Team:Caltech/week9

From 2014.igem.org

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<table>
<table>
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<tr><td colspan="2"> <h1>Notebook</h1> </td></tr>
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<tr> <td bgColor=#FFFFFF colspan = 3 height = 60px> <font size = +5> <center>Notebook </center> </td> </tr>
<tr>
<tr>
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<b><a href='week11'>Week 11</a></b><br><br>
<b><a href='week11'>Week 11</a></b><br><br>
<b><a href='week12'>Week 12</a></b><br><br>
<b><a href='week12'>Week 12</a></b><br><br>
 +
<b><a href='week13'>Week 13</a></b><br><br>
 +
<b><a href='week14'>Week 14</a></b><br><br>
 +
<b><a href='week15'>Week 15</a></b><br><br>
<!-- links end here -->
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</td>
</td>
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</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>Attempt to create a negative control for Western blotting:
 +
<ul><li>Ran colony PCR on colonies of A2 construct (pTet-GFP). Annealing temperature was 53&deg;C. Colonies were overloaded and smeared the lanes.</li>
 +
    <li>Reran colony PCR on A2 colonies. Annealing temperature was 51&deg;C. 2 presumed successes.</li>
 +
    <li>Set up 2 liquid cultures using the 2 colonies proposed to have A2.</li>
 +
</ul>
 +
LCMS analysis of lam system:
 +
<ul><li>Continued vacufuging LCMS acetonitrile pTG004-FLAG samples.</li></ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
 +
<ul><li>Miniprepped pMB001, 2, & 5 liquid cultures. Shipped them out for sequencing.</li>
 +
    <li>Resuspended colonies for pMB003, 4, & 6 and grew up overnight cultures of them.</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<br>agrBCDA system:
 +
<ul><li>Set up liquid cultures of pAA009 & pAA010 colonies from overnight.</li>
 +
    <li>Ran colony PCR on pAA009 & pAA010 colonies, adding DMSO & with annealing temperature of 51&deg;C. This yielded bright bands at correct length for 2 of the pAA010 colonies and some of the pAA009 colonies.</li>
 +
    <li>PCR-purified the pAA010 colony PCR products & shipped them for sequencing.</li>
 +
    <li>Miniprepped pAA009 liquid cultures & sent samples for sequencing.</li>
</ul>
</ul>
</td>
</td>
Line 121: Line 141:
</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>Attempt to create a negative control for Western blotting:
 +
<ul><li>Miniprepped yesterday's 2 liquid cultures of A2 and shipped them for sequencing</li></ul>
 +
LCMS analysis of lam system:
 +
<ul><li>Continued vacufuging LC/MS acetonitrile samples (lam-noFLAG)</li></ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<ul><li>Analyzed sequencing data, indicating that 2 of the pAA010 Gibson colonies contain the correct sequence.</li>
 +
    <li>Miniprepped liquid cultures of those colonies.</li>
 +
    <li>Since pAA009 construction was a failure, we ran another Gibson assembly constructing pAA009.</li>
 +
    <li>New Gibson assemblies were transformed into DH5&alpha;-Z1.</li>
</ul>
</ul>
</td>
</td>
Line 133: Line 162:
</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>Redo of cloning of agr system and fsr-FLAGafter:
 +
<ul><li>PCRed out backbones for pTG002,  pTG003, & pTG006.</li>
 +
    <li>DpnI digested and then purified PCR products.</li>
 +
    <li>Ran a gel to confirm isolation of backbones from above procedures.</li>
 +
    <li>Gibson assembly using constructed backbones and appropriate gblocks.</li>
 +
    <li>Gibson assembly products were transformed into JM109 cells. 2 transformations were performed for each product: one using 3 &mu;L of assembly product and the other using 11 &mu;L of assembly product.</li>
 +
    <li>Transformations werew plated on CARB plates & incubated overnight.</li>
 +
</ul>
 +
Preparation of fsr system for LC/MS analysis:
 +
<ul><li>Innoculated LB-CARB liquid culture with DH5&alpha; cells expressing pTG005 (fsr system with FLAG still included)</li></ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
 +
<ul><li>Miniprepped pMB003, pMB004, & pMB006 liquid cultures. Sent samples out for sequencing.</li>
 +
    <li>Resuspended pMB005 colonies and ran colony PCR.</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<ul><li>Ran colony PCR on colonies from the pAA009 Gibson assembly transformation yesterday.</li>
 +
    <li>Ran a gel of the colony PCR products.</li>
 +
    <li>PCR-purified promising PCR products and shipped them for sequencing.</li>
</ul>
</ul>
-
<br>
 
</td>
</td>
</tr>
</tr>
Line 146: Line 192:
</td>
</td>
<td valign="top">
<td valign="top">
 +
<b>Export Systems</b>
 +
<br>Redo of cloning of agr system and fsr-FLAGafter:
 +
<ul><li>Ran colony PCR on yesterday's transformations.</li>
 +
    <li>Colony PCR products were run on a gel.</li>
 +
    <li>8 LB-CARB liquid cultures were prepared and incubated overnight.</li>
 +
</ul>
 +
Attempt to create a negative control for Western blotting:
 +
<ul><li>Picked more colonies from A2 transformation last week and ran colony PCR on them.</li>
 +
    <li>Ran a gel on the colony PCR products.</li>
 +
    <li>Set up 4 LB-CARB liquid cultures and incubated them overnight.</li>
 +
</ul>
 +
Preparation of fsr system for LC/MS analysis:
 +
<ul><li>Set up 0 nM, 250 nM, & 500 nM aTc inductions of pTG005-expressing cells (fsr-FLAG) in MOPS media. 5 &mu;L aliquots of last night's liquid culture was used to start the cultures in MOPS.</li></ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
<ul>
<ul>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<ul><li>Analyzed sequencing results from yesterday. Bad results for all.</li>
 +
    <li>Some colonies that produced bands of right length from yesterday's colPCR weren't sequenced, so those colonies were miniprepped and then shipped for sequencing.</li>
</ul>
</ul>
</td>
</td>
Line 158: Line 222:
</td>
</td>
<td valign="top">
<td valign="top">
 +
<b>Export Systems</b>
 +
<br>Redo of cloning of agr system and fsr-FLAGafter:
 +
<ul><li>Miniprepped 3 of the 8 LB liquid cultures from last night.</li>
 +
    <li>Shipped samples out for sequencing.</li>
 +
</ul>
 +
Attempt to create a negative control for Western blotting:
 +
<ul><li>Miniprepped 3 of the 4 LB liquid cultures from last night.</li>
 +
    <li>Shipped samples out for sequencing.</li>
 +
</ul>
 +
Preparation of fsr system for LC/MS analysis:
 +
<ul><li>Started LB liquid culture of DH5&alpha; cells expressing pAA001 as a negative control in the LC/MS runs.</li>
 +
    <li>aTc-induced MOPS liquid cultures from yesterday were spun down, and then the supernatant was loaded into C18 columns, washed with 10% acetonitrile, and then eluted with 60% acetonitrile.</li>
 +
    <li>The pTG005 acetonitrile elutates were then left to evaporate in the fume hood overnight.</li>
 +
    <li>On Saturday, we began vacufuging the fsr-FLAG LC/MS acetonitrile samples.</li>
 +
    <li>On Saturday, we also set up 0 nM, 250 nM, & 500 nM aTc inductions of pAA001-expressing cells (negative control in LC/MS) in MOPS media. 5 &mu;L aliquots of last night's liquid culture was used to start the cultures.</li>
 +
    <li>On Sunday, we spun down the aTc-induced MOPS liquid cultures of pAA001 cells, and then loaded the supernatant into C18 columns, washed it with 10% acetonitirle, and then eluted it with 60% acetonitrile.</li>
 +
    <li>pAA001 acetonitrile samples were then left to evaporate in the fume hood overnight.</li>
 +
</ul>
 +
De-FLAGging pTG005:
 +
<ul><li>Attempted to PCR-out the 3xFLAG from pTG005.</li>
 +
    <li>DpnI digested and PCR-purified the pTG005 PCR product.</li>
 +
    <li>Ran round-the-horn ligation on the purified PCR product.</li>
 +
    <li>Transformed round-the-horn ligation product into DH5&alpha;</li>
 +
</ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
<ul>
<ul>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<ul><li>Analyzed sequencing results from yesterday's minipreps. Failed again.</li>
 +
    <li>Conducted Round-the-horn PCR using sequence-verified pAA010 plasmids to attempt to construct pAA009.</li>
 +
    <li>DpnI-digested for 1.5 hours and then PCR-purified PCR products.</li>
 +
    <li>QuickLigation reaction on purified PCR product. Ligation product was then transformed into JM109.</li>
</ul>
</ul>
</td>
</td>

Latest revision as of 00:35, 15 September 2014
Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions
Notebook
Overview

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week Nine

Monday, 8/11/14

 Export Systems
Attempt to create a negative control for Western blotting:
  • Ran colony PCR on colonies of A2 construct (pTet-GFP). Annealing temperature was 53°C. Colonies were overloaded and smeared the lanes.
  • Reran colony PCR on A2 colonies. Annealing temperature was 51°C. 2 presumed successes.
  • Set up 2 liquid cultures using the 2 colonies proposed to have A2.
LCMS analysis of lam system:
  • Continued vacufuging LCMS acetonitrile pTG004-FLAG samples.
lamBCDA & fsrABC Reception Systems
  • Miniprepped pMB001, 2, & 5 liquid cultures. Shipped them out for sequencing.
  • Resuspended colonies for pMB003, 4, & 6 and grew up overnight cultures of them.
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
  • Set up liquid cultures of pAA009 & pAA010 colonies from overnight.
  • Ran colony PCR on pAA009 & pAA010 colonies, adding DMSO & with annealing temperature of 51°C. This yielded bright bands at correct length for 2 of the pAA010 colonies and some of the pAA009 colonies.
  • PCR-purified the pAA010 colony PCR products & shipped them for sequencing.
  • Miniprepped pAA009 liquid cultures & sent samples for sequencing.

Tuesday, 8/12/14

 Export Systems
Attempt to create a negative control for Western blotting:
  • Miniprepped yesterday's 2 liquid cultures of A2 and shipped them for sequencing
LCMS analysis of lam system:
  • Continued vacufuging LC/MS acetonitrile samples (lam-noFLAG)
agrBCDA Reception System and Combinatorial Promoters
  • Analyzed sequencing data, indicating that 2 of the pAA010 Gibson colonies contain the correct sequence.
  • Miniprepped liquid cultures of those colonies.
  • Since pAA009 construction was a failure, we ran another Gibson assembly constructing pAA009.
  • New Gibson assemblies were transformed into DH5α-Z1.

Wednesday, 8/13/14

 Export Systems
Redo of cloning of agr system and fsr-FLAGafter:
  • PCRed out backbones for pTG002, pTG003, & pTG006.
  • DpnI digested and then purified PCR products.
  • Ran a gel to confirm isolation of backbones from above procedures.
  • Gibson assembly using constructed backbones and appropriate gblocks.
  • Gibson assembly products were transformed into JM109 cells. 2 transformations were performed for each product: one using 3 μL of assembly product and the other using 11 μL of assembly product.
  • Transformations werew plated on CARB plates & incubated overnight.
Preparation of fsr system for LC/MS analysis:
  • Innoculated LB-CARB liquid culture with DH5α cells expressing pTG005 (fsr system with FLAG still included)
lamBCDA & fsrABC Reception Systems
  • Miniprepped pMB003, pMB004, & pMB006 liquid cultures. Sent samples out for sequencing.
  • Resuspended pMB005 colonies and ran colony PCR.
agrBCDA Reception System and Combinatorial Promoters
  • Ran colony PCR on colonies from the pAA009 Gibson assembly transformation yesterday.
  • Ran a gel of the colony PCR products.
  • PCR-purified promising PCR products and shipped them for sequencing.

Thursday, 8/14/14

 Export Systems
Redo of cloning of agr system and fsr-FLAGafter:
  • Ran colony PCR on yesterday's transformations.
  • Colony PCR products were run on a gel.
  • 8 LB-CARB liquid cultures were prepared and incubated overnight.
Attempt to create a negative control for Western blotting:
  • Picked more colonies from A2 transformation last week and ran colony PCR on them.
  • Ran a gel on the colony PCR products.
  • Set up 4 LB-CARB liquid cultures and incubated them overnight.
Preparation of fsr system for LC/MS analysis:
  • Set up 0 nM, 250 nM, & 500 nM aTc inductions of pTG005-expressing cells (fsr-FLAG) in MOPS media. 5 μL aliquots of last night's liquid culture was used to start the cultures in MOPS.
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Analyzed sequencing results from yesterday. Bad results for all.
  • Some colonies that produced bands of right length from yesterday's colPCR weren't sequenced, so those colonies were miniprepped and then shipped for sequencing.

Friday, 8/15/14

 Export Systems
Redo of cloning of agr system and fsr-FLAGafter:
  • Miniprepped 3 of the 8 LB liquid cultures from last night.
  • Shipped samples out for sequencing.
Attempt to create a negative control for Western blotting:
  • Miniprepped 3 of the 4 LB liquid cultures from last night.
  • Shipped samples out for sequencing.
Preparation of fsr system for LC/MS analysis:
  • Started LB liquid culture of DH5α cells expressing pAA001 as a negative control in the LC/MS runs.
  • aTc-induced MOPS liquid cultures from yesterday were spun down, and then the supernatant was loaded into C18 columns, washed with 10% acetonitrile, and then eluted with 60% acetonitrile.
  • The pTG005 acetonitrile elutates were then left to evaporate in the fume hood overnight.
  • On Saturday, we began vacufuging the fsr-FLAG LC/MS acetonitrile samples.
  • On Saturday, we also set up 0 nM, 250 nM, & 500 nM aTc inductions of pAA001-expressing cells (negative control in LC/MS) in MOPS media. 5 μL aliquots of last night's liquid culture was used to start the cultures.
  • On Sunday, we spun down the aTc-induced MOPS liquid cultures of pAA001 cells, and then loaded the supernatant into C18 columns, washed it with 10% acetonitirle, and then eluted it with 60% acetonitrile.
  • pAA001 acetonitrile samples were then left to evaporate in the fume hood overnight.
De-FLAGging pTG005:
  • Attempted to PCR-out the 3xFLAG from pTG005.
  • DpnI digested and PCR-purified the pTG005 PCR product.
  • Ran round-the-horn ligation on the purified PCR product.
  • Transformed round-the-horn ligation product into DH5α
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Analyzed sequencing results from yesterday's minipreps. Failed again.
  • Conducted Round-the-horn PCR using sequence-verified pAA010 plasmids to attempt to construct pAA009.
  • DpnI-digested for 1.5 hours and then PCR-purified PCR products.
  • QuickLigation reaction on purified PCR product. Ligation product was then transformed into JM109.

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