Team:BroadRun-NorthernVA/Notebook

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Lab Notebook

Welcome to our Lab Notebook! Here, we have documented the work done in our project so we can see and keep track of how our project is progressing.

June

Week 1
  • Brainstormed ideas for projects. Decided on cells that could break down starch.
    • Week 2-4
  • Researched the best way to go about this solution.
  • Saccharomyces was decided as our organism, because of it ability to thrive in a variety of conditions, both aerobic and anaerobic.
  • Used alpha amylase as our target gene to be cloned
    •

    July

  • More literature research and worked on developing protocols. Ordered materials for the project, enzymes, reagents, buffers, kits, agar plates, LB broth, etc.
  • Experiment details were solidified and planned out in more detail.
  • Ordered materials for the project, enzymes, reagents, buffers, kits, agar plates, LB broth, etc.

    • August

    Week 1
    • Designed 3 amylase gene constructs to be synthesized through IDT’s offer. The final makeup of the gene constructs are listed below.

      Construct 1

    • Biobrick prefix
    • Promoterless
    • Kozak sequence (Part BBa_K165002)
    • Native secretion sequence, from Bacillus amyloliquefaciens
    • Alpha amylase coding sequence from Bacillus amyloliquefaciens
    • ADH1 Terminator (Part BBa_K392003)
    • Biobrick Suffix

      • Construct 2

    • Biobrick prefix
    • Minimal cyc promoter (Part BBa_K105027)
    • Kozak sequence (Part BBa_K165002)
    • Native secretion sequence, from Bacillus amyloliquefaciens
    • Alpha amylase coding sequence from Bacillus amyloliquefaciens
    • ADH1 Terminator (Part BBa_K392003)
    • Biobrick Suffix

      • Construct 3

    • Biobrick prefix
    • Minimal cyc promoter (Part BBa_K105027)
    • Kozak sequence (Part BBa_K165002)
    • Native secretion sequence, from Bacillus amyloliquefaciens
    • Alpha amylase coding sequence from Bacillus amyloliquefaciens
    • ADH1 Terminator (Part BBa_K392003)
    • Biobrick Suffix

    • No spacing was needed in between the composite parts, all constructs were optimized for S.cerevisiae, and an extra eight bases were added before the Ecor1 restriction site and after the pst1 restriction site, in order to increase the efficiency of the enzyme.

    • Designed primers for the three gene constructs, by hand using New England Biolabs Tm calculator. Primers were named p01, p02, p03, and p04.
    • p01- left primer for construct 1
    • p02 - right primer for construct 1
    • p03- left primer for construct 2 and 3
    • p01- right primer for construct 2 and 3

    Week 2

  • The gene constructs and primers were first resuspended, according to protocol.
  • Primer resuspension: Amount of water added to reach 100uM concentration, water was added and pipetted up and down to resuspend.
    • p01: 293 µl of water
    • p02: 336 µl of water
    • p03: 269 µl of water
    • p04: 345 µl of water
  • 100uM concentration was then diluted to 10uM concentration. Gene construct resuspension: 100 µl of TE buffer was added to each gene construct and pipetted up and down and vortexed to resuspend. Tubes were then incubated at 30 degrees Celsius for 20 minutes.

    Then we amplified the DNA with the primers using PCR.
      Amplification PCR
      100 µl per reaction
      Reaction #1
    primer 01 primer 02 Gene construct #1 2x Master Mix water
    5 5 2 50 38
      Reaction #2
    primer 03 primer 04 Gene construct #2 2x Master Mix water
    5 5 2 50 38
      Reaction #3
    primer 03 primer 04 Gene construct #3 2x Master Mix water
    5 5 2 50 38
      Negative Control #1
    primer 01 primer 02 Gene construct 2x Master Mix water
    5 5 0 50 40
      Negative Control #2
    primer 03 primer 04 Gene construct 2x Master Mix water
    5 5 0 50 40
  • Ran a gel to confirm size.
      • Gel Electrophoresis
    • 10µl of each PCR reaction was added into ten different tubes and 2µl of loading dye added.
    • 11µl was then loaded into each of the wells and ran for 15 minutes.
      • Week 3
        PCR Purification
    • 30 µl of PCR product out of 100 µl was purified, using a Quiagen PCR purification kit.
        • Restriction Digest
    • Total reaction volume was 50 µl
    • Concentrations of plasmids and PCR products
      • pSB1c3 25ng/µl
      • pRS426 129ng/µl
      • pAG36 900ng/µl
      • PCR products 100ng/µl
          • Restriction Digest of Plasmids
        pSB1c3 plasmid pAG36 yeast vector pRS426 yeast vector
        4µl DNA 7.8 µl DNA 1.1 µl DNA
        5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer
        1µl EcoR1 enzyme 1µl Kpn1 enzyme 1µl EcoR1 enzyme
        1µl Pst1 enzyme 1µl Spe1 enzyme 1µl Pst1 enzyme
        39 µl water 35.2 µl water 41.9 µl water
          Restriction Digest of PCR Products
        PCR product 1 (promoterless and native secretion sequence), cut with EcoR1 and Pst1 PCR product 2 (cyc promoter and native secretion sequence), cut with EcoR1 and Pst1 PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with EcoR1 and Pst1 PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with Kpn1 and Spe1
        10 µl DNA 10µl DNA 10µl DNA 10 µl DNA
        5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer
        1µl EcoR1 enzyme 1µl EcoR1 enzyme 1µl EcoR1 enzyme 1µl Kpn1 enzyme
        1µl Pst1 enzyme 1µl Pst1 enzyme 1µl Pst1 enzyme 1µl Spe1 enzyme
        33 µl water 33 µl water 33 µl water 33 µl water
    • After ligation, we completed a bacterial transformation and plated cultures on agar plates.

      • September

      Week 1

    • Colony PCR on bacterial cultures
    • Made liquid cultures of positive colonies
    • Miniprepped liquid cultures

    • Transformed DNA samples into S.cerevisiae

      Week 2

    • Tested genetically engineered cells for ability to produce amylase and hydrolyze starch
    • Cells were tested in known concentrations of soluble starch dissolved in water Iodine was used to determine starch concentrations
    • Then the yeast cells were tested in a sample of Armstrong's factory water

      • Week 3

    • Cells were heat killed at 80 degrees Celsius to inactivate enzymes.
    • Ran through a gel to verify the fragments
    • Ligation of gene after successful gel.

      Week 4