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 got colonies for pAK13CK, ran 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.