Difference between revisions of "Team:BroadRun-NorthernVA/Notebook"

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<h align="left" class="pageheading"<font size="3.8"><b>Week 1</b></font></h2>
 
<h align="left" class="pageheading"<font size="3.8"><b>Week 1</b></font></h2>
 
<font size="3.3"><li>
 
<font size="3.3"><li>
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      After hearing about the industrial waste water purification issue our corporate sponsor was dealing with, we began to brainstorm ideas for solutions. Our first few solutions were to break down butyric acid into butanol, prevent the pyruvate to butyric acid pathway by secreting a compound that would inhibit the enzymatic activity of acetyl-CoA-acetyl transferase, introducing water bears (tardigrades) to the water system that would feed on the microbes in the water, and eliminating the starch in the water so as to prevent the microbial growth.
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Brainstormed ideas for projects. Decided on cells that could break down starch.  
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Decided on the latter approach: it was more sustainable, efficient, and cost effective than the others.  
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     </li>  
 
     </li>  
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</li>
 
</li>
  
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<li>We decided on amylase, specifically alpha amylase, because of its ability to break down a variety of different starches. Alpha amylase can break down starches faster than other forms, because it can act in any substrate</li>
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<li>Used alpha amylase as our target gene to be cloned</li>
 
</font>
 
</font>
  
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<ul class="listitems"><font color="black">
 
<ul class="listitems"><font color="black">
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<font size="3.3"><li><span>Designed 3 amylase gene constructs to be synthesized through IDT’s offer. The amylase gene used for all three constructs was the alpha amylase coding sequence from Bacillus amyloliquefaciens. The Genbank accession number is J01542.1. First parts used were pCyc (medium) promoter (Part BBa_I766555), Kozak sequence (Part BBa_K165002), Alpha amylase coding sequence (Genbank Accession #J01542.1), and ADH1 Terminator (Part BBa_K801012).The alpha amylase coding sequence contained an EcoR1 restriction site, so GeneDesign was used to eliminate the site and optimize the construct for S.cerevisiae.
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<font size="3.3"><li><span>Designed 3 amylase gene constructs to be synthesized through IDT’s offer. The final makeup of the gene constructs are listed below.  
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However with this combination, IDT would not accept our design, due to repeats and sections with a low GC count. The constructs were redesigned, keeping the same coding sequence and Kozak sequence, but using the minimal cyc promoter (Part ) and minimal adh1 terminator (Part ). In order to test several variants on the expression of amylase, two promoters, and two secretion sequences were used. Plasma DNA was used to map restriction sites, and Gene design used to remove restriction sites. The final makeup of the gene constructs are listed below.  
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<p class="pageheading"> Construct 1  </p>
 
<p class="pageheading"> Construct 1  </p>
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<li><span>Designed primers for the three gene constructs, by hand using New England Biolabs Tm calculator. The first thirty bases from each construct were used as a starting point for the left primer and the last thirty a starting point for the right primer. Because construct 1 and 2 had the same promoter and terminator, the same primers could be used, thus only two sets of primers were needed. The primers were shortened at the correct ends until they had the same melting temperature. Then the reverse complement of the right primer was taken using a reverse complement online tool by bioinformatics.org. Primers were named p01, p02, p03, and p04.  
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<li><span>Designed primers for the three gene constructs, by hand using New England Biolabs Tm calculator. Primers were named p01, p02, p03, and p04.  
 
<li><span>p01- left primer for construct 1  
 
<li><span>p01- left primer for construct 1  
 
<li><span>p02 - right primer for construct 1  
 
<li><span>p02 - right primer for construct 1  
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<h2 class="pageheading"> Week 2</h2>
 
<h2 class="pageheading"> Week 2</h2>
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<font size="3.3"><li><span>The gene constructs arrived today, and the primers. The gene constructs and primers were first resuspended, according to protocol.  
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<font size="3.3"><li><span> The gene constructs and primers were first resuspended, according to protocol.  
 
<li><span>Primer resuspension:
 
<li><span>Primer resuspension:
 
Amount of water added to reach 100uM concentration, water was added and pipetted up and down to resuspend.  
 
Amount of water added to reach 100uM concentration, water was added and pipetted up and down to resuspend.  
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</table>
 
</table>
 
   
 
   
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<li>After amplification PCR, a gel was ran to confirm size. </li>
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<li>Ran a gel to confirm size. </li>
  
 
<ul>Gel Electrophoresis
 
<ul>Gel Electrophoresis

Revision as of 21:06, 10 September 2015

{{BroadRun-NorthernVA}}



>

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