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Team:Cornell/notebook
From 2014.igem.org
Notebook
Week 1 (June 2 - June 8)
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Wetlab Overview
Transformation efficiency and protocols were tested - heat shock did not work well, and we came to the conclusion to stick with electroporation. The first day of wetlab training began on the 7th! We started working on amplification of metallothionein genes - so far nixA and GST-PMT appear to be working.
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Drylab Overview
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Week 2 (June 9 - June 15)
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Wetlab Overview
Obtained transformants of pA14F and pC14T heat shocks and both E/B and E/D were successfully heat shocked into DH5a. Ran into problems later in the week with heat shocked plates from overgrowth and contamination; switched to electroporating of C+F and E+B.
Drylab Overview
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Week 3 (June 16 - June 22)
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Wetlab Overview
This week we ligated various parts (F+A, F+C, E+B, and E+D), transformed, ran colony PCRs, and submitted them for sequencing. Most of our attempts were unsuccessful either due to failed ligations or contamination. We also made T for future minipreps.
Drylab Overview
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Week 4 (June 23 - June 29)
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Wetlab Overview
Cloning and troubleshooting continues. E+D, F+C, E+D, E+B4, and E+D11 sequencing results came in negative. The restriction cocktail method was used on F+C and F+A because mRFP kept religating back into the backbone. Plates were successful, but colony PCRs showed they were negative. Repeat cloning. We are facing problems with contamination of lead transporter and R plate. Submitting F+C3 for sequencing. We are also working on transforming off the AA and AB kit plate.
Drylab Overview
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Week 5 (June 30 - July 6)
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Wetlab Overview
Transformants gave negative signals; many gels lacked proper bands in correct locations. Growth assay looked good for mercury although concentration needs to be increased for nickel.
Drylab Overview
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Week 6 (July 7 - July 13)
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Wetlab Overview
We gathered the data for an assay testing the growth of cells in different heavy metals. In addition, we ligated each the lead transporter (R), and nickel (AA) and mercury (AB) inducible promoters with H. Unfortunately, our transformations did not seem to work for reasons like incomplete digests and inefficient stocks.
Drylab Overview
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Week 7 (July 14 - July 20)
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Wetlab Overview
We are troubleshooting some problems with gel purification and smearing on the gel when we visualize it. Our new stocks of competent cells also appear to be bad. Sequencing of R+H returned a portion of a random promoter region of Enterococcus, so this will have to be remade. We are in the process of creating many different BioBrick parts, such as AB1+H, F, AC+H, AB2+H, AC1S+H, AB2S+H, AC+H, AA + H, which are all at various stages of the cloning process.
Lead Subteam
R was cultured, miniprepped and quantified. R and H were digested, gel purified and quantified. The pSB1C3 backbone was dephosporylated, and the R and H digests were ligated and transformed.
Mercury Subteam
Our first objective is to place the synthesized mercury gene pA14AG into the pSB1C3 backbone. This will be done by digesting pA14AG and pC14H (mRFP in pSB1C3 backbone) with EcoRI and PstI, ligating them, and transforming them. The resulting colonies should contain pA14AG+pC14H (pC14I). pC14I will then be placed upstream of the metallothionein construct pC14AI. This week liquid cultures of pC14H were made from glycerol stock, which were then miniprepped. pA14AG and pC14H were both digested with EcoRI-HF/PstI-HF and gel purified. The gel was good – the pA14AG band was around 800 bp and the pC14H band was around 2000 bp. The concentrations were around 10 ng/μL and they were not worth ligating. Next week we will try re-digesting and re-ligating.
Nickel Subteam
We started out by making glycerol stocks for cultures containing our plasmids pA14AF and pA14AG and miniprepping pA14AF for cloning into the psB1C3 backbone.
Reporters Subteam
The goal of the reporter subteam is to put the reporter (pc14H, which is mRFP) into pc14AA (the nickel inducible promter) and pc14AB (the mercury inducible promoter). Later, we will get a third vector (pc14AC, nixA) that we will also have to put our promoter inside. This week, we made glycerol stocks of pc14AA and pc14AB. We also used glycerol stocks to make cultures of pc14H (mRFP; this is our insert), and later miniprepped these cultures, as well as pre-existing cultures of pc14AA and pc14AB.
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Drylab Overview
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Week 8 (July 21 - July 27)
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Wetlab Overview
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Lead Subteam
T was miniprepped. New plates were made. Cultures of the putative transformants were made, but the transformation proved unsuccessful. R and H cultures were grown and miniprepped, but the concentrations were low. New cultures of R and H were subsequently made. A culture of T was made.
Mercury Subteam
The double digests of pA14AG and pC14H were redone and new pC14H minipreps made. We increased the amount of DNA used in each digest in hopes of increasing the concentration of the gel purified bands. The gel did not turn out well so we had to re-digest. These newly digested pA14AG and pC14H were run on a gel and gel purified. In the meantime, the gel purified pA14AG and pC14H from last week were ligated (even though the concentrations for each were low) to see if the ligation would work. This ligation was transformed but once plated, there was a lawn of cells – it appears the antibiotic had degraded. The most recent digests turned out to have good concentrations so the backbone was dephosphorylated and ligated with the insert. Transformation and plating of this new ligation produced colonies! Colony cells were grown in liquid cultures, miniprepped, digest screened, and sent to sequencing.
Nickel Subteam
We went through numerous iterations of miniprepping pA14AF and pC14H, digesting both with EcoRI and PstI, gel purifying the insert from pA14AF and backbone from pC14H, dephosphorylating the backbone, ligating, and transforming. By the end of the week, one of the transformations yielded colonies! Here’s hoping…
Reporters Subteam
This week we almost completed one iteration of the cloning cycle. We digested, cleaned, and ligated pc14AA, pc14AB, and pc14H. We transformed the ligations for a total of 6 plates plates (4 pc14AA +pc14H and 6 pc14B + pc14H). We were able to culture 3 colonies from each of the pc14AA+pc14H plates for a total of 12 cultures, and we miniprepped these cultures for digest screening.
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Drylab Overview
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Week 9 (July 28 - August 3)
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Wetlab Overview
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Lead Subteam
T and S cultures were made and miniprepped. T and R were gel purified. The T digest was dephosphorylated and ligated with R. The S ligation was transformed.
Mercury Subteam
The sequencing did not go well – we think the miniprep might have been contaminated. The minipreps of pC14I were redone and another pA14AG and pC14H ligation was done which we will transform if the sequencing of the new minipreps is again not successful. Sequencing came back as GST-YMT which is not what we were hoping for. Thus, we transformed the new ligation and redid the minipreps of pA14AG which were digested with EcoRI-HF/PstI-HF. When loading dye was added to the digested pA14AG prior to running a gel, the digest turned brown…pA14AG was digested again with EcoRI-HF/PstI-HF in two tubes and with EcoRI-HF/SpeI in two tubes but still turned brown when dye was added. We think the EB from the miniprep might have been contaminated and so grew up more pA14AG insert which were miniprepped. The first pA14AG double digest which turned brown was run on a gel. The gel did not look good however, the bands were too long.
Nickel Subteam
This week we grew up, miniprepped, and digest screened a couple colonies from the previous week’s pC14H+pA14AF transformation. The bands on the gel looked to be in the correct location, so off to sequencing they go!
The rest of the week was spent culturing, miniprepping, and digesting pC14T with EcoRI and SpeI. Our pA14AF EcoRI/SpeI digest will be the insert into the digested pC14T backbone.Reporters Subteam
Only one of the plates of pc14AB + pc14H had colonies, so we made three cultures and miniprepped them. These minipreps had very low concentrations, so we didn’t digest them, but the minipreps of pc14AA + pc14H from last week had much better concentrations, so we digested them and ran a digest screen. The digest screen was successful, so we submitted a sample for sequencing. The results of the sequencing weren’t what we were looking for, so we cultured a red culture from the plate to prepare for sequencing in order to see if there is a problem with the sequence of the promoter. We miniprepped this RFP culture, and sent it in for sequencing. Meanwhile, we also made overnight ligations of pc14AB + pc14H and transformed them into more cells using heat shock. Unfortunately, nothing grew on the pc14AB + pc14H plates so we threw them out and worked on cleaning and purifying previous pc14AB and pc14H digests.
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Drylab Overview
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Week 10 (August 4 - August 10)
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Wetlab Overview
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Lead Subteam
Attempts to transform the ligations continued, without much success.
Mercury Subteam
We just realized that the pA14AG culture we had been using was growing in LB without ampicillin, so we grew up another culture overnight, with antibiotic in the LB this time. The pA14AG digested with EcoRI-HF/PstI-HF and with EcoRI-HF/SpeI from last week were run on a gel, the insert bands looked around .7kb so we gel purified. The concentrations were very low – 13 ng/μL for the EcoRI-HF/PstI-HF digests and 5 ng/μL for the EcoRI-HF/SpeI digests. However, the pA14AG digests with EcoRI-HF/PstI-HF were still ligated with pC14H from the metallothionein task force group and transformed. Unfortunately, the plates did not have colonies. pA14AG was again miniprepped and double digested with EcoRI-HF/PstI-HF and EcoRI-HF/SpeI. These digests were run on a gel and gel purified. The gel purified pA14AG digests had good concentrations, were ligated to the pC14H backbone, and transformed/plated. There was one colony on this plate – a LB culture was made of this colony in chloramphenicol and will be miniprepped next week. Four colonies were picked from the plate from earlier this week and LB cultures were made. These will also be miniprepped and sent for sequencing to see if we have successfully transformed pC14I.
Nickel Subteam
Sequencing came back for pC14H+pA14AF. Unfortunately it was not correct, so we needed to go back and reclone those. More cultures of pC14T were miniprepped.
Reporters Subteam
We made ligations from the digests of pc14AB and pc14H – however, we transformed them incorrectly and were unable to plate any cells. Because we ran out of ligations, we made more cultures of pc14AA and pc14AB from the glycerol stocks. We also learned that pc14H is a constitutive promoter, meaning that it will always be on (the cells will always turn red, regardless of whether or not heavy metal is present). Because of this, we started working with a new promoter this week – pc14AJ. The new promoter is a constitutive promoter (amcilCP, and it is blue!). We made a glycerol stock of pc14AJ and cultured from this stock. However, we were unable to continue with the minipreps of pc14AA, pc14AB, and pc14AJ until the beginning of the next week because the lab ran out of supplies.
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Drylab Overview
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Week 11 (August 11 - 17)
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Wetlab Overview
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Lead Subteam
The process of reculturing and religating R and T was repeated. The transformation, however, was a failure.
Mercury Subteam
pC14I cultures #1 and #3 were miniprepped and a small portion digested with EcoRI-HF/PstI-HF for a digest screen. The pA14AG insert appeared too long so this pC14I is probably not what we want. New pA14AG liquid cultures were made and miniprepped, digested with EcoRI-HF/Psti-HF, and run on a gel which did not turn out well. We had to begin the miniprepping, digesting, and gel purifying process again for pA14AG. Additionlly, new pA14 minipreps were made in case we need them again next week.
Nickel Subteam
After many, many cultures, we were finally able to get a couple of minipreps of pC14T that had good concentration. We proceeded to digest portions with EcoRI/SpeI and EcoRI/PstI for future ligations.
Reporters Subteam
Once the miniprep supplies arrived, we started making 16 minipreps…which we then lost (on the second-to-last step of the process) to the centrifuge when it refused to open. (Author’s note: as of right now, early October, the centrifuge is still sealed shut and our minipreps are still inside it). The setbacks of the previous week (losing the ligations, having to use a completely different reporter, and the stuck centrifuge) have put the reporter subteam back in square one. We miniprepped 2 cultures of pc14AA, 2 cultures of pc14AB, and 4 cultures of pc14AJ, and then digested, ligated and transformed the ligations. However, once only one plate (pc14AB + pc14AJ) had growth on it, so we made two cultures. Neither of the cultures grew, so nothing could be miniprepped.
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Drylab Overview
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Week 12 (August 18 - August 24)
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Wetlab Overview
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Lead Subteam
The process of reculturing and religating R and T was repeated. The transformation, however, was a failure.
Mercury Subteam
The new pA14AG minipreps were double digested with EcoRI-HF/PstI-HF and gel purified. The gel purified pA14AG from last week was ligated with pC14H which already had been double digested with EcoRI-HF/PstI-HF and dephosphorylated. This ligation was transformed into cells and plated. This process was again repeated with the newly digested pA14AG minipreps from earlier this week. There was one colony that grew on the pC14I plate which was minprepped.
Nickel Subteam
Ligations of pA14AF and pC14T were made multiple times, trying out different combinations of restriction enzymes too (EcoRI/SpeI for both and EcoRI/PstI for both). All were transformed, but colonies only seemed to grow for the EcoRI/PstI digestion enzyme combination. Regardless, colonies were grown to see if it worked.
Reporters Subteam
There was a hiatus this week because all subteam members went back home for a week of summer vacation.
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Drylab Overview
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Week 13 (August 25 - August 31)
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Wetlab Overview
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Mercury Subteam
The pC14I miniprep was digested with EcoRI-HF/PstI-HF for a digest screen and run on a gel. pC14I was then sequenced confirmed! This new biobrick consists of mercury MerT and MerP genes, Anderson promoter, and the pSB1C3 backbone. Now our objective is to place pC14I upstream of the metallothionein construct pC14AI and transform the ligated product in BL21 cells. The miniprep of pC14I that was sequence confirmed was transformed into new cells and plated to make glycerol stocks. New LB cultures of pC14AI were grown and were miniprepped.
Nickel Subteam
pC14T+pA14AF colony cultures were miniprepped and digest-screened. Unfortunately, the gel test was inconclusive, so more colonies were cultured, miniprepped, and digest-screened; this second round also led to inconclusive results.
Reporters Subteam
We transformed and plated using ligations of pc14AA + pc14AJ, pc14AB + pc14AJ, and pc14AC +pc14AJ that we’d made prior to leaving for break. Only the pc14AA + pc14AJ plate had any growth on it, despite all the plates being left in the incubator for two days, so we made a culture of this (hoping that it wasn’t contamination that we were culturing). We also cultured some more pc14AA, pc14AB, pc14AC, and pc14AJ from glycerol stocks, and were able to make cleaned digests of the four.
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Drylab Overview
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Week 14 (September 1 - September 7)
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Wetlab Overview
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Lead Subteam
Cultures of R and T were miniprepped. PCR’s of R were run, one with Q5 and one with Phusion. The PCR’s did not show up in a gel. A PCR of varying dilutions of R Miniprep (1 ul, 3 ul, 5 ul, 8ul - each in 200uL H2O) was run. All appeared in a gel. The PCR product was subsequently cleaned.
Mercury Subteam
The miniprepped pC14AI was digested with EcoRI-HF/PstI-HF and pC14I digested with EcoRI-HF/SpeI. pC14AI was gel purified, pC14I was dephosphorylated and was ligated to pC14AI. The length of the insert is 1076bp and backbone is about 2761bp. The pC14AI+pC14I ligation was transformed into BL21 cells and plates on chloramphenicol plates. The plates did not show colonies and the ligation was redone and transformed/plated. Liquid cultures were made of the colonies.
Nickel Subteam
We decided to redigest pA14AF and ligate with pC14T, maybe this time it will work? These ligations were then transformed and colonies grown for sequencing. Much to our pleasure, sequencing came back with positive results! We were able to construct pC14K (pC14T+pA14AF)! Glycerol stocks were made of pC14K and pC14H was religated with pA14AF.
Reporters Subteam
We digested the pc14AA + pc14AJ cells and screened them, but the first gel was inconclusive because it was warped, and the second gel showed that the bands were the incorrect lengths (the first, pc14AA was around 2000 base pairs, as it should be, but the second band, pc14AJ was around 1000 bp when it should have been closer to 700 bp). We also made 2 plates of pc14AA + pc14AJ, 3 plates of pc14AB + pc14AJ, and 1 plate of pc14AC + pc14AJ we were able to get 11 cultures from these plates. We miniprepped theses 11 cultures, and we made 12 ligations from cleaned digests left from the previous week. Towards the end of the week we plated a few pc14AA + pc14AJ and pc14AC + pc14AJ transformations.
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Drylab Overview
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Week 15 (September 8 - September 14)
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Wetlab Overview
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Lead Subteam
Cultures of T were made from glycerol stock. Quantification of the PCR yielded poor results. The PCR was subsequently restarted. Cultures of T were miniprepped, and the R PCR was redone. The column purification of the R PCR yielded positive results. The R PCR and the T miniprep were subsequently digested. R and T were ligated and transformed. The ligations were plated with chloramphenicol. No growth appeared, so the ligation was repeated.
Mercury Subteam
pC14AI+pC14I from the plates was miniprepped and digested with EcoRI-HF/PstI-HF to run a digest screen to see if the ligation was truly of pC14AI and pC14I. The gel showed the insert to be very short and we think it may only be the mercury construct in pSB1C3. The ligation was redone, transformed/plated, and liquid cultures were made of the colonies. pC14I was also miniprepped for future use.
Nickel Subteam
Cultures were made of pC14AI and pC14K for miniprepping and future cloning. pC14AI and pA14AH were digested, but the gel for gel purification came out oddly - bands were not coming out where we would have expected them. We also digested pC14K for more cloning, but none of the digestions had measurable DNA.
Reporters Subteam
We made four cultures from the plates we made last week – 2 of pc14AA + pc14AJ and 2 of pc14AC + pc14AJ. We ran digest screens on the digested minipreps, but all the bands were the wrong length. There were a few digests remaining from the previous week, so we cleaned, dephosphorylated and ligated them to make 4 ligations (2 of pc14AB + pc14AJ, and 2 of. However, the quality of the ligations is a bit suspect because the digest concentrations were on the lower end. We transformed, plated, and cultured cells using these 4 ligations, but we were unable to miniprep them because the lab ran out of 1.5 mL tubes. We also transformed using a pc14AB + pc14AJ ligation from earlier that we’d found in the box.
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Drylab Overview
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Week 16 (September 15 - September 21)
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Wetlab Overview
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Lead Subteam
Colony growth was observed on the transformant plate. They were cultured, and a colony PCR was run.
Mercury Subteam
Many minipreps were made of the pC14AI+pC14I liquid cultures, digested with EcoRI-HF/PstI-HF, and run on a gel. Successful ligation would show a band at around 1.8kb and one of our bands did appear in that area – this band was gel purified and sent for sequencing. In the meantime, pC14I was miniprepped, digested with EcoRI-HF/SpeI, and column purified.
Nickel Subteam
In the process of constructing pC14K+pC14AI, we experienced a bit of difficulty. After multiple failed cloning experiments, we tried synthesizing the part using pC14K+pA14AH. Hopefully the second approach will work!
Reporters Subteam
Tubes arrived in the lab, so we miniprepped and digested the cultured cells from last week. The gel screen of the digests was textbook perfection for all three of pc14AA + pc14AJ, pc14AB + pc14AJ, and pc14AC + pc14AJ. We sent in samples of the minipreps for sequencing, but the results were strange – sequencing reported that only pc14AJ was in the plasmid, and that none of pc14AA, pc14AB, and pc14AC were present. Luckily earlier in the week, we’d made several ligations of all three that we’d already transformed, plated and cultured so we could still continue with miniprepping these cultures.
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Drylab Overview
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Week 17 (September 22 - September 28)
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Wetlab Overview
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Lead Subteam
Gel of colony PCR was run, yielding poor results. Minipreps of some of the cultures were prepared. Colony PCR’s of the R+T cultures were also run. Cultures of relevant strains were remade, and a new colony PCR was run. The cultures of these colonies were miniprepped, and one tube was sent for sequencing. The sequencing yielded unsatisfactory results.
Mercury Subteam
The miniprep of pC14AI+pC14I we had sent for sequencing ended up being Neema’s reporter and we think our whole plate was contaminated. We made more pC14AI+pC14I minipreps and digests from the same plate and ran another gel, but we still observed bands in the same region as where Neema’s reporter band was so our hypothesis was virtually confirmed. Thus, we began the entire process of miniprepping and digesting pC14AI and pC14I, gel purifying pC14AI, column purifying and dephosphorylating pC14I, ligating them together and transforming into BL21.
Nickel Subteam
Inserting pA14AH into pC14K did not seem to work judging from the digest-screen. After preparing more pC14AI for cloning, we also were going to try inserting pC14K into pC14AI. However, the gel purification step for that was bizarre and indicated our parts were shorter than what they were. Digest screens of pC14K+pA14AH minipreps also were inconclusive.
Reporters Subteam
This week, we decided to change up our digest protocol a bit – rather than digesting 3000 ng of DNA at a time and incubating the digests for 3 hours, we decided to stick to 1500 ng of DNA and incubate for 1.5 hours maximum because we thought that star activity was causing the DNA to not be digested properly when it was incubated for long periods of time. We made 2 cultures each of pc14AA, pc14AB, pc14AC, and pc14AJ, miniprepped, and digested them for the shorter period of time. When cleaning these digests, however, they smeared in the gel, so we made more cultures of the four plasmids using glycerol stocks. Additionally, we ran digest screens on the cultures from last week, but they were indicative of contamination. By the end of the week, we were able to produce 6 ligations of pc14AC + pc14AJ (we transformed and plated all 6 of these for a total of 12 plates), and we’d started up another batch of cultures from glycerol stocks in case these ligations were not any good. Unfortunately, on the last day of this week, we ran out of Pst1-HF enzyme and were unable to continue with digests because neither of our buffers worked well with both Pst1-HF and Spe1-HF.
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Drylab Overview
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Week 18 (September 29 - October 5)
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Wetlab Overview
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Lead Subteam
A digest of an R miniprep was prepared. A PCR of an old R miniprep was run. Cleanup of the PCR yielded poor results, possibly suggesting ethanol contamination.
Mercury Subteam
pC14AI digests were run on a gel last week but the ladder did not appear so we could not tell what the length of the insert was (and therefore we could not be sure if the band was what we wanted). Therefore, we began the process again for pC14AI. pC14AI and pC14AK were also miniprepped so they could be turned into iGEM HQ. pC14AI and pC14I were again ligated and transformed into BL21 and plated.
Nickel Subteam
The minipreps of pC14K+pA14AH were sequenced just to see if they were correct. Sadly, they were not - drat! More cultures of pC14K were prepped and quantified. These were then digested for inserting into pC14AI. Here’s hoping the construct works!
Reporters Subteam
We made a total of 21 digests using a various combination of buffers that we didn’t normally use because Pst1-HF hadn’t arrived yet. Over the course of the week, we made 6 pc14AA + pc14AJ ligations, 6 pc14AB + pc14AJ ligations, and 4 pc14AC + pc14AJ ligations. Transforming using the pc14AC + pc14AJ ligations, we were able to make 4 plates, which yielded 3 successful cultures. We spun down the cultures and stored them in the fridge because the lab had once again run out of tubes. Towards the end of the week, we also made 20 more digests (7 pc14AA, 10 pc14AB, and 3 pc14AC).
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Drylab Overview
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Week 19 (October 6 - October 12)
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Wetlab Overview
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Mercury Subteam
The remainder of the ligations and new ligations were transformed into BL21 and hopefully these will yield colonies!
Nickel Subteam
Can anyone sing, “The Final Countdown?” This is it! We iterated through the process of transforming, culturing, miniprepping, and sequencing many times. Sadly, none of the constructs came out correctly and we ran out of time. We put in a great effort, but biology took this round from us.
Reporters Subteam
Over the course of the weekend, we realized we only had three days to get our final constructs in. Resigning ourselves to the fact that tubes were unlikely to arrive anytime soon, we ligated and transformed using all the digests we had. We’d also had some ligations from previous weeks of pc14AC + pc14AJ that we transformed with early in this final week and sent in for sequencing, after a successful digest screen. Additionally, we finished dephosphorylating the digests we already have to make a second batch of ligations. As our final Hail Mary, we transformed using every single ligation we had, which yielded an astounding 18 plates. Shockingly enough, there was growth on every single plate, which meant we had well over 30 samples to prep so that they could be sent for sequencing. There wasn’t enough time to screen all the samples prior to sequencing, so we selected the samples with the highest concentrations (in the 500 ng/mL range) and sent ten of those in for sequencing. Unfortunately, sequencing results returned exactly what they had in previous times – that only pc14AJ was in the plasmid of the cell. We thought this was because the cell stock had accidentally been made using cells that had pc14AJ as the backbone, and that this problem had been rectified by making new cell stocks, but apparently that was not the case.
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Drylab Overview
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Week 20 (October 13 - October 19)
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Wetlab Overview
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Drylab Overview
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