Team:Caltech/week8

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>
<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>
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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 de-FLAG pTG004 to create lam system in its "native state" for use in LCMS:
 +
<ul><li>Ran a colony PCR on 4 colonies picked from a CARB plate that had been plated with a 2nd Round-the-horn reaction.</li>
 +
    <li>A gel was run on the products, and a liquid culture was set up for the one colony suggesting it contains a properly formed pTG004-noFLAG plasmid.</li>
 +
</ul>
 +
Attempt to create a negative control for Western blotting:
 +
<ul><li>Reattempted transformation of Anton's B2 pTet_GFP GG-assemblies into JM109. Plated on Cm plates & incubated overnight.</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<ul><li>Transformed JM109 cells with the pAA009 & pAA010 Gibson assembly products from Friday.</li>
 +
    <li>Transformed DH5&alpha;-Z1 cells with the pAA002 Gibson assembly products from Friday.</li>
 +
</ul>
 +
Beginning of a new, TXTL characterization project:
 +
<ul><li>We began characterization of a constitutive promoter family designed by Berkeley iGEM 2006 (biobrick parts BBa_J23100-J23118). We transformed JM109 cells with the re-suspended plasmids.</li>
 +
    <li>Some parts were not included with the kit mailed to us, so we might request those at a later date.</li>
</ul>
</ul>
</td>
</td>
Line 121: Line 138:
</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>LCMS pTG004-noFLAG (lam system in its native configuration without the 3xFLAG) preparation:
 +
<ul><li>Miniprepped the liquid culture of pTG004-noFLAG incubated last night. However, the final DNA conc. was too low to send for sequenceing, so the liquid culture was grown up again for another try tomorrow.</li>
</ul>
</ul>
 +
Western blot negative control preparation:
 +
<ul><li>Ran colony PCR on colonies transformed with A2 (pTet_GFP from Anton).</li>
 +
    <li>Set up liquid cultures with carbenicillin for picked colonies.</li>
 +
</ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
 +
<ul><li>Reran colony PCR using the new primers that we ordered.</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<br>agrBCDA system:
 +
<ul><li>Only 1 colony was resulted from yesterday's transformation of pAA009. Set up a liquid culture of this colony.</li>
 +
    <li>Ran gradient PCR on pAA009 backbone using 6 temperatures from 50-70&deg;C.</li>
 +
    <li>DpnI digested pAA009 backbone for 2 hours and stored at 4&deg;C overnight.</li>
 +
</ul>
 +
Combinatorial promoters project:
 +
<ul><li>Picked 6 colonies from the plate transformed yesterday with pAA002 and set up liquid cultures.</li></ul>
 +
TXTL characterization project:
 +
<ul><li>Miniprepped transformed liquid cultures grown up last night</li></ul>
</td>
</td>
</tr>
</tr>
Line 133: Line 169:
</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>LCMS pTG004-noFLAG (lam system in its native configuration without the 3xFLAG) preparation:
 +
<ul><li>Redid miniprep of pTG004-noFLAG liquid culture.</li>
 +
    <li>Sent sample for sequencing</li>
 +
    <li>Transformed that pTG004-noFLAG miniprep into DH5&alpha;-Z1</li>
 +
</ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
 +
<ul><li>Set up overnight liquid culture of pMB006-transformed colony that looks promising based on yesterday's colPCR</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<br>agrBCDA system:
 +
<ul><li>Ran a colPCR on the single colony that obtained from Monday's transformation, but didn't get the right band.</li>
 +
    <li>Ran a gel on the pAA009 backbones that we PCR'ed yesterday, confirming bands that were around the size.</li>
 +
    <li>Did Gibson assembly with the backbones for pAA009/10, and then transformed DH5alpha-Z1 cells with them.</li>
 +
</ul>
 +
Combinatorial Promoters:
 +
<ul><li>Ran a colony PCR on colonies from earlier transformations. 5 of then were of correct length.</li>
 +
    <li>Shipped out colPCR products for sequencing.</li>
 +
</ul>
 +
TXTL characterization project:
 +
<ul><li>Set up and ran TXTL reactions on the plasmids containing the family of constitutive promoters.</li>
</ul>
</ul>
-
<br>
 
</td>
</td>
</tr>
</tr>
Line 146: Line 201:
</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>LCMS pTG004-noFLAG (lam system in its native configuration without the 3xFLAG) preparation:
 +
<ul><li>Set up liquid culture of 1 colony of DH5&alpha; with pTG004-noFLAG (transformed yesterday).</li></ul>
 +
LCMS analysis of lam system:
 +
<ul><li>Induced expression of pTG004 in pTG004-FLAG constructs by adding varying amounts of aTc to 3 separate liquid cultures (MOPS media).
 +
    <ul><li>Concentrations of aTc induction: 0 nM, 250 nM, 500 nM</li></ul></li>
 +
</ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
 +
<ul><li>Miniprepped overnight liquid culture of pMB006. Shipped to Eurofin for sequencing.</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<br>agrBCDA system:
 +
<ul><li>Ran colony PCR on yesterday's transformations. No successes.</li></ul>
 +
Combinatorial promoters:
 +
<ul><li>Analyzed sequencing results, suggesting that 2 of the pAA002 colonies have desired plasmid.</li>
 +
    <li>Started overnight liquid culture for experiments on the pAA002 combinatorial promoter.</li>
</ul>
</ul>
</td>
</td>
Line 158: Line 228:
</td>
</td>
<td valign="top">
<td valign="top">
-
<ul>
+
<b>Export Systems</b>
 +
<br>Continued cloning of unconstructed plasmids:
 +
<ul><li>Re-DpnI-digested PCRed backbones created on July 30th, by adding 2.5 uL 10x CutSmart Buffer and 0.5 uL of a newly ordered stock of DpnI and incubating for 6 hours.</li>
 +
    <li>PCR-purified DpnI digests, yielding much lower DNA concentrations from what was measured on July 30.</li>
 +
    <li>Ran Gibson Assemblies of pTG002/3/6 using the new backbones & appropriate geneblock inserts</li>
 +
    <li>Assemblies were transformed into JM109</li>
 +
</ul>
 +
LCMS analysis of lam system:
 +
<ul><li>Took pTG004-FLAG liquid cultures induced with aTc and prepared the samples via the LCMS protocol with acetonitrile & vacufuging.</li>
 +
    <li>Set up 3 more liquid cultures but with pTG004-noFLAG (sequencing-verified) with varying aTc induction (0 nM, 250 nM, 500 nM).</li>
 +
</ul>
 +
Western blot negative control preparation:
 +
<ul><li>Again tried transforming A2 plasmid (pTet_GFP) from Anton into JM109. Plated on carbenicillin plate.</li>
 +
</ul>
 +
<b>lamBCDA & fsrABC Reception Systems</b>
 +
<ul><li>Analyzed sequencing results. They were messy and revealed nothing about the sequence.</li>
 +
    <li>Resuspended colonies and grew overnight cultures for pMB001, 2, & 5</li>
 +
</ul>
 +
<b>agrBCDA Reception System and Combinatorial Promoters</b>
 +
<br>Combinatorial Promoters:
 +
<ul><li>Tested B83 combinatorial promoter (within pAA002 plasmids) in tbAA002 cells using IPTG & aTc induction.
 +
    <ul><li>([IPTG] in &mu;M, [aTc] in ng/mL): (0,0); (5,0); (50,0); (500,0); (0,25); (5,25); (50, 25); (500,25); (0,50); (5,50); (50,50); (500,50); (0,100); (5,100); (50,100); (500,100)</li></ul>
 +
    </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 Eight

Monday, 8/4/14

 Export Systems
Attempt to de-FLAG pTG004 to create lam system in its "native state" for use in LCMS:
  • Ran a colony PCR on 4 colonies picked from a CARB plate that had been plated with a 2nd Round-the-horn reaction.
  • A gel was run on the products, and a liquid culture was set up for the one colony suggesting it contains a properly formed pTG004-noFLAG plasmid.
Attempt to create a negative control for Western blotting:
  • Reattempted transformation of Anton's B2 pTet_GFP GG-assemblies into JM109. Plated on Cm plates & incubated overnight.
agrBCDA Reception System and Combinatorial Promoters
  • Transformed JM109 cells with the pAA009 & pAA010 Gibson assembly products from Friday.
  • Transformed DH5α-Z1 cells with the pAA002 Gibson assembly products from Friday.
Beginning of a new, TXTL characterization project:
  • We began characterization of a constitutive promoter family designed by Berkeley iGEM 2006 (biobrick parts BBa_J23100-J23118). We transformed JM109 cells with the re-suspended plasmids.
  • Some parts were not included with the kit mailed to us, so we might request those at a later date.

Tuesday, 8/5/14

 Export Systems
LCMS pTG004-noFLAG (lam system in its native configuration without the 3xFLAG) preparation:
  • Miniprepped the liquid culture of pTG004-noFLAG incubated last night. However, the final DNA conc. was too low to send for sequenceing, so the liquid culture was grown up again for another try tomorrow.
Western blot negative control preparation:
  • Ran colony PCR on colonies transformed with A2 (pTet_GFP from Anton).
  • Set up liquid cultures with carbenicillin for picked colonies.
lamBCDA & fsrABC Reception Systems
  • Reran colony PCR using the new primers that we ordered.
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
  • Only 1 colony was resulted from yesterday's transformation of pAA009. Set up a liquid culture of this colony.
  • Ran gradient PCR on pAA009 backbone using 6 temperatures from 50-70°C.
  • DpnI digested pAA009 backbone for 2 hours and stored at 4°C overnight.
Combinatorial promoters project:
  • Picked 6 colonies from the plate transformed yesterday with pAA002 and set up liquid cultures.
TXTL characterization project:
  • Miniprepped transformed liquid cultures grown up last night

Wednesday, 8/6/14

 Export Systems
LCMS pTG004-noFLAG (lam system in its native configuration without the 3xFLAG) preparation:
  • Redid miniprep of pTG004-noFLAG liquid culture.
  • Sent sample for sequencing
  • Transformed that pTG004-noFLAG miniprep into DH5α-Z1
lamBCDA & fsrABC Reception Systems
  • Set up overnight liquid culture of pMB006-transformed colony that looks promising based on yesterday's colPCR
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
  • Ran a colPCR on the single colony that obtained from Monday's transformation, but didn't get the right band.
  • Ran a gel on the pAA009 backbones that we PCR'ed yesterday, confirming bands that were around the size.
  • Did Gibson assembly with the backbones for pAA009/10, and then transformed DH5alpha-Z1 cells with them.
Combinatorial Promoters:
  • Ran a colony PCR on colonies from earlier transformations. 5 of then were of correct length.
  • Shipped out colPCR products for sequencing.
TXTL characterization project:
  • Set up and ran TXTL reactions on the plasmids containing the family of constitutive promoters.

Thursday, 8/7/14

 Export Systems
LCMS pTG004-noFLAG (lam system in its native configuration without the 3xFLAG) preparation:
  • Set up liquid culture of 1 colony of DH5α with pTG004-noFLAG (transformed yesterday).
LCMS analysis of lam system:
  • Induced expression of pTG004 in pTG004-FLAG constructs by adding varying amounts of aTc to 3 separate liquid cultures (MOPS media).
    • Concentrations of aTc induction: 0 nM, 250 nM, 500 nM
lamBCDA & fsrABC Reception Systems
  • Miniprepped overnight liquid culture of pMB006. Shipped to Eurofin for sequencing.
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
  • Ran colony PCR on yesterday's transformations. No successes.
Combinatorial promoters:
  • Analyzed sequencing results, suggesting that 2 of the pAA002 colonies have desired plasmid.
  • Started overnight liquid culture for experiments on the pAA002 combinatorial promoter.

Friday, 8/8/14

 Export Systems
Continued cloning of unconstructed plasmids:
  • Re-DpnI-digested PCRed backbones created on July 30th, by adding 2.5 uL 10x CutSmart Buffer and 0.5 uL of a newly ordered stock of DpnI and incubating for 6 hours.
  • PCR-purified DpnI digests, yielding much lower DNA concentrations from what was measured on July 30.
  • Ran Gibson Assemblies of pTG002/3/6 using the new backbones & appropriate geneblock inserts
  • Assemblies were transformed into JM109
LCMS analysis of lam system:
  • Took pTG004-FLAG liquid cultures induced with aTc and prepared the samples via the LCMS protocol with acetonitrile & vacufuging.
  • Set up 3 more liquid cultures but with pTG004-noFLAG (sequencing-verified) with varying aTc induction (0 nM, 250 nM, 500 nM).
Western blot negative control preparation:
  • Again tried transforming A2 plasmid (pTet_GFP) from Anton into JM109. Plated on carbenicillin plate.
lamBCDA & fsrABC Reception Systems
  • Analyzed sequencing results. They were messy and revealed nothing about the sequence.
  • Resuspended colonies and grew overnight cultures for pMB001, 2, & 5
agrBCDA Reception System and Combinatorial Promoters
Combinatorial Promoters:
  • Tested B83 combinatorial promoter (within pAA002 plasmids) in tbAA002 cells using IPTG & aTc induction.
    • ([IPTG] in μM, [aTc] in ng/mL): (0,0); (5,0); (50,0); (500,0); (0,25); (5,25); (50, 25); (500,25); (0,50); (5,50); (50,50); (500,50); (0,100); (5,100); (50,100); (500,100)

Retrieved from "http://2014.igem.org/Team:Caltech/week8"