Team:Cornell/notebook/wetlab/august

Daily Details:

August 1st, Wednesday

In the morning, Dylan noted that 5 of the 7 transformations from Tuesday produced plates with colonies. Those that worked were three Anderson series constitutive promoters (with downstream mRFP) in pBBRBB and the versions of our two arsenic reporters with mRFP downstream of mtrB. In the evening, Swati and Dylan prepared overnight cultures from all transformants and JG700, both to set up conjugations to get plasmid into Shewanella and to screen isolated plasmids for successful ligation. We will use these constructs to characterize the activity of the arsenic-sensitive promoter in Shewanella with respect to known constitutive promoter strengths. The two transformations that failed were versions of the salicylate reporter. After staring at plates, Dylan ran the nah+p29, oriT+p17c, p31c, and nah operon (from p20) digestions on a gel, extracted the fragments of interest, and then dephosphorylated the backbone. The isolated digestion products were used later used by Steven, who set up two overnight ligations – one to put the nah operon, with a constitutive promoter, on a pSB3C5 backbone with an added oriT sequence, and one to put the nah operon into the MCS of pSB1C3 for subsequent site directed mutagenesis. While Dylan ran his gel, Caleb miniprepped from the JG700+SAL and S31 cultures. After quantification, SAL plasmid isolated from JG700 was submitted for sequencing in order to confirm successful conjugation. Meanwhile, Mark decided that he enjoyed making DH5a electrocompetent stocks on Tuesday so much that he had to make more WM3064 electrocompetent stocks today. Starting from subcultures that Dylan had prepared, he prepared the stocks, and was assisted by Danielle.
From left: ladder, oriT+p17c digest (E+X), nah + p29 digest (E+S), p31c digest (E+S), p20 PCR digest (E+S):


August 2nd, Thursday

Caleb and Dylan created conjugation plates of p14RFP, p16RFP, and p25-29BB as well as miniprepped from the same plasmids in WM3064 (see: strain list). Dylan, Mark, and Tina then desalted and transformed SAL2 and SALRFP into WM3064 as well as p31c_nah and oriT_nah_p17c into DH5a. Tina and Dylan then quantified the minipreps from earlier in the day. Dylan also digested pBBRBB and p33k with EcoRI & PstI to put lac inducible mtrB into the pBBRBB backbone.



August 3rd, Friday

After running a digestion of p33k, the lac inducible mtrB part from the parts registry, we discovered that the part does not include mtrB! We should have seen a ~3.6kb band on our gel after digesting with EcoRI and PstI, but instead isolated a band ~1.5kb. After checking the sequencing on the parts registry we discovered this was because the part didn't include mtrB, so we won't be able to use it as a positive control for inducible mtrB. Sequencing of p37k-p41k from WM3064 came back good, meaning we can put these control parts, with RFP downstream of our various reporters, into Shewanella via conjugation. Hence, conjugation was carried out.
Digests of p11 and p33 (E+P):




August 4th, Saturday

This week, it was confirmed that RFP was successfully inserted downstream of mtrB in our arsenic reporter in Shewenella. Site-directed mutagenesis of the nah operon failed. We continued trying to insert the nah operon into the mobile backbone (OriT) to prepare for conjugation into Shewanella.

Daily Details:

August 5th, Sunday

Swati yet again was saddled with a massive number of minipreps , since her magic fingers are able to cast yield-increasing spells on minipreps. She miniprepped: SAL2 from WM3064, p37-41k from JG700 (one arsenic reporter with cut sites flanking the RBS, another arsenic reporter without the cut sites, and three different Anderson series promoters with mRFP1 downstream on a pBBRBB backbone), oriT p29nah_p17c from DH5a, and nah_p31c from DH5a. And indeed her yields were impressive, massive, gargantuan! Good job Sorceress Swati. We will sequence SAL2 and oriT p29nah_p17c to see if we should continue to conjugation, and sequence nah_p31c to see if we can start site-directed mutagenesis to get a biobrick-compatible nah operon on the biobrick backbone. We will sequence or PCR to confirm p37k-p41k's conjugation into Shewanella.



August 6th, Monday

Caleb and Dylan amplified the two different arsenic promoters (p37k and p38k) and ran them on a gel alongside p14 and p16 (the arsenic reporters without mRFP1 downstream) to confirm successful insertion of RFP downstream of mtrB in our arsenic reporter parts in Shewenella. Band lengths appeared in expected places. Dylan is worried that we will not be able to use PCR to definitely confirm that p39-41k worked, so we submitted these for sequencing . Unfortunately, sequencing failed, perhaps because random gunk from Shewanella added noise to the process. Tina did a PCR cleanup of the nah operon PCR. Dylan also ran a gel of nah operon PCRs to make sure that there was no mispriming. The gel looked good, so he submitted the PCRs for sequencing.
Successful insertion of RFP downstream of mtrB in arsenic reporter strains in Shewanella:




August 7th, Tuesday

JG1531 overnight culture didn't grow. We suspect that the plate Dylan picked from is dead. We'll have to go back to the glycerol stocks if we want to play with mtrE. Dylan set up a continuous flow M4 reactor in morning. Caleb started a liquid culture of MR-1 with which to inoculate the reactor. Checked sequencing results of nah stuff. oriT thing was bad. nah_p31c was good. Caleb and Dylan proceeded with site-directed mutagenesis of nah_p31c, attempting to get rid of the first PstI cutsite in the nah operon. After digestion with DpnI, we purified using the E.Z.N.A. MicroElute kit and quantified. DNA was split into three directions: First, Danielle and Dylan set up another mutagenic PCR using the digestion product as template to get rid of the second internal cut site (we expect lower mutation efficiency because template DNA is not methylated). Second, we transformed DH5a with the mutated plasmid. Third, Danielle and Chie digested both the purified – and hopefully mutated – plasmid and un-mutated plasmid with PstI. Dylan ran these digestions on a gel, along with supercoiled plasmid as a control. Unfortunately, the (hopefully) mutated plasmid never showed up on the gel. In the evening, we didn't observe growth in the MR-1 culture, so Dylan set up another culture just in case Shewanella was dead and not lazy.
Looking for mutagenesis – from left – ladder, unmutated nah, mutated nah, supercoil control:




August 8th, Wednesday

Caleb and Dylan performed another digest to check if the second site-directed mutagenesis (that was supposed to get rid of the second PstI internal cut site within the nah operon) worked. Unfortunately, there wasn't enough DNA to see anything when visualized on a gel. They decided to proceed with electroporation into DH5a just in case - however, to use Caleb's terminology, cells exploded - there were probably too many salts in the solution, which causing arcing and a PBBHTTTZZZ! of cells all over the cuvette. Dylan re-digested p29 nah and oriT p17c to redo a ligation that would allow for conjugation into Shewanella later on. Dylan also re-digested p27 (Anderson series promoter with RFP downstream) and SAL to redo a ligation to create a plasmid with SAL-RFP. Dylan also submitted several samples for sequencing to make sure that three Anderson promoters with RFP downstream in JG700 and SAL2 (the salicylate reporter without the BAMHI cut site) in WN3064 and JG700 were in the correct sequence.
Seeing if mutagenesis worked; nothing there:




August 9th, Thursday

Today Dylan chilled with his mom. Well, he tried to. :) Despite the eternal bonds that bind mother and son, plus the 3 billion miles she flew to see him, he still couldn't resist coming in to lab to open packages! And then got sucked into a long discussion of what had to be done for the rest of the day - scientific endeavors taking him away yet again from filial duties. Swati and Claire bonded over dry ice, and Caleb regaled us with tales of dry ice bombs gone awry. Swati and Caleb miniprepped the first mutagenesis of nah_p31c that had been transformed into DH5a yesterday. They then digested it with PstI-HF to see if the mutation was successful. Unfortunately the mutagenesis failed! We will start over from nah_p31c and lower the annealing temperature at 60degC. The Stratagene kit, which uses PFU ultra, asks for 60degC, but because we are using our own boot-leg protocol with Phusion we did the first try at 65degC, as Phusion usually calls for a higher annealing temperature than the theoretically calculated value. For this second try we will stick to the 60degC suggested by Stratagene and see if we get better results. Also, called NEB to find out if after PCR with Phusion, DpnI will still have activity in the following digestion step in Buffer 4, or if we need to clean up the PCR before digesting with DpnI - they said PCR clean up isn't needed. We also ran digestions for making the nah operon on a backbone with a mobility gene and the salicylate reporter (w/ BamHI cutsite) with RFP downstream. We cut our mobile backbone, OriT in p17c (pSB3C5), with EcoRI and XbaI, while cutting the nah operon (p29nah) with EcoRI and SpeI. The nah operon with mobility gene must be constructed so that we may conjugate into Shewy and start testing our salicylate reporter. The salicylate reporter and p27a (from the Anderson series), with RFP, were cut with SpeI and PstI. The salicylate/RFP part will be used for troubleshooting the salicylate reporter. Digestion products were run on a gel, extracted, and quantified. Swati then dephosphorylated backbones and ligated both parts. Sequencing for p39-41k didn't look good, and neither did the salicylate reporter w/BamHI cutsite miniprepped from JG700. The sequencing for salicylate reporter w/ BamHI in WM3064, however, looked good, suggesting that conjugation may not have been as efficient as we had hoped. After a pow-wow we decided not to sequence more colonies, as we are hoping some may be good, and more importantly that if we use qPCR to get quantitative characterization data, we won't need to use the RFP parts. The plates will stay in the fridge as a back-up plan. In other news: Claire cried because it was difficult to update the notebook with a week's worth of work. Mark's dedication to the notebook is laudable and impressive. Good job team for doing so much! My head can't even comprehend the magnitude of your endeavors.
Unsuccessful mutagenesis (test with Pst1 cutsite):

Digestion products from left – ladder, p27, p29nah, oriTp17c, SAL:




August 10th, Friday

Caleb miniprepped a plasmid with mtrE from JG1531, and just for kicks, miniprepped from the "exploded cells" from August 8th. Suprisingly, he ended up getting decent yields for both, showing that the electroporation worked despite arcing. Caleb then digested the nah operon of the miniprep with PSTI and NotI to check if the mutagenesis was a success. It was run on a gel alongside a PCR of the Anderson series promoters with RFP downstream and SAL2 from Shewanella (to check if SAL2 and the promoters were sucessfully conjugated after sequencing on Monday failed). Unfortunately, bands did not appear where we expected them to. Steven and Spencer performed a PCR to get mtrE out of the Gralnick (JG700) plasmid.
From left lane: ladder, p39 PCR, p40 PCR, p41 PCR, nah-p31c (P+N digestion), SAL2 PCR:




August 11th, Saturday

Spencer and Steven checked the nah operon PstI and NotI digest (because yesterday's gel ran weirdly) and their mtrE PCR from the previous day on a gel. Unfortunately, bands did not appear where we expected them to.
mtrE PCR product (left) and checking mutagenesis again (right):

Daily Details:

August 12th, Sunday

Weekend overview: It looks like the mtrE primers are actually working to amplify the part out of JG1531. We'll be putting mtrE in p31c, both so that we may submit the novel part to the registry, and to begin site-directed mutagenesis. We'll use mtrE mutagenesis as something of a control for nah operon mutagenesis (to see if the large size of the plasmid and nah operon is the problem). It also looks like nah operon mutagenesis hasn't worked. We'll put this on hold, and continue with ligation of p29nah into oriT_p17c once desalting paper arrives. Also, we'll have to redo things to confirm p39-41, SAL2 in JG700. What was done over the weekend looks weird.



August 13th, Monday

Dylan ran four simultaneous Phusion PCRs with an annealing temperature of 67degC and an extension time of 45sec to amplify mtrE. The gel looked good, with no mispriming and one band ~2.2kb. Claire did PCR clean up of the product, which will eventually be cut and ligated into p31c (see: strain list). Because the results from the weekend's screenings were confusing, we also ran PCRs of p39-41k (miniprepped from JG700 strains) to confirm whether conjugation into Shewanella was successful. These plasmids contain mRFP under the control of constitutive promoters of varying strengths. After confirmation, we will use these parts to characterize our inducible promoters in Shewanella. Dylan also started a liquid culture of JG700 + SAL to be inoculated into a continuous flow reactor with M4 media. With this setup, we will begin characterizing our reporter strains, initially in response to salicylate. Dylan performed a Phusion PCR to amplify mtrE, and ran a gel to check for product. There was a single band so no mispriming was occurring. Claire performed a double digest of the p14k and p16k with XbaI and PstI HF. This is for the construction of our final biobricks to be submitted to the registry.
PCR amplification of mtrE:

p39 and p41 verification:




August 14th, Tuesday

Steven performed a double digest of mtrE (EcorI & SpeI), Sal (SpeI & PstI), Sal2 (SpeI & PstI), and p26a (SpeI & PstI). Steven performed a ligation of p29nah + oriTp17c, and also a ligation of p27 digest + Sal. Dylan transformed colonies with the ligation.



August 15th, Wednesday

Dylan ran a gel and Claire performed gel extraction of mRFP to put downstream of Sal reporters. They also checked to see if site-directed mutagenesis was successful for the nah-p31c. Found the digest of mRFP and p26 looked good, but the mutagenesis looked strange. Dylan performed a taq PCR to confirm the presence of Sal2 in Shewanella. However no band was present in the gel ran afterwards, so perhaps colony PCR is the next best step. Dylan also ran a gel of the p14k, p16k p31 digests (all cut with XbaI and PstI) for the set-up for forming the biobricks. Steven performed a colony Phusion PCR of four colonies containing the p4k plasmid to again test for the presence of mtrE. Spencer then did PCR clean up Spencer performed a double digest of p4kdlg and p27a cutting with EcoRI & SpeI followed by running a gel and extracting the digests. Spencer picked colonies from the previous ligations of the Sal, Sal2, p31c biobrick plasmids for miniprepping on Thursday. This morning Dylan noted that we may be getting a detectable basal level of current - around 6mA - in a continuous flow setup. This would make it possible to bypass the addition of mtrE to the salicylate sensing part, and still be able to distinguish Shewanella not detecting salicylate from dead Shewanella. He then added salicylate to the reactor innoculated with our salicylate reporting strain, bringing the concentration to 10uM. Later in the day, he noted that the current had risen to around 9mA, which is promising: our strain may be working! Claire set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. Dylan also transformed a salicylate reporter with mRFP downstream into DH5alpha and WM3064, as well as the nah operon with the p29 promoter in a p17c backbone with an oriT. The second of these could be conjugated into Shewanella if transformed successfully. The first, the salicylate reporter with mRFP downstream, is a fluorescent version of the salicylate reporter. The fluorescent versions of our reporters is an alternative to qPCR to measure the relative expression level of mtrB: if we know what mRFP under the control of a constitutive promotor in Shewanella looks like, we can add arsenic or naphthalene/salicylate to our fluorescent reporter parts until reaching the same level of expression. Then, we can correlate this concentration of arsenic or salicylate to a certain strength of induced expression. In case the fluorescent SAL reporter was not successfully ligated, we are preparing more mRFP, SAL, and SAL2: - In the morning Claire also cleaned up the digestions of SAL, SAL2 and mtrE PCR. However, due to a silly mistake on her part involving wash buffer and absolute ethanol (absolutely missing, to be precise) we will redo the digestions in order to get more DNA for putting these reporters into the iGEM backbone, and to put mRFP downstream. She then set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. - We digested mRFP with SpeI and PstI to make the part with mRFP downstream of mtrB in the salicylate reporters. We have already successfully put mRFP downstream of mtrB in our arsenic reporters, but it did not seem to work in the arsenic reporter. The gel for mRFP looked as expected, with the band for the insert slightly shorter than 900bp. However, on the same gel the the mutagenesis trial of the nah operon, digested with PstI and NotI, showed only one band. If mutagenesis had not worked, we would expect more than two bands, and if it had worked we would still expect two bands. Therefore we are unsure how to interpret this result, which we have seen twice now, but are going to start over with site-directed mutagenesis of the nah operon. We will also visualize the unmutated nah operon, digested with PstI and NotI, on a gel to see if that looks like we expect it to. Finally, it should be noted with jubilance that Claire was reunited with her umbrella today! The joy in the lab at this event was palpable and will be remembered for years to come.
mRFP digest and additional (failed) attempt at confirming mutagenesis:

From left – ladder, failed SAL2, p14, p16, p31digestions:




August 16th, Thursday

Dylan performed a miniprep of the Sal, Sal2, and p31c reporters for us to do sequencing for confirmation of successful ligations. These will hopefully be the biobricks we send in. In the afternoon Dylan performed a dephosphorylation of the p31c (cut with XbaI, PstI) followed by a gel of the mtrE cut with EcoRI & SpeI as well as mRFP cut with SpeI and PstI. A 4 hour digestion was also performed by Claire to get Sal parts into pSB1C3 for the SAL, SAL2, and p31 parts. Steven then ran a gel of the digest products. Steven also performed a colony Taq PCR of the Sal2 reporter to confirm its presence in Shewanella. Today, we continued work to get our engineered reporters into pSB1C3 for submission to the parts registry. In the morning, Dylan miniprepped both versions of our salicylate reporters, along with more pSB1C3 from overnight cultures of the corresponding DH5a strains. Following quantification, he set up digestions of each miniprep with XbaI and PstI. (We decided to cut with XbaI because we discovered an extra base pair between the NotI and XbaI cutsites in the normal BioBrick prefix, an artifact of a previous team's work). Caleb also dephosphorylated previously isolated pSB1C3 backbone to prevent self-ligation, while Steven gel extracted the salicylate reporter inserts and more pSB1C3 backbone (to be quantified Friday morning).
mtrE and mRFP digests:

Digestions of SAL, SAL2, and p31:




August 17th, Friday

Claire performed a ligation for the construction of the Sal_RFP, and Sal2_RFP plasmids. Transformation was then performed immediately after.

Daily Details:

August 19th, Sunday

In the morning, Dylan came to lab to look at the transformant plates from Saturday. There were colonies on all but one of the plates: It looks like either the ligation to put mtrE into p31c or the subsequent electroporation failed. It is of note that the competent DH5α cells that Dylan used for the mtrE electroporation were from an older stock – one which we'd previously determined to confer lower transformation efficiency. Because we used up all of the newer stock – with markedly better efficiency – we will make more electrocompetent DH5α in the coming week. Because all other transformations seemed to work, we grew up overnight cultures from single colonies on each plate. We will miniprep from these cultures and screen the minipreps for the correct insert. If confirmed, we will have successfully have put both versions of our arsenic reporters, as well as both versions of our salicylate reporters inside p31c (i.e., pSB1C3), as is required for registry submission. We will also have successfully put mRFP downstream of mtrB on SAL2, which will be used for characterization of the salicylate sensitive promoter in S. oneidensis.
Gel showing failure of nahoriT p17c:




August 22th, Wednesday

Conjugating nah into Shewy: Caleb, Tina, and a guest Louis miniprepped 5 plasmids containing nah operons with constituitive promoters from a WM3064 plate and miniprepped one plasmid that we previously confirmed as having the nah operon (without a promoter) to use as a positive control in the future. Claire performed miniprep of grown cultures of the Sal_RFP and Sal2_RFP to submit for sequencing.



August 24th, Friday

Jim performed the digestion of the nah operon+ p29a and the p31c backbone for ultimate ligation in the creation of the last biobrick. Ligation had to be redone, as sequencing results of last week's ligations looked great except for the nah operon biobrick. Conjugating nah into Shewy: We wanted to accomplish two things: • Amplify the nah operon with a promoter using primers with appended cut sites. • Optimize colony PCR for future Cornell iGEMers (past attempts have failed.) To accomplish both of these objectives, we did Phusion PCRs using plasmids containing nah operons with constituitive promoters as a template and a Phusion colony PCR using the same colonies we miniprepped the plasmid from. Because we expect the miniprep PCRs to work, doing the colony PCRs in parallel will allow us to determine whether our colony PCR technique is working or not. For our colony PCR purposes, we want to be able to screen for colonies while being certain we can go back and miniprep the same PCRed colony. Our colony PCR method is as follows: NOTE: We later determined there was a cataclysmic flaw in the following method, can you figure it out? (wink wink nudge nudge...) • Label colonies of interest and correspondingly label microfuge tubes • Add 50 uL ddH2O to each microfuge tube • Using pipette tip, dip into colonies of interest then dip into labelled microfuge tubes and swirl around to release cells • Use the the ddH2O+cell mixture as the DNA template for a PCR - instead of adding plain ddH2O to the PCR to bring it up to volume, add the ddH2O+cell mixture • Confirm your PCR had desired results with a DNA gel, then use the ddH2O+cell mixture from the corresponding microfuge tube the PCR was done on to inoculate a culture or plate.



August 25th, Saturday

Jim performed an overnight ligation of the nah operon pSB1C3 for transformation on Sunday. Conjugating nah into Shewy: Caleb, Tina, and a guest Louis ran a gel of the miniprep and colony parallel Phusion PCRs. All of the miniprep PCRs worked, but only a couple colony PCRs worked. We believe this is due to not having added enough cells to the colony PCR tube because some of the colonies were very tiny.
Top: Positive control from colony, colony test 1, colony test 2, colony test 3, colony test 4, colony test 5, miniprep 5, Benchtop 1kb; Bottom: Benchtop 1kb, miniprep 1, miniprep 2, miniprep 3, miniprep 4, positive control miniprep: