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

 
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<h2 align="center" class="pageheading">Lab Notebook</h2>
 
<h2 align="center" class="pageheading">Lab Notebook</h2>
 
<p align="center"> 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. </p>
 
<p align="center"> 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. </p>
−
<h2 align="left" class="subheading">June</h2>
 
  
 +
<h2 align="left" class="pageheading"<font size="4.3"><b>June</b></h2>
 +
 +
<h align="left" class="pageheading"<font size="3.8"><b>Week 1</b></font></h2>
 +
<font size="3.3"><li>
 +
Brainstormed ideas for projects. Decided on cells that could break down starch.
 +
 +
    </li>
 +
</font>
 +
 +
<h align="left"class="pageheading" <font size="3.8"><b>Week 2-4</b></font></h2>
 +
<font size="3.3"><li>Researched the best way to go about this solution.</li>
 +
<li>Saccharomyces was decided as our organism, because of it ability to thrive in a variety of conditions, both aerobic and anaerobic.
 +
</li>
 +
 +
<li>Used alpha amylase as our target gene to be cloned</li>
 +
</font>
 +
 +
 +
<h2 align="left" class="pageheading"<font size="4.3"><b>July</b></h2>
 +
 +
<font size="3.3"><li>More literature research and worked on developing protocols.
 +
Ordered materials for the project, enzymes, reagents, buffers, kits, agar plates, LB broth, etc.
 +
 +
</li>
 +
<li>Experiment details were solidified and planned out in more detail.
 +
</li>
 +
 +
<li>Ordered materials for the project, enzymes, reagents, buffers, kits, agar plates, LB broth, etc. </li>
 +
</font>
 +
 +
 +
 +
<h2 align="left" class="pageheading"<font size="4.3"><b>August</b></h2>
 +
 +
<b><h align="left" class="pageheading"<font size="3.8">Week 1</b></h2></font>
  
−
<h2 align="left" class="subheading">August 8</h2>
 
 
<ul class="listitems"><font color="black">
 
<ul class="listitems"><font color="black">
−
<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.
+
<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.  
−
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.  
+
  
 
<p class="pageheading"> Construct 1  </p>
 
<p class="pageheading"> Construct 1  </p>
Line 239: Line 269:
 
<li><span> ADH1 Terminator (Part BBa_K392003)</span></li>
 
<li><span> ADH1 Terminator (Part BBa_K392003)</span></li>
 
<li><span>Biobrick Suffix </span></li>
 
<li><span>Biobrick Suffix </span></li>
 +
<p></p>
  
 +
<li><span>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.
 +
</span></li>
  
  
−
</font></ul>
+
<p></p>
 +
<p></p>
 +
 
 +
 
 +
<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>p02 - right primer for construct 1
 +
<li><span>p03- left primer for construct 2 and 3
 +
<li><span>p01- right primer for construct 2 and 3  </span></li>
 +
</font>
 +
 
 +
</ul>
  
 
<!-- week 2 -->
 
<!-- week 2 -->
  
 
<h2 class="pageheading"> Week 2</h2>
 
<h2 class="pageheading"> Week 2</h2>
 +
<font size="3.3"><li><span> The gene constructs and primers were first resuspended, according to protocol.
 +
<li><span>Primer resuspension:
 +
Amount of water added to reach 100uM concentration, water was added and pipetted up and down to resuspend.
  
−
<!-- week 1 -->
+
<ul>
 +
      <li>p01: 293 µl of water
 +
<li>p02: 336 µl of water
 +
<li>p03: 269 µl of water
 +
<li>p04: 345 µl of water </li>
 +
    </ul>
 +
<li><span>100uM concentration was then diluted to 10uM concentration.
  
−
<h2 class="subheading" > Wet Lab </h2>
+
Gene construct resuspension: 100 µl of TE buffer was added to each gene construct and pipetted up and down and vortexed to resuspend.  
−
<ul class="listitems">
+
Tubes were then incubated at 30 degrees Celsius for 20 minutes.  
−
<li>Isolated DNA from the transformed plasmids, for use in PCR later in the week. </li>
+
<p></p>
−
<li> PCR used to amplify and isolate our desired DNA from their plasmids, using the plasmids we designed in week one.</li>
+
Then we amplified the DNA with the primers using PCR.
−
<li>PCR products then run on a gel to confirm they were of the correct size.  Desired DNA was isolated for later use using gel extraction.</li>
+
<ul><b>Amplification PCR</b> </ul>
−
<li>Primers designed for GvpA , GvpC, and for the purposes of isolating FliC from the E. Coli genome.  This will be used later on in the project to study the effect of the flagella in the presence of gas vesicles.</li>
+
−
<li>Bacteria transformed with plasmids taken from the distribution.  The parts used contained useful things such as GFP, a promoter, and vectors that will be used at a later stage.  The useful DNA from these transformations was isolated and digested using EcoR1 and Pst1, to ensure the sizes were correct.</li>
+
−
<li>One transformation was done using 2 different vectors containing RFP (pSB6A1 and pSB3C5), and two plasmids containing GFP, each with a different RBS.
+
−
Another used instead the vector pSB1C3 and another containing the promoter J23100(CHECK) – both expressing RFP.  This later transformation will be run on a gel in week 3.</li>
+
−
<li> We had chosen 2 samples of our miniprepped DNA from out first transformation (one with GvpA, the other with GvpC) to be sent off for sequencing.  At the end of the week, this sequenced data was analysed, and proved that out isolated DNA contained the desired sequences.</li>
+
−
<li> An important step was taken this week: the ligation of our PCR-produced GvpA and GvpC - containing prefix, suffix and desired RBS – into separate pSB1C3 plasmid vectors.  In order to do this, the PCR products and the vector underwent a restriction digest using EcoR1 and Pst1.  The resultant DNA fragments were run on a gel and the fragments containing GvpA and GvpC and pSB1C3 were cut out and the DNA purified using gel extraction.
+
−
GvpA was then ligated into pSB1C3.  Seperately, GvpC was ligated into pSB1C3.  The pSB1C3 was obtained by digesting the GFP-0034 BioBrick part from the distribution.</li>
+
−
<li>As the above was taking place, FliC was also digested with EcoR1 and Pst1.  The resultant gel showed a restriction site within FliC – a problem which must be resolved. </li>
+
  
 +
<ul>100 µl per reaction </ul>
 +
<ul>Reaction #1</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>primer 01
 +
<td>primer 02
 +
<td>Gene construct #1
 +
<td>2x Master Mix
 +
<td>water</td>
 +
 +
   
 +
  </tr>
 +
  <tr>
 +
 
 +
 +
  <td>5
 +
  <td>5
 +
  <td>2
 +
  <td>50
 +
  <td>38</td>
 +
  </tr>
 +
</table>
 +
 +
<ul>Reaction #2</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>primer 03
 +
<td>primer 04
 +
<td>Gene construct #2
 +
<td>2x Master Mix
 +
<td>water</td>
 +
  </tr>
 +
  <tr>
 +
  <td>5
 +
  <td>5
 +
  <td>2
 +
  <td>50
 +
  <td>38</td>
 +
  </tr>
 +
</table>
 +
 +
<ul>Reaction #3</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>primer 03
 +
<td>primer 04
 +
<td>Gene construct #3
 +
<td>2x Master Mix
 +
<td>water</td>
 +
  </tr>
 +
  <tr>
 +
  <td>5
 +
  <td>5
 +
  <td>2
 +
  <td>50
 +
  <td>38</td>
 +
  </tr>
 +
</table>
 +
 +
<ul>Negative Control #1</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>primer 01
 +
<td>primer 02
 +
<td>Gene construct
 +
<td>2x Master Mix
 +
<td>water</td>
 +
  </tr>
 +
  <tr>
 +
  <td>5
 +
  <td>5
 +
  <td>0
 +
  <td>50
 +
  <td>40</td>
 +
  </tr>
 +
</table>
 +
 +
<ul>Negative Control #2</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>primer 03
 +
<td>primer 04
 +
<td>Gene construct
 +
<td>2x Master Mix
 +
<td>water</td>
 +
  </tr>
 +
  <tr>
 +
  <td>5
 +
  <td>5
 +
  <td>0
 +
  <td>50
 +
  <td>40</td>
 +
  </tr>
 +
</table>
 +
 +
<li>Ran a gel to confirm size. </li>
 +
 +
<ul>Gel Electrophoresis
 +
<li> 10µl of each PCR reaction was added into ten different tubes and 2µl of loading dye added.
 +
<li>11µl was then loaded into each of the wells and ran for 15 minutes. </li>
 +
 +
 +
<h align="left" class="pageheading"<font size="3.8"><b>Week 3</b></font></h2>
 +
<font size=3.3>
 +
<ul> <b> PCR Purification </b></ul>
 +
<li>30 µl of PCR product out of 100 µl was purified, using a Quiagen PCR purification kit. </li>
 +
 +
<ul> <b> Restriction Digest </b></ul>
 +
<li> Total reaction volume was 50 µl
 +
<li>Concentrations of plasmids and PCR products </li>
 +
<ul>
 +
<li>pSB1c3 25ng/µl
 +
<li>pRS426 129ng/µl
 +
<li>pAG36 900ng/µl
 +
<li>PCR products 100ng/µl</li>
 +
 +
<ul>Restriction Digest of Plasmids</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>pSB1c3 plasmid
 +
<td>pAG36 yeast vector
 +
<td>pRS426 yeast vector</td>
 +
  </tr>
 +
  <tr>
 +
  <td>4µl DNA
 +
<td>7.8 µl DNA
 +
<td>1.1 µl DNA</td>
 +
  </tr>
 +
 +
<tr>
 +
  <td>5 µl 10x NEB Cut Smart Buffer
 +
<td>5 µl 10x NEB Cut Smart Buffer
 +
<td>5 µl 10x NEB Cut Smart Buffer</td>
 +
  </tr>
 +
<tr>
 +
  <td>1µl EcoR1 enzyme
 +
<td>1µl Kpn1 enzyme
 +
<td>1µl EcoR1 enzyme
 +
</td>
 +
  </tr>
 +
 +
<tr>
 +
  <td>1µl Pst1 enzyme
 +
<td>1µl Spe1 enzyme
 +
<td>1µl Pst1 enzyme
 +
</td>
 +
  </tr>
 +
<tr>
 +
  <td>39 µl water
 +
<td>35.2 µl water
 +
<td>41.9 µl water
 +
</td>
 +
  </tr>
 +
</table>
 +
 +
 +
<ul>Restriction Digest of PCR Products</ul>
 +
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>PCR product 1 (promoterless and native secretion sequence), cut with EcoR1 and Pst1
 +
<td>PCR product 2 (cyc promoter and native secretion sequence), cut with EcoR1 and Pst1
 +
<td>PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with EcoR1 and Pst1 
 +
<td>PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with Kpn1 and Spe1</td>
 +
  </tr>
 +
  <tr>
 +
  <td>10 µl DNA
 +
<td>10µl DNA
 +
<td>10µl DNA
 +
<td>10 µl DNA</td>
 +
  </tr>
 +
 +
<tr>
 +
  <td>5 µl 10x NEB Cut Smart Buffer
 +
<td>5 µl 10x NEB Cut Smart Buffer
 +
<td>5 µl 10x NEB Cut Smart Buffer</td>
 +
  </tr>
 +
 +
<tr>
 +
  <td>1µl EcoR1 enzyme
 +
<td>1µl EcoR1 enzyme
 +
<td>1µl EcoR1 enzyme
 +
<td>1µl Kpn1 enzyme
 +
</td>
 +
  </tr>
 +
 +
<tr>
 +
  <td>1µl Pst1 enzyme
 +
<td>1µl Pst1 enzyme
 +
<td>1µl Pst1 enzyme
 +
<td>1µl Spe1 enzyme
 +
</td>
 +
  </tr>
 +
<tr>
 +
  <td>33 µl water
 +
  <td>33 µl water
 +
  <td>33 µl water
 +
  <td>33 µl water
 +
</td>
 +
  </tr>
 +
</table>
 
</ul>
 
</ul>
  
−
<br>
+
<li> After ligation, we completed a bacterial transformation and plated cultures on agar plates. </li>
  
 +
<h4><b>Week 4</b></h4>
 +
<b></b>
 +
<p class="page heading"><h6><span> <ul> The PCR mix components were combined in a large microcentrifuge tube with:</ul></h6>  </p>
 +
<li>100µl of 2x master mix  </span></li>
 +
<li>16µl of VF2 primer  </span></li>
 +
<li>13 µl of VR primer </span></li>
 +
<li> 71 µl of sterile water </span></li>
 +
<li>20µl of the mix was added into each of nine PCR tubes. </span></li>
  
 +
<h6><span><ul>Three colonies were chosen from each of the pSB1c3 ligations that had been transformed into the E.coli, labeled 1A,1B,1C, 2A, so forth. Each colony was mixed into the PCR mix and then streaked onto a master plate.</ul></h6>
  
−
    
+
<li>10µl of the finished PCR product was mixed with 2µl of loading dye and loaded into a agarose gel.
 +
 
 +
<ul><h6>The results confirmed that our insert size was correct</h6></ul>
 +
 
 +
<h6>Next, we mini-prepped our liquid cultures using Quiaprep mini prep kit. There were eighteen cultures total, three colonies for each of the five transformations.</h6>
 +
 
 +
<p>After the mini prep, we needed to check the size of the DNA and the DNA had not been lost during the miniprep.
 +
To linearize the DNA, 5µl was added to 45 µl of a master mix.</p>
 +
 
 +
<h6>Two master mixes were used, one with EcoR1 and one with Spe1. The ligations with the pSB1C3 plasmid and yeast vector pRS426 contain a single EcoR1 restriction site and the pAG36 yeast vector contains a Spe1 restriction site. Master mix with Ecor1 was made for 15 reactions and the Spe1 master mix for 5 reactions.</h6>
 +
<p>Master mix:</p>
 +
<br></br>
 +
<p>Ecor1 mix:</p>
 +
<li>75 µl 10x New England Bio Labs Cut Smart Buffer
 +
<li>15 µl EcoR1 enzyme
 +
<li>585 µl distilled water
 +
<li>Spe1 mix:
 +
<li>25 µl 10x New England Bio Labs Cut Smart Buffer
 +
<li>5 µl Spe1 enzyme
 +
<li>195 µl distilled water
 +
 
 +
<h6>DNA was incubated at 37° Celsius for one hour.</h6>
 +
<h6>10 µl of loading dye was added, the solution was pipetted up and down to mix thoroughly.</h6>
 +
<h6>20µl was loaded into each well in addition to a 1kb DNA ladder.</h6>
 +
<p>
 +
 
 +
<h4><b>Week 5</b></h4>
 +
<h5>Master Mix 1:</h5>
 +
<table style="width:80%">
 +
<tr>
 +
  <td>Primer 1
 +
<td>Primer 2
 +
<td>std taq
 +
<td>dntps
 +
<td>taq
 +
<td>h2o
 +
</td>
 +
   </tr>
 +
<tr>
 +
  <td>10
 +
  <td>10
 +
  <td>20
 +
  <td>4
 +
<td>2
 +
<td>134
 +
</td>
 
</tr>
 
</tr>
 
</table>
 
</table>
  
 +
<h5>Master Mix 2:</h5>
 +
 +
<table style="width:80%">
 +
<tr>
 +
  <td>Primer 2
 +
<td>Primer 3
 +
<td>std taq
 +
</td>
 +
  </tr>
 +
<tr>
 +
  <td>10
 +
  <td>10
 +
  <td>20
 +
</td>
 +
</tr>
 +
</table>
 +
 +
<h6>colony pcr gel</h6>
 +
<li>10 fold dilution(2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)
 +
<li>100 fold dilution (2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)
 +
<li>prs426 positive control
 +
<li>pag36 pc
 +
<li>psb1c3 nc
 +
 +
<h2>Project Timeline</h2>
 +
 +
<p><b>Week 1</b></p>
 +
 +
<li>Colony PCR on bacterial cultures
 +
<li>Made liquid cultures of positive colonies
 +
<li>Miniprepped liquid cultures
 +
<br></br>
 +
<li>Transformed DNA samples into <i> S.cerevisiae</i>
 +
<p><b>Week 2</b></p>
 +
<li>Tested genetically engineered cells for ability to produce amylase and hydrolyze starch
 +
<li>Cells were tested in known concentrations of soluble starch dissolved in water
 +
<lli>Iodine was used to determine starch concentrations
 +
<li> Then the yeast cells were tested in a sample of Armstrong's factory water</li>
 +
 +
<p><b>Week 3</b><p>
 +
 +
 +
<li>Cells were heat killed at 80 degrees Celsius to inactivate enzymes.
 +
<li>Ran through a gel to verify the fragments
 +
<li>Ligation of gene after successful gel.
 +
 +
<p><b>Week 4</b></p>
 +
 +
<li>Today we ran colony PCR to confirm the insert size. Our bacterial transformations went well, and we had both liquid cultures and agar plates with the colonies. The synthesized primers from IDT for the yeast vectors have not arrived, so colony PCR was done only on the three transformations with the pSB1c3 vector.
 +
 +
<p><b>Week 5</b></p>
 +
 +
<li>Colony PCR
 +
<li>Starch Analysis
 +
<li> psb13 plasmid results
 +
 +
 +
 +
 +
</font>
 
</div>
 
</div>
 
</html>
 
</html>

Latest revision as of 02:58, 19 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
    • After ligation, we completed a bacterial transformation and plated cultures on agar plates.

      • Week 4

        The PCR mix components were combined in a large microcentrifuge tube with:

    • 100µl of 2x master mix
    • 16µl of VF2 primer
    • 13 µl of VR primer
    • 71 µl of sterile water
    • 20µl of the mix was added into each of nine PCR tubes.
        • Three colonies were chosen from each of the pSB1c3 ligations that had been transformed into the E.coli, labeled 1A,1B,1C, 2A, so forth. Each colony was mixed into the PCR mix and then streaked onto a master plate.
    • 10µl of the finished PCR product was mixed with 2µl of loading dye and loaded into a agarose gel.
        The results confirmed that our insert size was correct
      Next, we mini-prepped our liquid cultures using Quiaprep mini prep kit. There were eighteen cultures total, three colonies for each of the five transformations.

      After the mini prep, we needed to check the size of the DNA and the DNA had not been lost during the miniprep. To linearize the DNA, 5µl was added to 45 µl of a master mix.

      Two master mixes were used, one with EcoR1 and one with Spe1. The ligations with the pSB1C3 plasmid and yeast vector pRS426 contain a single EcoR1 restriction site and the pAG36 yeast vector contains a Spe1 restriction site. Master mix with Ecor1 was made for 15 reactions and the Spe1 master mix for 5 reactions.

      Master mix:



      Ecor1 mix:

    • 75 µl 10x New England Bio Labs Cut Smart Buffer
    • 15 µl EcoR1 enzyme
    • 585 µl distilled water
    • Spe1 mix:
    • 25 µl 10x New England Bio Labs Cut Smart Buffer
    • 5 µl Spe1 enzyme
    • 195 µl distilled water
      DNA was incubated at 37° Celsius for one hour.
      10 µl of loading dye was added, the solution was pipetted up and down to mix thoroughly.
      20µl was loaded into each well in addition to a 1kb DNA ladder.

      Week 5

      Master Mix 1:
      Primer 1 Primer 2 std taq dntps taq h2o
      10 10 20 4 2 134
      Master Mix 2:
      Primer 2 Primer 3 std taq
      10 10 20
      colony pcr gel
    • 10 fold dilution(2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)
    • 100 fold dilution (2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)
    • prs426 positive control
    • pag36 pc
    • psb1c3 nc

      Project Timeline

      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

    • Today we ran colony PCR to confirm the insert size. Our bacterial transformations went well, and we had both liquid cultures and agar plates with the colonies. The synthesized primers from IDT for the yeast vectors have not arrived, so colony PCR was done only on the three transformations with the pSB1c3 vector.

      Week 5

    • Colony PCR
    • Starch Analysis
    • psb13 plasmid results