From 2014.igem.org

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Revision as of 17:09, 15 October 2014

Cornell iGEM

Notebook

Week 1 (June 2 - June 8)

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.
Drylab Overview

Today was the first drylab meeting for the new project! We discussed the mechanics and optimization of general filtration systems. We also started looking for the best way to make hollow fiber reactors the focal point of our filtration system.

Week 2 (June 9 - June 15)

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

We began the design process for our filtration system by choosing our ideal target: runoff streams that feed contaminated water into natural rivers. By basing our assumptions around this, we were able to sketch out some functional requirements for our system, such as minimum flow rate, power consumption, materials, etc. We divvied up the system into subparts to research and report back next week.

Week 3 (June 16 - June 22)

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

After researching hollow fiber reactors, we found that our assumptions from the last meeting were too liberal. We scaled down all our numbers around a slower flow rate. We discussed our findings on tubing materials, pipe fittings, power sources, and pump options. We sketched out a rough sketch of our system, which included a pump, multiple fiber reactors, larger filters to prevent debris from entering, and a solar panel and battery as power sources.

Week 4 (June 23 - June 29)

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

After contacting several fiber reactor manufacturers for product quotes, we quickly discovered our hypothetical multi-reactor system was insanely expensive. Since we didn’t want Eric to have to sell an organ to pay for our filtration system, we scaled it down again to a one or two fiber reactor system so it was more feasible.

Week 5 (June 30 - July 6)

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

Dan Levine, a drylab member from the 2012 Cornell iGEM team, met with us to help plan our project and pass down ancient drylab design wisdom. He had a unique technique for brainstorming, which was to spend five minutes writing down any ideas you have, regardless of how farfetched, and then share them with the rest of the team. This approach worked extremely well. We had an extensive, positive discussion about our project and decided to completely overhaul our original design. The overall structure of our system was changed to some sort of drum to collect contaminated water, which would then be moved to and filtered in an attached box. We also changed the targeted water source to outflow from a factory, which would fall into the collecting drum.

Week 6 (July 7 - July 13)

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

We found a pump deep in the box of drylab components that have accumulated over the years and decide to test it out. We gutted “the Box” from the 2012 team’s project in order to use it as housing for our own filtration system. Unfortunately, the flow was too slow for us to salvage it for our project, but we decided to order new parts in order to begin assembling our own Box.

Week 7 (July 14 - July 20)

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.

Metallothionein Subteam

This week, AH insert was rehydrated and tranformed, to be prepared in the future.
Drylab Overview

We ordered our fiber reactor!

Week 8 (July 21 - July 27)

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.

Metallothionein Subteam

Miniprepped the cultures made from the colonies of the transformed cells. After multiple digestion attempts with EcoRI and PstI, we decided to stagger our efforts. We were going to start running multiple processes of Miniprep/digestion/ligation to increase our potential for success. By the end of the week, our first ligation samples were ready for heat shocking. Luckily, the heat shocked contained colonies and we crossed our fingers that these colonies contained was we needed.

Drylab Overview

We received most of our ordered parts earlier this week, including the fiber reactor! We tested the fiber reactor with our new pump, and the flow rate was about 0.2 mL/min. After testing the fiber reactor, we brainstormed orientations for our collecting drum and ideas for how to compactly fit the filter and piping into a protected and accessible casing.

Week 9 (July 28 - August 3)

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.

Metallothionein Subteam

The colonies were miniprepped and sent in for sequencing… ‘lo and behold, it worked! Our construct was a complete success. The nest construct, AHH, was renamed AI. Our next task is to digest out the T7 promoter, and then subsequently submitted. Cultures of AI were made, then miniprepped. The minipreps were then digested with KpnI to cut out the T7 promoter. Again, we staggered out multiple processes. We are hoping to be able to verify a successful digestion next week with a digest screen.

Drylab Overview

We decided to collaborate with the Outreach group to create a portfolio of future applications for our filtration system. We had a lot of fun brainstorming cool designs, the collective favorite being the idea of a water roomba. The ideas were distributed among the drylab team and each person was tasked with making a CAD model of theirs.

Week 10 (August 4 - August 10)

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.

Metallothionein Subteam

AI-T7 was digest screened with SacI, a few of the bands looked good, so they were purified and then sent in for sequencing. Sequencing came back a success and we had our construct AI-T7, which was renamed AK. With the quick success of this task force, we were transferred to work on creating the lead construct. Thus, we started with the same process with R, the lead insert, and H, the psB1C3 backbone. We miniprepped both pieces this week.

Drylab Overview

We made progress on our CAD models.

Week 11 (August 11 - 17)

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.

Metallothionein Subteam

This week comprised not only of struggling to attain successful minipreps for digestions and ligations, but also to hope that the ensuing ligations will successfully have colonies. After multiple attempts at ligations and transformations, we finally had a single colony that grew that we cultured then digest screened and sent in for sequencing. Like the previous construct, these are being digested with EcoRI and PstI.

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Team:Cornell/notebook

From 2014.igem.org

Revision as of 17:09, 15 October 2014

Notebook

Week 1 (June 2 - June 8)

Wetlab Overview

Drylab Overview

Week 2 (June 9 - June 15)

Wetlab Overview

Drylab Overview

Week 3 (June 16 - June 22)

Wetlab Overview

Drylab Overview

Week 4 (June 23 - June 29)

Wetlab Overview

Drylab Overview

Week 5 (June 30 - July 6)

Wetlab Overview

Drylab Overview

Week 6 (July 7 - July 13)

Wetlab Overview

Drylab Overview

Week 7 (July 14 - July 20)

Wetlab Overview

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 8 (July 21 - July 27)

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 9 (July 28 - August 3)

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 10 (August 4 - August 10)

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 11 (August 11 - 17)

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 12 (August 18 - August 24)

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 13 (August 25 - August 31)

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 14 (September 1 - September 7)

Lead Subteam

Mercury Subteam

Nickel Subteam

Reporters Subteam

Metallothionein Subteam

Drylab Overview

Week 15 (September 8 - September 14)

Lead Subteam