Difference between revisions of "Team:Virginia/Notebook"

 
(31 intermediate revisions by 5 users not shown)
Line 3: Line 3:
 
<head>
 
<head>
 
<style>
 
<style>
 +
 +
 +
.gelimage {
 +
width:400px;
 +
margin-left:50px;
 +
margin-top:10px;
 +
}
  
 
table {
 
table {
Line 37: Line 44:
 
bottom:-25px;
 
bottom:-25px;
 
left:-50px;
 
left:-50px;
−
right:500px;
+
right:470px;
 
border-radius:0 0 5px 5px;
 
border-radius:0 0 5px 5px;
 
background-color:white;
 
background-color:white;
Line 47: Line 54:
 
width:480px;
 
width:480px;
 
min-height:100px;
 
min-height:100px;
−
margin-left:600px;
+
margin-left:640px;
 
}
 
}
  
Line 63: Line 70:
  
 
.date {
 
.date {
−
background-color:#99e6ff;
+
background-color:#4690b0;
 
}
 
}
  
 
.date:hover {
 
.date:hover {
 
cursor:pointer;
 
cursor:pointer;
−
background-color:#1ac6ff !important;
+
background-color:#9fb140 !important;
 
}
 
}
  
Line 86: Line 93:
 
<div id="menuspace"></div>
 
<div id="menuspace"></div>
 
<div class="footerspace">
 
<div class="footerspace">
−
<a href="https://2016.igem.org/Team:Virginia/Safety" class="footerleft">&larr; Safety</a>
+
<a href="https://2016.igem.org/Team:Virginia/Safety" class="footerright">Safety &rarr;</a>
−
<a href="https://2016.igem.org/Team:Virginia/Attributions" class="footerright">Attributions &rarr;</a>
+
 
</div>
 
</div>
  
Line 94: Line 100:
  
 
<!---------------------------Top image on page------------------------------------------>
 
<!---------------------------Top image on page------------------------------------------>
−
<!--<div class="imagearea">
 
−
<img class="mainimage" src="http://s3.amazonaws.com/content.washingtonexaminer.biz/David%20Fitzsimmons.jpg"/>
 
−
</div>-->
 
  
−
<div class="imagearea" style="background-color:#ffa366">
+
<div class="imagearea">
−
<h1 style="line-height:230px">NOTEBOOK</h1>
+
            <img src="https://static.igem.org/mediawiki/2016/e/e2/T--Virginia--Notebook_header.png">
 
</div>
 
</div>
−
 
−
 
−
<!---------------------------Page title area-------------------------------------------->
 
−
<!--<div class="pagetitle">
 
−
<h1 style="color:#66b3ff">Insert title of page here!</h1>
 
−
</div>-->
 
−
 
  
  
Line 384: Line 380:
 
<li>XL1-blue cells transformed with LeuS-ampR plasmid - plated and incubated at 10:45pm</li>
 
<li>XL1-blue cells transformed with LeuS-ampR plasmid - plated and incubated at 10:45pm</li>
 
</ul>
 
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/7/7e/5gelimage.jpeg"/>--->
 
</span>
 
</span>
  
Line 408: Line 405:
 
<li>Performed confirmatory digests on LeuS and terminator plasmid DNA</li>
 
<li>Performed confirmatory digests on LeuS and terminator plasmid DNA</li>
 
</ul>
 
</ul>
 +
<!--- <img class="gelimage" src="https://static.igem.org/mediawiki/2016/9/94/8gel.jpeg"/>--->
 
</span>
 
</span>
  
Line 461: Line 459:
 
<li>Performed a ligation reaction for T1 and T2</li>
 
<li>Performed a ligation reaction for T1 and T2</li>
 
</ul>
 
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/4/4c/T--Virginia--the16thofjuly.jpeg"/>--->
 
</span>
 
</span>
  
Line 573: Line 572:
 
<li>Performed ligation reactions between the LeuS gene and the T1 and T2 genes</li>
 
<li>Performed ligation reactions between the LeuS gene and the T1 and T2 genes</li>
 
</ul>
 
</ul>
 +
<!--- <img class="gelimage" src="https://static.igem.org/mediawiki/2016/e/e9/T--Virginia--august2gel.jpeg"/>--->
 
</span>
 
</span>
  
Line 609: Line 609:
 
<li>Began preparing competent JW strain <i>E. coli</i> cells</li>
 
<li>Began preparing competent JW strain <i>E. coli</i> cells</li>
 
</ul>
 
</ul>
 +
<!--- <img class="gelimage" src="https://static.igem.org/mediawiki/2016/6/61/T--Virginia--august19gel.jpeg"/>--->
 
</span>
 
</span>
  
Line 660: Line 661:
 
<li>Performed a PCR purification</li>
 
<li>Performed a PCR purification</li>
 
</ul>
 
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/0/00/T--Virginia--august15gel.jpeg"/>--->
 
</span>
 
</span>
  
Line 670: Line 672:
 
<li>PCR of LeuS gene</li>
 
<li>PCR of LeuS gene</li>
 
</ul>
 
</ul>
 +
<!--- <img class="gelimage" src="https://static.igem.org/mediawiki/2016/c/c6/T--Virginia--august16gel.jpeg"/>--->
 
</span>
 
</span>
  
Line 687: Line 690:
 
<li>Redoing competent JW cells</li>
 
<li>Redoing competent JW cells</li>
 
</ul>
 
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/b/b1/T--Virginia--august18gel.jpeg"/>--->
 
</span>
 
</span>
  
 +
<span class="targetspan" id="sa22">
 +
Lab work:
 +
<ul>
 +
<li>Took nanodrop concentrations of minipreppred ligation reaction</li>
 +
<li>Restriction digests</li>
 +
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/d/d1/T--Virginia--august22gel.jpeg"/>--->
 +
</span>
  
−
</div>
+
<span class="targetspan" id="sa23">
 +
Lab work:
 +
<ul>
 +
<li>Made primer solutions</li>
 +
<li>SDM of Pst 1</li>
 +
<li>Transformation and sequencing</li>
 +
</ul>
 +
</span>
  
 +
<span class="targetspan" id="sa26">
 +
Lab work:
 +
<ul>
 +
<li>Performed digests of T1</li>
 +
<li>Digest PCR LeuS</li>
 +
<li>Ligated T1 and LeuS</li>
 +
<li>Transformed cells</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa27">
 +
Lab work:
 +
<ul>
 +
<li>Preculture of term-leuS lig</li>
 +
<li>3 colonies of 6:1 ligation and 1 colony of 8:1 ligation</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa28">
 +
Lab work:
 +
<ul>
 +
<li>Miniprep of leuS term ligation</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa29">
 +
Lab work:
 +
<ul>
 +
<li>Ran a gel</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa31">
 +
Lab work:
 +
<ul>
 +
<li>Performed PCR</li>
 +
<li>Performed transformation</li>
 +
</ul>
 +
</span>
 +
</div>
  
 
<div id="nbbottom">
 
<div id="nbbottom">
 +
<!-------------------------------------------September---------------------------------------------------->
 +
<span class="targetspan" id="ss1">
 +
Lab work:
 +
<ul>
 +
<li>Preculture of Pst sdm</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss2">
 +
Lab work:
 +
<ul>
 +
<li>Performed miniprep</li>
 +
<li>Ran confirmatory digest</li>
 +
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/4/46/T--Virginia--september2gel.jpeg"/>--->
 +
</span>
 +
 +
<span class="targetspan" id="ss5">
 +
Lab work:
 +
<ul>
 +
<li>Performed restriction digests</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss6">
 +
Lab work:
 +
<ul>
 +
<li>Performed ligations</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss7">
 +
Lab work:
 +
<ul>
 +
<li>Preculture for transformations</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss8">
 +
Lab work:
 +
<ul>
 +
<li>3A assembly of zeo-E-Kan backbone</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss13">
 +
Lab work:
 +
<ul>
 +
<li>Sequencing</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss16">
 +
Lab work:
 +
<ul>
 +
<li>Performed restriction digest</li>
 +
<li>Performed ligation</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss17">
 +
Lab work:
 +
<ul>
 +
<li>Performed restriction digest</li>
 +
<li>Performed ligation</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss18">
 +
Lab work:
 +
<ul>
 +
<li>Nanodrop used to determine concentrations</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss20">
 +
Lab work:
 +
<ul>
 +
<li>Nanodrop to determine concentrations and sequencing performed</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss26">
 +
Lab work:
 +
<ul>
 +
<li>Ran term and enzyme gels</li>
 +
<li>Digest of term performed</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss27">
 +
Lab work:
 +
<ul>
 +
<li>Miniprep of term and pro</li>
 +
<li>Performed digest</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="ss29">
 +
Lab work:
 +
<ul>
 +
<li>Digest of terminator</li>
 +
<li>Success! Got term in lane 5</li>
 +
<li>Ligation performed</li>
 +
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/6/6d/T--Virginia--september29gel.jpeg"/>--->
 +
</span>
 +
 +
<span class="targetspan" id="ss30">
 +
Lab work:
 +
<ul>
 +
<li>PCR of mutant LeuS</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="so1">
 +
Lab work:
 +
<ul>
 +
<li>PCR purifed mut LeuS</li>
 +
<li>Digest performed</li>
 +
<li>Ligations performed</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="so3">
 +
Lab work:
 +
<ul>
 +
<li>Miniprep x 27</li>
 +
<li>Digest MT 7.1 &rarr; CIP</li>
 +
<li>Digest piece A and controls</li>
 +
<li>Ligations of mut term and piece A performed</li>
 +
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/d/d5/T--Virginia--october3gel.jpeg"/>--->
 +
</span>
 +
 +
<span class="targetspan" id="so6">
 +
Lab work:
 +
<ul>
 +
<li>Miniprep of HA and mut and term</li>
 +
<li>Restriction digest of enz-term and promoter</li>
 +
<li>Preculture JW cells</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="so7">
 +
Lab work:
 +
<ul>
 +
<li>Competent cell procedure for JW</li>
 +
<li>Confirmation test followed by plating</li>
 +
</ul>
 +
</span>
 +
 +
 +
<span class="targetspan" id="so8">
 +
Lab work:
 +
<ul>
 +
<li>Restriction digest</li>
 +
</ul>
 +
<!---<img class="gelimage" src="https://static.igem.org/mediawiki/2016/b/b4/T--Virginia--october8gel.jpeg"/>--->
 +
</span>
 +
 +
        <span class="targetspan" id="so9">
 +
Lab work:
 +
<ul>
 +
<li>JW5807 competent cell protocol</li>
 +
<li>Miniprepped promoter-enzyme-terminator plasmid</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="so10">
 +
Lab work:
 +
<ul>
 +
<li>Confirmatory digest of promoter-enzyme-terminator plasmid miniprepped on 10/9</li>
 +
<li>Sequencing of promoter-enzyme-terminator plasmid miniprepped on 10/9</li>
 +
<li>Made minimal medium plates with nothing, 3mM leucine, or 3mM CBZ-leu</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="so12">
 +
Lab work:
 +
<ul>
 +
<li>digest, gel, ligation of parts into CAM backbones</li>
 +
</ul>
 +
</span>
 +
 +
 
</div>
 
</div>
 +
 +
  
 
<!----------------------------------------------------------May---------------------------------------------------------------->
 
<!----------------------------------------------------------May---------------------------------------------------------------->
Line 924: Line 1,171:
 
</tr>
 
</tr>
 
</tbody>
 
</tbody>
−
</table
+
            </table>
  
 
<!----------------------------------------------------------August-------------------------------------------------------------->
 
<!----------------------------------------------------------August-------------------------------------------------------------->
Line 980: Line 1,227:
 
<tr>
 
<tr>
 
<td>21</td>
 
<td>21</td>
−
<td>22</td>
+
<td class="date" id="a22">22</td>
−
<td>23</td>
+
<td class="date" id="a23">23</td>
 
<td>24</td>
 
<td>24</td>
 
<td>25</td>
 
<td>25</td>
−
<td>26</td>
+
<td class="date" id="a26">26</td>
−
<td>27</td>
+
<td class="date" id="a27">27</td>
 
</tr>
 
</tr>
  
  
 
<tr>
 
<tr>
−
<td>28</td>
+
<td class="date" id="a28">28</td>
−
<td>29</td>
+
<td class="date" id="a29">29</td>
 
<td>30</td>
 
<td>30</td>
−
<td>31</td>
+
<td class="date" id="a31">31</td>
 
<td></td>
 
<td></td>
 
<td></td>
 
<td></td>
Line 999: Line 1,246:
 
</tr>
 
</tr>
 
</tbody>
 
</tbody>
−
</table
+
            </table>
 
</div>
 
</div>
  
Line 1,026: Line 1,273:
 
<td></td>
 
<td></td>
 
<td></td>
 
<td></td>
−
<td>1</td>
+
<td class="date" id="s1">1</td>
−
<td>2</td>
+
<td class="date" id="s2">2</td>
 
<td>3</td>
 
<td>3</td>
 
</tr>
 
</tr>
Line 1,034: Line 1,281:
 
<tr>
 
<tr>
 
<td>4</td>
 
<td>4</td>
−
<td>5</td>
+
<td class="date" id="s5">5</td>
−
<td>6</td>
+
<td class="date" id="s6">6</td>
−
<td>7</td>
+
<td class="date" id="s7">7</td>
−
<td>8</td>
+
<td class="date" id="s8">8</td>
 
<td>9</td>
 
<td>9</td>
 
<td>10</td>
 
<td>10</td>
Line 1,046: Line 1,293:
 
<td>11</td>
 
<td>11</td>
 
<td>12</td>
 
<td>12</td>
−
<td>13</td>
+
<td class="date" id="s13">13</td>
 
<td>14</td>
 
<td>14</td>
 
<td>15</td>
 
<td>15</td>
−
<td>16</td>
+
<td class="date" id="s16">16</td>
−
<td>17</td>
+
<td class="date" id="s17">17</td>
 
</tr>
 
</tr>
  
  
 
<tr>
 
<tr>
−
<td>18</td>
+
<td class="date" id="s18">18</td>
 
<td>19</td>
 
<td>19</td>
−
<td>20</td>
+
<td class="date" id="s20">20</td>
 
<td>21</td>
 
<td>21</td>
 
<td>22</td>
 
<td>22</td>
Line 1,067: Line 1,314:
 
<tr>
 
<tr>
 
<td>25</td>
 
<td>25</td>
−
<td>26</td>
+
<td class="date" id="s26">26</td>
−
<td>27</td>
+
<td class="date" id="s27">27</td>
 
<td>28</td>
 
<td>28</td>
−
<td>29</td>
+
<td class="date" id="s29">29</td>
−
<td>30</td>
+
<td class="date" id="s30">30</td>
 
<td></td>
 
<td></td>
 
</tr>
 
</tr>
 
</tbody>
 
</tbody>
−
</table
+
            </table>
  
 
<!----------------------------------------------------------October---------------------------------------------------------------->
 
<!----------------------------------------------------------October---------------------------------------------------------------->
Line 1,103: Line 1,350:
 
<td></td>
 
<td></td>
 
<td></td>
 
<td></td>
−
<td>1</td>
+
<td class="date" id="o1">1</td>
 
</tr>
 
</tr>
  
Line 1,109: Line 1,356:
 
<tr>
 
<tr>
 
<td>2</td>
 
<td>2</td>
−
<td>3</td>
+
<td class="date" id="o3">3</td>
 
<td>4</td>
 
<td>4</td>
 
<td>5</td>
 
<td>5</td>
−
<td>6</td>
+
<td class="date" id="o6">6</td>
−
<td>7</td>
+
<td class="date" id="o7">7</td>
−
<td>8</td>
+
<td class="date" id="o8">8</td>
 
</tr>
 
</tr>
  
  
 
<tr>
 
<tr>
−
<td>9</td>
+
<td class="date" id="o9">9</td>
−
<td>10</td>
+
<td class="date" id="o10">10</td>
 
<td>11</td>
 
<td>11</td>
−
<td>12</td>
+
<td class="date" id="o12">12</td>
 
<td>13</td>
 
<td>13</td>
 
<td>14</td>
 
<td>14</td>
Line 1,178: Line 1,425:
 
$(document).ready(function(){
 
$(document).ready(function(){
 
$(".date").click(function(){
 
$(".date").click(function(){
−
$(".date").css("backgroundColor", "#99e6ff");
+
$(".date").css("backgroundColor", "#4690b0");
−
$(this).css("backgroundColor", "#1ac6ff");
+
$(this).css("backgroundColor", "#9fb140");
 
});
 
});
 
});
 
});

Latest revision as of 02:27, 20 October 2016

Safety →
Lab work:
  • Shelf organization: Acids, Proteins, Buffers, Gels, Agarose, Salts, Carbs
  • Competent Cell Formation (K12)
 Agenda:
  • PrG Determination (1-4)
  • Orthogonality Research
  • Competent Cell Formation Cont. (K12)
  • Growing K12 cells and making buffer (sterilized transformation buffer)
    • Plans to grow another batch with XL1 Blue tomorrow
Modeling:
  • Research docking software and test
Lab work:
  • Competent cells: Beginning competent cell preparation for E. coli XL1-Blue and K-12
 Lab work:
  • Continued preparing competent cells
Protecting group:
  • Phenyl acetyl protecting group, cleaved by penicillin g acylase
  • Truncated from 5 PGs to 3
  • Dipeptide (maybe leu-leu), with a D aa on N-terminus
  • A protease w/ cleavage activity acting on D aa is being researched for increased specificity
Modeling:
  • AD4 returned zero errors w/ synthetase and leucine as ligand
  • Still unsuccessful AG4 and AD4 returned
  • Researching alternatives to direct another approach
  • Unpacked Rosetta ligand modeling
  • IGEM dock successful modeling; concerns about synthetase model
CRISPR
  • Found biobrick to insert plasmid
Funding
  • Anders finished half application for grant
 Lab work:
  • Continuing competent cell preparation
 Lab work:
  • Competency test
Decided on 4 different PrGs:
  • Cbz leu, pro leu, phenyl acetyl leu, N-methoxy leu
Modeling:
  • Corrected enzyme mistake, now using correct configuration of enzyme
 Lab work:
  • Competency test control plated at 4:50pm
 Lab work:
  • No growth on control plates
 Lab work:
  • JW cells grown in overnight liquid culture at 37°C shaking
 Lab work:
  • JW cells frozen in glycerol stock at -80°C (5 tubes)
 Lab work:
  • Prepared growth media for pr-leu uptake test
 Lab work:
  • Began pr-leu uptake test
  • Test for lawn growth or individual colonies on a spread plate (for CRISPR)
 Lab work:
  • Took OD measurements for uptake test
  • CRISPR plate - lawn produced
 Lab work:
  • Pr-leu uptake test failed, bacteria were fixed by ethanol overnight and could not be lysed
  • Restarted test, XL1-blue innoculated in LB at 5:20pm
Policies and Practices:
  • Meeting with Rivanna river waste treatment plant
  • Meeting with open bio labs
 Tested spectrophotometer in the PLSB lab - Not consistent with results in Prof. Kozminski's lab Lab work:
  • Pr-leu uptake test failed again, cells did not lyse
  • Started a new pr-leu uptake procedure
Wiki team meeting - wiki design Lab work:
  • Created standards for LC-MS
 Lab work:
  • Transformed XL1-blue with Bba-K1218011 CRISPR Cas9 plasmid
  • Transformed XL1-blue with BBa-B0010 forward terminator plasmid
  • Transformed XL1-blue with RFP control plasmid
 Lab work:
  • Growth on CRISPR, terminator, and RFP plates, no growth on controls
  • Cultures of transformed bacteria prepared for glycerol storage
 Lab work:
  • Optical densities taken for second round of pr-leu uptake and enzyme tests
 Lab work:
  • Optical densities taken again for second round of pr-leu uptake and enzyme tests
 Lab work:
  • Sample taken from enzyme reaction and frozen in -20°C freezer
 Lab work:
  • Second sample taken from enzyme reaction and frozen in -20°C freezer
 Lab work:
  • Redoing growth uptake test with aerated growth conditions
 Lab work:
  • OD measurements taken for growth test
  • Extracted and purified E. coli genomic DNA via a minikit
  • Began PCR on genomic DNA to obtain the leuS gene DNA
 Lab work:
  • Purified the PCR product
  • Ran a gel to confirm product - bands ran together
  • Took OD measurements for growth test
Lab work:
  • Extractions performed for enzyme test
  • Redo growth uptake test again
  • Growth uptake test results came out as expected - No growth on plates with pr-leu, but growth present on plates supplemented with leucine
  • Enzyme test was inconclusive due to poor sensitivity of the LC/MS - Suspected due to buffer contamination
 Lab work:
  • PCR performed on LeuS gene, product was purified
 Lab work:
  • Confirmatory digest performed on LeuS PCR product
  • XL1-blue cells transformed with LeuS-ampR plasmid - plated and incubated at 10:45pm
 Lab work:
  • Excessive growth on control transformation plate from 7/5 - Ligation likely did not succeed
  • Inoculated precultures of LeuS and terminator transformed cells for miniprep and sequencing
 Lab work:
  • Redoing enzyme test (again) with a new protocol
  • Performed a miniprep to obtain LeuS and terminator plasmids
 Lab work:
  • Sent off minipreped LeuS and terminator plasmid DNA for sequencing
  • Performed confirmatory digests on LeuS and terminator plasmid DNA
 Lab work:
  • Sequencing of LeuS and terminator plasmid failed due to poor quality of DNA samples
  • Performed PCR and PCR purification on E. coli genomic DNA to obtain LeuS gene
 Lab work:
  • Talked with Professor Kozminski about PCR
  • Set up another PCR reaction and PCR purification to obtain LeuS gene
  • Transformed XL1-blue cells with terminator plasmid
 Lab work:
  • No colonies grew on the terminator transformation plate
  • Restarted enzyme efficacy test
  • Transformed XL1-blue cells with RFP, T1, and T2 plasmids
 Lab work:
  • Removed and stored a sample of the enzyme test
  • Observed growth on the RFP, T1, and T2 plates, no growth on control
  • Started precultures with colonies from the terminator 1 and 2 plates
  • Performed PCR on LeuS gene (genomic DNA)
 Lab work:
  • Performed gel electrophoresis on LeuS PCR product, obtained wrong product
  • Extracted DNA from gel
  • Performed minipreps
 Lab work:
  • Confirmatory digest of minipreps from 7/15
  • Performed a ligation reaction for T1 and T2
 Lab work:
  • Transformed XL1-blue cells with T1, T2, and an RFP control
  • Extracted digested products from agarose gel
  • Plated again using transformed cells from 12am at 1:30pm
  • Transformed and plated new cells at 3:30pm
  • Ligation reaction performed for T1 and T2 using DNA extracted from gel
 Lab work:
  • Transformed XL1-blue cells with the 2 ligation products (T1 with PCR, T2 with PCR)
  • Began a preculture of XL1-blue cas9 cells in LB+cam at 5:45pm
 Lab work:
  • Performed a restriction digest
  • Cells transformed and incubated at 37°C at 9:45am
  • T1, T2, RFP, and control removed and plated at 11:15am
  • Started uptake and enzyme activity tests with N-methoxy-leu
 Lab work:
  • Continued performing uptake test for N-methoxy-leu
 Lab work:
  • Continued performing uptake test for N-methoxy-leu - lysed cells and stored cell lysate
  • Performed minipreps and confirmatory digests - No result from digests because no EtBr was in the gel
 Lab work:
  • Redoing digests from 7/21 and running a new gel
  • Received sgRNA from biobasic, prepared and stored the sgRNA at -20°C
  • Digested CRISPR RNA insert and Cas9 vector
 Lab work:
  • Performed digests with BSA1 on the CRISPR vector and insert
  • Performed a ligation reaction between the vector and insert
 Lab work:
  • Realized that the primers used to PCR the LeuS gene were incorrect - Therefore, our T1 and T2 biobricks did not contain the correct DNA sequences
 Lab work:
  • Continued the PrG uptake test - purification of the cell lysate
  • Performed a miniprep for the T1 and T2 terminators (for biobricking)
 Lab work:
  • Transformed and plated KanR gene from kit
  • Streaked pS1M27 cells from gel stab to Tet plate
 Lab work:
  • Re-transformed and plated KanR gene on CAM plates
  • Re-streaked pS1M27 plate
 Lab work:
  • Miniprep performed for pS1M27
  • Re-transformed KanR DNA
  • Performed a PCR reaction to obtain LeuS gene from genomic DNA
 Lab work:
  • Confirmatory digest performed on PCR product (LeuS gene)
  • Restriction digest for biobrick #1
  • Performed ligation reactions between the LeuS gene and the T1 and T2 genes
 Lab work:
  • Performed a ligation reaction between CRISPR Cas9 genes and sgRNA sequence
  • Minipreped ZeoR, KanR, T1, and T2 plasmids
  • Confirmatory digests performed for T1 and T2 plasmids
  • Performed a ligation reaction with T1 and T2 genes, transformed products
 Lab work:
  • PCR performed to confirm CRISPR/Cas9 and sgRNA gene ligation
 Lab work:
  • Digests performed to redo LeuS and terminator ligation
 Lab work:
  • Ligation reaction performed
  • Received IDT DNA: Penicillin G Acylase pieces 1 and 2
  • Sequencing of Cas9 plasmid
  • Digested PCRed LeuS product
  • Transformed cells with T1 LeuS lig and T2 LeuS lig
  • Began preparing competent JW strain E. coli cells
 Lab work:
  • Restriction digest of pac1, pac2, kan, and zeo resistance genes
  • Restriction digest of terminators followed by cipping
  • Terminator digest Ex + Sip gel
  • LeuS PCR purification
 Lab work:
  • Performed transformations using the following genes:
    • Promoter from iGEM distribution kit
    • RFP for cell competency check
    • T1-LeuS ligation reaction products
    • T2-LeuS ligation reaction products
 Lab work:
  • Restriction digests on pac1 and pac2
  • Ligation reaction for pac1-pac2-terminator
 Lab work:
  • Miniprepped 9 terminator-LeuS ligation plasmids
  • Transformed pac-terminator ligation
  • Precultured promoter into pac vector
 Lab work:
  • Minipreped Pro1 and Pro2
 Lab work:
  • Performed PCR on CRISPR plasmid
  • Performed a restriction digest to confirm LeuS-terminator plasmid
  • Performed a PCR purification
 Lab work:
  • Performed restriction digests on terminator 1, terminator 2, LeuS from PCR, Cas9 plasmid, and on sgRNA insert
  • Performed sequencing on CRISPR insert
  • Miniprepped 5 pac-term
  • PCR of LeuS gene
 Lab work:
  • Completed PCR purification
  • Performed ligation on zeo and piece E
  • Performed a transformation of zeo-E-tet
 Lab work:
  • Restriction digest of LeuS
  • Redoing competent JW cells
 Lab work:
  • Took nanodrop concentrations of minipreppred ligation reaction
  • Restriction digests
 Lab work:
  • Made primer solutions
  • SDM of Pst 1
  • Transformation and sequencing
 Lab work:
  • Performed digests of T1
  • Digest PCR LeuS
  • Ligated T1 and LeuS
  • Transformed cells
 Lab work:
  • Preculture of term-leuS lig
  • 3 colonies of 6:1 ligation and 1 colony of 8:1 ligation
 Lab work:
  • Miniprep of leuS term ligation
 Lab work:
  • Ran a gel
 Lab work:
  • Performed PCR
  • Performed transformation
Lab work:
  • Preculture of Pst sdm
 Lab work:
  • Performed miniprep
  • Ran confirmatory digest
 Lab work:
  • Performed restriction digests
 Lab work:
  • Performed ligations
 Lab work:
  • Preculture for transformations
 Lab work:
  • 3A assembly of zeo-E-Kan backbone
 Lab work:
  • Sequencing
 Lab work:
  • Performed restriction digest
  • Performed ligation
 Lab work:
  • Performed restriction digest
  • Performed ligation
 Lab work:
  • Nanodrop used to determine concentrations
 Lab work:
  • Nanodrop to determine concentrations and sequencing performed
 Lab work:
  • Ran term and enzyme gels
  • Digest of term performed
 Lab work:
  • Miniprep of term and pro
  • Performed digest
 Lab work:
  • Digest of terminator
  • Success! Got term in lane 5
  • Ligation performed
 Lab work:
  • PCR of mutant LeuS
 Lab work:
  • PCR purifed mut LeuS
  • Digest performed
  • Ligations performed
 Lab work:
  • Miniprep x 27
  • Digest MT 7.1 → CIP
  • Digest piece A and controls
  • Ligations of mut term and piece A performed
 Lab work:
  • Miniprep of HA and mut and term
  • Restriction digest of enz-term and promoter
  • Preculture JW cells
 Lab work:
  • Competent cell procedure for JW
  • Confirmation test followed by plating
 Lab work:
  • Restriction digest
 Lab work:
  • JW5807 competent cell protocol
  • Miniprepped promoter-enzyme-terminator plasmid
 Lab work:
  • Confirmatory digest of promoter-enzyme-terminator plasmid miniprepped on 10/9
  • Sequencing of promoter-enzyme-terminator plasmid miniprepped on 10/9
  • Made minimal medium plates with nothing, 3mM leucine, or 3mM CBZ-leu
 Lab work:
  • digest, gel, ligation of parts into CAM backbones
May
S M T W T F S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
June
S M T W T F S
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
July
S M T W T F S
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
August
S M T W T F S
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
September
S M T W T F S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30
October
S M T W T F S
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31