Team:Cornell/testing/notebook/wetlab/2

Daily Details:

July 1st, Sunday

Dylan and Caleb set up two gels for electrophoresis. Caleb's was 1% agarose in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green, containing NEB 100 bp ladder, NEB 2-log ladder, Promega 1 kb ladder and run at 100V. Dylan's was 1% agarose in Owl box using ethidium bromide, containing the nah operon PCR product from previous night, and run at 55 V. Our plates of DH5a transformed with p21 ligated into pBBRBB with mtrB grew only one colony, possibly contamination. Dylan ran a colony PCR and got a smear, suggesting that the PCR of p21 or the ligation did not work. Because we learned that our SYBR Green was causing ladder to run strangely, Dylan decided to redo a Vent PCR to amplify the salicylate reporter region out of p21. Also, a liquid culture of JG700 was prepared, as well as replating of p21, p22, JG700, JG1220, JG1537, JG1219. (See: Strain List)



July 2nd, Monday

Today, Dylan and Caleb ran a electrophoresis of a Vent PCR of p21 (the PCR product being the salicylate reporter) at 55 V. Additionally, Caleb decided to run a control gel at 100 V to determine whether higher voltage was a factor in our previous ladder problems (in addition to the SYBR Green stock). While we determined that the higher voltage did not cause our ladder issues, we did not see any bands from the p21 PCR (gel image not included). Dylan prepared electrocompetent cells using the Myers and Myers protocol. Modified protocol using two 5mL cultures @ 4000g for 10 min. Washed with 2 mL sorbitol, resuspended in 100 uL sorbitol. First electroporation ts = 2.32 ms, second ts = 2.02 ms. Added 1 uL of plasmid (575.5 ng) to each cuvette. First used .60 V, then 0.55 V, both with R = 400 ohms. Mark and Danielle started liquid cultures of S1, S9, S10, S11, S15, S16, S18, S27 (See: Strain List)



July 3rd, Tuesday

Caleb miniprepped S16 (p15), S22 (p21), S9 (p8), S10 (p9), S11 (p10), S18 (p17), and S15 (p14). (See: Strain List ) Instead of a single EB elution, Caleb did two elutions, 30 ul each. The double 30 ul elution turned out to be effective at recovering a usable amount of DNA in the second elution, so we're including it on our miniprep protocol for future minipreps. The rerun of our Gibson sequencing failed again, so we tried a more roundabout method of determining whether our 3 Gibson transformants have complete nah operons or not. We digested them each with BamHI, expecting specific fragment lengths on a gel electrophoresis. When Dylan and Mark were attempting to determine what volume of ethidium bromide should be used in the gel, they came to the conclusion that better images may result from staining the gel in dilute ethidium bromide after running the gel, rather than including the stain in the gel. This experimental first staining used 200 mL of water conaining 20 uL of 10 mg/mL ethidium bromide, with gentle agitation for 1 hour. This stained the bands well, but also provided a large amount of background and took a very long time. We only saw one band on each, all of them at about 3.6kb. Because no transformations from our previous competent freezer stocks were successful, Dylan decided to make a starter culture of Shewanella strain JG 700 in preparation for making new competent stocks using the PNNL Protocol . Swati and Tina used this starter culture to complete the preparation.



July 4th, Wednesday

In the morning, Dylan, Swati, and Danielle prepared p8, p9, p10, p14, and p15 for sequencing (from the minipreps that Caleb preformed the previous day). The team took the rest of the day off and had a barbeque at Buttermilk Falls. There was an excess of food, spontaneous song, and fireflies.



July 5th, Thursday

Upon arriving to the lab, Dylan dropped off the sequencing tubes we'd set up on the morning of the 4th for analysis. He also decided to do a Colony PCR of the potential transformant with the salicylate reporter plasmid. However, when setting up this reaction, Dylan realized that we'd used the wrong sequencing primers (we'd used the standard VF2 BioBrick primer instead of a custom primer for the pBBRBB backbone). Consequently, Mark and Dylan resubmitted p14 and p15 for sequencing while the colony PCR was ongoing. After visualizing the PCR products using DNA gel electrophoresis, Dylan concluded that the colony we'd screened did not have our plasmid of interest, and was the result of contamination. Similarly, Dylan noticed small, evenly spaced colonies on all of the JG700 transformant plates from the Myers and Myers procedure we'd undertaken on the 2nd of July. Because none of the colonies looked red (as they should have if they were replicating pSB3C5), Dylan concluded that the chloramphenicol on the plates had degraded, and the colonies were resultant from untransformed cells. Because the colony PCR of the potential salicylate reporter didn't yield good results, Dylan and Youjin set up another Phusion PCR to amplify the salicylate-sensing region from p21. Simultaneously, Mark made SOB media for recovery after future transformations, as we'd just received the ingredients for the broth. Because we needed to submit for sequencing twice (because we used the wrong sequencing primers), Swati et al. set up liquid cultures of p21, p8, p9, p10, p17, and p14 in order to have more DNA in the freezer. We like having DNA in the freezer. (See: Strain List)



July 6th, Friday

Caleb miniprepped p21, p8, p9, p10, p17 from the liquid cultures set up the previous night. (See: Strain List ) We couldn't miniprep p14, since – after checking notes from the previous night – we learned that the culture was made with the wrong antibiotic (chloramphenicol instead of kanamycin). Some sequencing results came back, however the Gibson results are still confusing. Also, a gel was run for the p21 PCR product.

Daily Details:

July 9th, Monday

Dylan performed a Phusion PCR with a single annealing temperature of 59 degrees Celsius using primers 27 and 28 for the final time. In the future, new primers will be used. The purpose of this PCR was to amplify p21 (See: Strain List ). The PCR product was analyzed using agarose gel electrophoresis to determine if the desired 1.127 kb fragment is present. We talked to Dr. Jeff Gralnick about electroporating Shewanella, and his recommendation was to try conjugation instead. To transform our plasmids into Shewanella with conjugation, we had asked Dr. Jeff Gralnick for WM3064 E. coli for which plates containing 2,6-Diaminopimelic Acid (DAP) are required. In preparation to receive this strain, the plates with DAP and DAP/kanamycin were prepared.



July 10th, Tuesday

Caleb miniprepped strains containing Gibson products and the arsenic promotors without BamHI cutsites: plasmids p8, p9, p10, p15, and p16 (See: Strain List ). Also, our new freezer arrived. We cleaned and welcomed our new friend. The WM3064 came in and we plated from the agar stab. Dylan set up two 18 mL/hr continuous flow reactors using 10x diluted LB and innoculated one of the reactors with JG700 and one with wild type Shewanella oneidensis MR-1.



July 11th, Wednesday

Dylan started a WM3064 subculture in the morning before meeting Swati, Shweta, Caleb, and Danielle at the Boyce Thompson Institute for Plant Research to give a presentation both on our project and on the broader applications of synthetic biology to the energy industry to a group of high school teachers from New York state. After the presentation, Dylan and Danielle prepared plates containing DAP/chloramphenicol and DAP/ampicillin to grow transformed WM3064 on, while Mark continued preparing electrocompetent stocks of WM3064 with the subculture that Dylan had started. We transformed things later that day.



July 12th, Thursday

Shweta prepared a glycerol stock of WM3064 for future use, and Dylan plated some remaining transformed WM3064 hoping to get more colonies of the successfully transformed cells. Dylan and Mark submitted p8, p9, and p10 for sequencing using two different primers for each plasmid. The previous night's p21 PCR seemed to have failed, even with the new primers. Based on the gel run by Claire, then analyzed by Dylan and Caleb.



July 13th, Friday

Today began with Dylan and Caleb setting up conjugations between our transformed donor E. coli and S. oneidensis. The sequencing results for p8, p9 and p10 all came back as failed, so it was concluded that the Gibson assembly failed. There were five locations on the nah operon at which sequencing could begin, of which we considered all but the fourth. Sequencing at the first location resulted in a sequence that corresponded to the fifth sequencing location, with some sequence before it and all of the expected sequence after it. The fifth sequencing location produced the expected sequence. Then the middle two regions which we considered all resulted in failed sequencing. This has led us to the conclusion that segments that are required in the operon are missing, and thus the assembly failed. Dylan and Caleb began attempting to look into the possibility of this being a cause, and some potential solutions. In discussing why p21 PCR seems to continue failing, the idea of having p21 sequenced arose. Dylan prepared the sample and submitted it for sequencing. Meanwhile, Mark set up yet another Phusion PCR of p21, this time with a higher annealing temperature of 61 degrees Celsius and a decreased extension time of 18 seconds. The goal was to prevent the mispriming which seemed to have occurred in the previous day's attempt. Later that day, Dylan set up yet another PCR with a decreased annealing temperature of 55 degrees Celsius in order to empirically narrow-in on the optimal annealing temperature for the amplification of the salicylate sensing region from p21.



July 14th, Saturday

Swati ran a gel of 5uL of the p21 PCR that Mark set up yesterday. After staining, the DNA ladder was well visualized but there were no bands in the p21 lane, suggesting that the PCR did not work. In a fit of rage, Swati yelled at Maneesh for going to get a drink of water - yet another innocent victim of failed PCR. Then, after a full team meeting, we all came together for a dumpling party. Noodles, dumplings, and gnocchi of all kinds were eaten by the voracious team. In the spirit of true, unwavering scientific inquiry, we conducted a novel experiment whilst feasting on these delectable treats. The result: fried Dorito dumplings are delicious.

Daily Details:

July 15th, Sunday

Dylan noticed that we had colonies on the plates re-streaked from the original plates of conjugated S. oneidensis. He started overnight cultures of S20 (See: Strain List ) in kanamycin so that we can sequence and make glycerol stocks. He also started overnight cultures of w.t. S. oneidensis to inoculate into new reactors in Riley Robb, which will be used as positive controls.



July 16th, Monday

Dylan ran another Phusion PCR of the entire nah operon, since we got a lot of mispriming the first time we ran it; we believe this was because we had used the optimal annealing temperature for Vent Polymerase (55C) with Phusion Polymerase. Dylan set up the new PCR correctly, with an annealing temperature of 66C and a lengthened final extension time of 15 minutes to account for the size of the nah operon (~10kb). Caleb ran a gel of the PCR and visualized a single band ~9.5kb. PCR of the nah operon out of p20, P. putida (See: strain list) was successful! Tina and Swati gel extracted and quantified, extracting two samples at 38.8 ng/uL and 27.2 ng/uL. Spencer miniprepped p14, our arsenic reporter part (arsR + mtrB w/ BamHI cutsite), from S20 (See: Strain List ) for sequencing, recording yields of 44.3ng/uL & 35.8ng/uL (colony 1) and 50.6ng/uL & 53.5ng/uL (colony 2) from quantification. Dylan and Tina set up transformations of p15 and p16 (See: strain list), the arsenic reporter parts without a BamHI cutsite, so conjugations with S. oneidensis can be done in the next few days. They also set up a transformation of the miniprepped BBa_J01003 with the oriT mobility gene. Because of our lack of success with electroporation, we are planning on trying conjugation of our constructs into S. oneidensis. However, this requires that all our plasmids have the mobility gene. pSB3C5, the plasmid we are going to use for the nah operon has no mobility gene. Thus, we are going to try two things: to clone oriT from iGEM kit plates into pSB3C5, and to amplify the mobility gene out of one of our own plasmids and clone that into pSB3C5.



July 17th, Tuesday

Shweta set up a Phusion PCR of p21 (nahR and Psal) to try and extract the salicylate-sensitive promoter again. Sequencing of p21 showed a stem loop sitting upstream of the ENX biobrick cutsites, which was not expected and likely the reason for our unsuccessful PCRs. Before redesigning primers, we are going to try PCR with the standard iGEM forward sequencing primer and the same reverse primer that we designed for p21.



July 18th, Wednesday

Dylan set up p15k, p16k conjugation plates from overnight cultures of transformed WM3064 and JG700. Dylan let these plates incubate for 8 hours, and then streaked for single colonies on kan plates with E.coli and Shewanella controls. Caleb miniprepped p24a (BioBrick part with oriT) from overnight cultures. He then desalted an overnight ligation of the nah operon in pSB1C3 and transformed into DH5a. Dylan also trekked over to Riley Robb to set up a reactor for inoculation tomorrow. That night, Spencer set up overnight cultures of p25a, p28a, and p31c.



July 19th, Thursday

Only observed 2 possible colonies from transformants of yesterday's ligation (nah operon in pSB1C3). We restreaked from one of these colonies along with a DH5a control. We got single colonies on the JG700+p15 and JG700+p16 plates, and no growth on control plates. Thus, our tentative/optimistic conclusion is that the conjugation was successful. Dylan and Caleb made reference plates of the picked colonies, and grew up liquid cultures from same colonies for sequencing. Dylan and Caleb then miniprepped p25a, p28a, p31c from overnight cultures set up by Spencer and made glycerol stocks of S25, S28, and S31. That afternoon, Dylan also inoculated a reactor in Riley Robb with S20, our engineered arsenic reporter strain. We then set up a digestion of the p21 PCR product along with our miniprepped p14 with EcoRI and AscI. We purified the p21 PCR digestion with Omega Bio-Tek E.Z.N.A. MicroElute DNA Clean-Up Kit. We dephosphorylated p14 digestion with Antarctic Phosphatase, and ran the entire mixture on a gel. (Picture of gel). That night, we gel extracted the 6kb band with our Qiagen gel extraction kit and set up overnight cultures of a p26a, 027a, p29a, p30a (our Anderson series constitutive promoters), and p14k.



July 20th, Friday

This morning Shweta and Tina miniprepped p26a, p27a, p29a, p30a (Anderson series constitutive promoters), and p14k from overnight cultures. Dylan set up a 30 minute room temperature ligation to connect the salicylate reporter with digested p21 PCR product and p14 (isolated yesterday, quantified today). Because Shewanella grows more slowly than E.coli, Claire did a second miniprep of p15k and p16k from JG700 in order to submit the plasmid for sequencing to confirm that conjugation was successful. In anticipation of the failure of Dylan's ligation, Swati set up 3 Phusion PCR reactions in parallel, each with the same template (p21), and repeated the parameters that had proven successful (no mispriming) previously. Dylan also set up overnight cultures of the strain with the nah operon in pSB1C3, S18 (DH5a + pSB3C5/p17c), and WM3064+p14.

Daily Details:

July 22nd, Sunday

Danielle and Dylan digested p14(Arsenic reporter) and p21(Salicylate sensing region) for ligation. Swati ran a Phusion PCR to amplify p21. Then Dylan ran a gel electrophoresis and did gel extraction to isolate p21 PCR digest and p14 backbone.



July 23rd, Monday

Dylan quantified the gel extractions, quite successfully with a newly invented protocol. Then he dephosphorylated p14, and submitted some DNA for sequencing . At one point, the Magical Graduated Cylinder of Elmira (500 mL) fell from the sky and shattered in the sink. Some voodoo was clearly in the air. It appears that the ligation products from the day before were either not created or not successfully transformed, as the plates contained no colonies. Caleb and Tina prepared more kanamycin and chloramphenicol plates while Mark desalted Dylan's ligation product. Then Dylan, Mark, and Tina transformed DH5α with the ligation product, p33k, p14k, and p31c (See: Strain List).



July 24th, Tuesday

In the morning, Dylan did Phusion PCR of p20(nah operon). Then Caleb and Dylan did a double digestion of p20 PCR and p17c for ligation. Dylan ran a DNA gel electrophoresis to confirm that the previous PCR was successful. Steven performed gel extraction to isolate p17c backbone fragment.



July 25th, Wednesday

Mark set up two ligations of a p20 PCR product and p17c, one at standard concentrations and one at very high concentrations. (See: Strain List ) p20 PCR was cut at Xbio1 and Spe1, which have compatible sticky ends. This means that p20 PCR can be ligated into backbone in two directions, only one of which is useful. These were desalted and transformed by Mark and Dylan, along with p33k. Meanwhile, Tina and Chie set up double digestions of p24a, p20 PCR product, and single digestions of p25a, p27a, and p29a. Dylan ran two gels of the double and single digestions. The single digestions of the Anderson series promoters showed single bands, which ran slightly faster than the supercoiled DNA control. This may be because the supercoiled DNA was nicked. The gel of the oriT double digestion showed more long bands than expected – possibly a result of star activity – but had a band ~400bp which was interpreted to be the insert of interest.



July 26th, Thursday

Caleb gel purified the digestion products of p24a, p20 PCR, p25a, p27a, and p29a. Danielle dephosphorylated products of p25a, p27a and p29a in preparation for ligations .



July 27th, Friday

In the morning, Claire miniprepped p11k, p17c, and p33k from the overnight cultures that Dylan had set up. Meanwhile, Dylan heat killed the ligase from the overnight ligations (p25a, p27a and p29a) (See: Strain List ), while Caleb prepared to desalt the ligation mixtures before electroporation via drop dialysis. Shortly thereafter, Dylan and Caleb electroporated the four desalted mixtures into DH5α, allowing the cells to recover one hour in SOC before plating. If these transformations are successful, we will have cells that carry plasmids expressing the nah operon under the control of three different constitutive promoters with varying strength, as well as a cell line that carries a plasmid that confers chloramphenicol resistance, has a p15a origin of replication, and an origin of transfer. Eventually, the nah operon will be ligated as an insert into such a backbone. In the evening, Dylan and Tina started 30 mL overnight cultures of S24, S25, S27, S29, S15, and S17 to miniprep from the next morning. S24 carries a plasmid with an oriT, which will be miniprepped in case our previous ligation of an oriT into p17c failed. S25,27, and 29 all carry plasmids with Anderson series constitutive promoters of varying strength, which will be used to set up fluorescent controls to monitor mtrB expression levels. S15 and S17 carry our engineered arsenic reporter plasmids, which will be further modified to facilitate control studies in mtrB expression. After setting up the cultures, Dylan and Swati left Weill and headed over to Riley Robb where they assembled three new reactor setups in the Angenent lab. Two of these reactors will be run in continuous flow operation with a BioLogic potentiostat, while the other will be run in batch mode with a CH Instruments potentiostat. Tomorrow, one continuous flow and one batch reactor will be inoculated with wildtype Shewanella oneidensis MR-1, while the remaining continuous flow reactor will be inoculated with our engineered arsenic reporter strain, S20. The purpose of running the continuous flow reactors is to better define the maximum current output we can expect from our induced strains in response to analyte, and to repeat an experiment carried out on the CH potentiostat for basal activity from the uninduced arsenic reporter. Because we were unsure of the veracity of the data from our previous experiment using the CH potentiostat, we are running the batch reactor to see if the observed current response is what we'd expect from wildtype Shewanella.



July 28th, Saturday

Today, Swati did six minipreps in the morning and it took her four hours...and it paid off! The nanodrop told us that our highest yield was 812.6 ng/uL. While Swati was devoting her soul to the E.Z.N.A kit, Dylan continued to set up reactors in the Angenent Lab, making reference electrodes for the three reactors, and getting the pumps set up for the continuous flow reactors. After our weekly team meeting, he inoculated the reactor. Swati prepared overnight cultures and reference plates, while Danielle prepared kanamycin plates and did autoclave. In the evening, Dylan and Swati did digestions for RFP controls and SAL2 reporter. Also, SAL reporter was transformed into WM3064.