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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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Mercury Subteam
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Nickel Subteam
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Reporters Subteam
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Drylab Overview
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