricin does nothing to bacteria and we could not blend the fungi under sterile conditions. We will put ampicillin in later cultures to kill bacteria, which shouldn’t affect G. lucidum because only bacteria will have peptidoglycan cell walls. We did a PCR clean up for pC13A. We ran a digest screen for pC13I, pC13AF and pC13CV, but only pC13AF (colony #2) appeared to work. We then ran a PCR with Vent for pC13I, pC13AF, pC13CV and pC13L, but they all failed…

We got colonies for pAK13CK and ran a colony PCR. The PCR didn’t show anything, but the primers are not ideal, so we will miniprep and digest screen. We digested PCRs of pC13BR, pC13BQ, pC13CD, and pC13Y with KpnI, HindIII. They were all cleaned. We digested miniprep of pC13BN with KpnI and HindIII. This was cleaned and dephosphorylated. We prepped pC13BN miniprep for sequencing with primer 31. Miniprepped pC13AY, pC13BF, pA13AM, and pA13AW cultures and will quantify tomorrow. We prepared pA13DC colonies 3 and 4 and pA13DB colonies 1 and 4 for sequencing Tuesday morning. We then reran a Vent PCR for pC13CB and grew up liquid cultures of the same colonies. We ran a gel for pC13A which showed that the PCR failed. We then redid a Vent PCR with the addition of MgSO4 for pC13AF, pC13CV, pC13I and minipreps of pC13L. We made 5 pAh13G cultures from glycerol stocks, and we electroporated pC13BJ, pC13BK, pC13BL, and pC13BM to make glycerol stocks. All transformations except pC13BM arced, but we are going to plate them anyway. We also attempted protoplasting again, using fresh enzyme osmoticum and STC. We couldn't really see mycelium at the beginning, and we could not see cells when we attempted to count them, possibly due to our mycelium being too old or having depleted its media. We made a new culture with a blended cell mass and ampicillin. We also made more CYM plates and media, and prepared a plan to test the sensitivity of G. lucidum to antibiotics and antifungals.

We quantified digestions and ligated pC13BQ + pC13BN, pC13Y + pC13BN, pC13BR + pC13BN, and pC13CD + pC13BN. We electroporated the ligations. We miniprepped pC13CK cultures and ran digest screen, which was successful. The construct is correct. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with holin, which was unsuccessful. The pC13AY, pA13AM, and pA13AW preps were digested with EcoRI and PstI and run on a gel. Results indicate that pC13AY and some of the pA13AW colonies worked. The gel of Vent PCR of pC13CB did not turn out too well. We got a bunch of lanes with smeared rather than distinct bands. We then ligated and transformed T7 promoter with mRFP to obtain pAK13AO and PgpdA with GFP to create pC13CF. We ran a Q5 PCR and gel for pC13AF, pC13CV, pC13I and pC13L. Sadly, they all failed except pC13AF from colony #2. We are concerned with what’s going on. The PCR for pC13A failed again too. We miniprepped our pAh13G cultures. Our new fungal culture doesn’t have nearly as much mass in it, leading us to believe that the old cultures were almost definitely contaminated with bacteria. Our transformed pC13BK and pC13BM grew into masses covering the plates rather than colonies, and the others didn’t grow. We restreaked pC13BK and pC13BM hoping for single colonies. We attempted a diagnostic protoplasting, determining that pellets formed are likely from contaminations, as the new cultures with ampicillin did not produce pellets. We also tried to manually extract mycelium and enzyme digest it, but microscope visualization did not show signs of protoplasting having occurred. Even after filtering through one layer of cheesecloth and nylon, there was no appearance of protoplasts.

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.

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 Cochliobolus 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 G. lucidum transformation in the future. We will also check for homology between the promoter gpd in G. lucidum and Cochliobolus to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after Cochliobolus 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.

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 G. lucidum homologous regions. We spun down the cultures of pC13BC, pC13BV, and pC13CE and stored them in the fridge.

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.

We got transformants from the afp1 and Cht1_2 Gibsons. There were a lot of red colonies and very few white ones, and an attempted pick and streak of the white colonies was performed. We got transformants for pC13CO, pC13BN + pC13CD, pC13BN + pC13Y, pC13BN + pC13BQ, and pC13BN + pC13BR. A colony PCR was performed, and a gel of the PCR products looked good for only pC13CO colony 2. No positive inserts were present, so maybe the lox construct is not correct. To check, we will sequence it tomorrow. Afterwards, we miniprepped pC13BF, pC13P, and pC13Q and digested pC13BF. We ran the gel for pC13A and did a PCR clean up for it, as well as for pC13J and pC13L. We also digested pSB1C3 with XbaI and PstI, and miniprepped pC13CV, pC13I, pC13AH and pC13AG. We attempted to run another gel of our PCR product to create Gibson assembly parts, which looked even worse than yesterday. We also did a PCR cleanup of pC13BJ, pC13BK, pC13BL, and pC13BM.

We ran a digest screen on pC13F with KpnI (mRFP has 2 internal KpnI cut sites). No digestion occurred since the enzyme was degraded. We submitted sequencing for pC13CK and pC13BN. We miniprepped pC13CO, pC13BA, pC13CL cultures and need to quantify and prep for sequencing. We ran a colony PCR on streaked pC13BA and pC13C plates and got a definite correct insert on pC13BA, and a lot of nonspecific amplification on pC13CL. We will run a digest screen and sequence. pC13BF colonies were quantified and gel screened and PgpdA was amplified from G. lucidum DNA, digested, and gel screened. The results were good, thus it will be Gibson assembled. We redid the PCR for pC13A with new materials but it unfortunately failed again. We also digested pC13L with XbaI and PstI and dephosphorylated the pSB1C3 digest from yesterday. We made an overnight culture of Cochliobolus in the Turgeon lab, as well as preparing fresh solutions of enzyme osmoticum and regeneration medium. We miniprepped the cultures of pC13BC, pC13BV, and pC13CE from the weekend. We added antibiotics to CYM plates, and intended to inoculate them for the growth experiment for testing G. lucidum’s antibiotic sensitivity, but the plates were too wet for reliable inoculation (that would spread in a circle of constant diameter), and inoculation was postponed so the plates could dry.

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 Cochliobolus 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 Cochliobolus 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 G. lucidum and inoculated each of the plates for the antibiotic sensitivity test with 20 μL aliquots, without spreading.

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 Cochliobolus. We also ran our Gibson assembly of homology regions in G. lucidum. 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).

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 Aspergillus Niger, 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.

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 Aspergillus plate. This preliminary test will see whether or not there is activity of afp1 on the Aspergillus. We are hoping for a ring of death on the Aspergillus 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.

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.

We plated 100μL of afp1 culture on fresh Aspergillus plates to demonstrate its effectiveness. We ran a PCR for pC13AI with primers 45/52 and 48/51 to add homologous regions for Gibson. The PCR protocol was a 1-step PCR with a 60C annealing temperature and 1:10 extension using GC buffer. We picked colonies from new pC13CO plates. We miniprepped pC13CL colonies 3 and 4, and pC13BN colonies 5 and 6 although the results of the gel was not clear. We redid the PCR for pC13BN. We also redid the PCR to append cut sites to pC13F for creation of pC13BF. We did a PCR clean up for the inserts in pC13BG, pC13P and pC13AF. We ran a PCR for pC13J and pC13BG. We prepared a double digestion for pC13AF and pC13P, and redid the ligation of rbs + crtI (from pC13L) and vector pSB1AK8. We ran gel of the colony PCR of our Gibson products, looking 4-5kb fragments. It appears that pC13BK + pC13BM + pC13BQ colony 1 was the only possibly successful reaction, so we made cultures of it, as well as various other cultures. We prepared pC13BC, pC13BQ, pC13BV, pC13CD, and pC13CE for part submission.

We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of G. lucidum 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.

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 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.

We used less hygromycin for transformation because Cochliobolus concentrations are less than G. lucidum concentrations. We are recording more and more data with regards with fungal resistance! We found a new contaminant on another plate but we will keep an eye out for it and make sure our measurements will not include its small area of contact with G. lucidum. 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.

We quantified the holin minipreps. Both were around 48ng/uL, which is too 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. We took photos of the plates for the experiment on G. lucidum’s growth in the presence of antibiotics, to capture the differences in appearance in addition to colony size.

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.

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. We transformed T7 + hph (pC13BV) into BL21 cells and plated on chloramphenicol plates. Unfortunately they arced, so we will need to repeat that with clean electroporation cuvettes. We poured more CYM plates.

We did a PCR cleanup of the pC13AI Gibson pieces, electroporated (time constant was 2.88 ), and plated the cells. We continued taking masses for Aspergillus with afp1 growth assay and began a growth assay with inoculating discs. We measured the colonies of G. lucidum (control, and in the presence of various antibiotics) one final time, as they reached the maximum possible sizes for the petri dishes they were growing in.

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 E. coli. A culture of G. lucidum 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.

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.

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 continued to take measurements for growth assays. We put a lot of Aspergillus around the inoculating discs for gathering more data. We set up cultures of BL21, with no plasmid and with pC13BC and pC13BV, and then standardized the optical densities of the cells between the three cultures. We spread 50 μL aliquots onto LB, adding discs dipped in 10X, 1X, and 0X concentrations of geneticin and hygromycin.

There was a clear zone of inhibition around the discs dipped in 10X hygromycin, no inhibition around the discs dipped in sterile water, for the wild type BL21 E. coli. However, there was normal growth around the 10X hygromycin discs for the BL21 E. coli transformed with our pC13BV construct. For the geneticin tests, the antibiotic appeared relatively ineffective, possibly due to degradation, and that experiment will need to be repeated.