Team:Cornell/notebook

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Revision as of 14:24, 15 October 2014

Cornell iGEM

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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

    Text goes here.



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

    Text goes here.



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

    Text goes here.

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

    Text goes here.

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

    Text goes here.

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

    Text goes here.

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

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 8 (July 21 - July 27)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 9 (July 28 - August 3)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 10 (August 4 - August 10)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 11 (August 11 - 17)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 12 (August 18 - August 24)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 13 (August 25 - August 31)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 14 (September 1 - September 7)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 15 (September 8 - September 14)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 16 (September 15 - September 21)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 17 (September 22 - September 28)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 18 (September 29 - October 5)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 19 (October 6 - October 12)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    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

    Text

  • Drylab Overview

    Text goes here.

Week 20 (October 13 - October 19)


  • Wetlab Overview

    Text

    Lead Subteam

    Text

    Mercury Subteam

    Text

    Nickel Subteam

    Text

    Reporters Subteam

    Text

  • Drylab Overview

    Text goes here.