Team:The Tech Museum/Notebook

From 2014.igem.org

(Difference between revisions)
 
(11 intermediate revisions not shown)
Line 7: Line 7:
<tr>
<tr>
-
<td width="45%" valign="top">  
+
<td colspan="3" valign="top">  
-
<p>You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. </p>
+
<p><b>Timeline of major events:</b><br></p>
 +
<p><UL><LI>May: Basic concept research and design<br>
 +
<LI>June: Software development initiated; plasmid design started and promoter-RBS’s picked<br>
 +
<LI>July: Wetlab testing of possible color protein combinations<br>
 +
<LI>August 10: Design of complete tri-color plasmids finalized<br>
 +
<LI>August 24: Assembly of tri-color plasmid pools completed (DNA2.0)<br>
 +
<LI>September 10: Tri-color plasmids optimized on museum wetlab setup<br>
 +
<LI>September 29: Software fully integrated with hardware on mobile exhibit<br>
 +
<LI>September 30 - October 3: Prototyping of mobile exhibit with museum staff and visitors<br>
 +
<LI>October 6 - October 17: Data collection on the museum floor with visitor!</UL></p><br>
 +
 
 +
<p><b>Biology Details:</b></p>
 +
<p>Design of tri-color plasmid pool <br>
 +
<UL><LI>9 promoter-rbs pairs of varying strength from Kosuri et al paper (2013):</UL>
 +
<table border="1" cellpadding="0" cellspacing="0">
 +
<tr>
 +
<td width="25"></td>
 +
<td>Promoter - RBS Pairs</td>
 +
<td valign="top">Sequence</td>
 +
</tr>
 +
<br>
 +
<tr>
 +
<td>1</td>
 +
<td>BBa_J23117 - BBa_J61112</td>
 +
<td>TTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGCCAATCTCTAGAGAAAGAGGTGACATAC</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>BBa_J23104 - BBa_J61107</td>
 +
<td>TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCTTACGTCTAGAGAAAGAAGAGACTCAC</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>apFAB78 - apFAB954</td>
 +
<td>TTGACATTTATCCCTTGCGGCGATAGATTTAACGTATGACGGATCTTAATCTAGCTCAGGACAATTT</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>apFAB76 - apFAB927</td>
 +
<td>TTGACATTTATCCCTTGCGGCGATATAATAGATATCTTAATCTAGCCCGGGAGTTTTTTCATTCCGGATCTTAATCTAGCTGGGGACTGTTT</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<td>apFAB70 - apFAB844</td>
 +
<td>TTGACATCGCATCTTTTTGTACCTATAATGTGTGGATAGAGTATCTTAATCTAGCAGGGGACACTTT</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>BBa_J23104 - apFAB909</td>
 +
<td>TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCTTACGATCTTAATCTAGCGAAGGATAGTTT</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<td>apFAB45 - apFAB901</td>
 +
<td>AAAAAGAGTATTGACTTCGCATCTTTTTGTACCTATAATGTGTGGATAGCGG</td>
 +
</tr>
 +
<tr>
 +
<td>8</td>
 +
<td>apFAB92 - apFAB863</td>
 +
<td>AAAAAATTTATTTGCTTTCAGGAAAATTTTTCTGCATAATTATTTCATGGAGCATCTTAATCTAGCGGGGGAGCGTTT</td>
 +
</tr>
 +
<tr>
 +
<td>9</td>
 +
<td>apFAB71 - salis-4-10</td>
 +
<td>TTGACATCGCATCTTTTTGTACCTATAATAGATTCATGATGAAATCTCTTTTATCAAATATAAGCAGGAT</td>
 +
</tr>
 +
</table>
 +
<br>
 +
 
 +
<p>Selection of colored reporter proteins <br>
 +
<UL><LI>Co-transformation testing of multiple color combinations of proteins from DNA2.0<br>
 +
<LI>Transformations on Kanamycin and IPTG</UL><br></p>
 +
<center><img src="https://static.igem.org/mediawiki/2014/6/6c/Tech_Museum_Chromo-Testing.png" width="800"><br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/1/12/Tech_Museum_Fluoro-Testing.png" width="800"><br><br></center>
 +
<p>Final tri-color plasmid designs, assembled by DNA2.0:</p>
 +
<p>Chromogenic plasmid pool <br>
 +
<UL><LI>Blitzen (blue) <br>
 +
<LI>Kringle (yellow)<br>
 +
<LI>Paprika (red)</UL></p>
 +
<p>Fluorescent plasmid pool<br>
 +
<UL><LI>CindyLouCFP (400/495)<br>
 +
<LI>KringleYFP (520/542)<br>
 +
<LI>PaprikaRFP (554/590)</UL></p>
 +
 
 +
<p>Optimization of plasmid pools with visitor-accessible museum wetlab set up. Transformation condition:<br>
 +
<UL><LI>6cm plates<br>
 +
<LI>400 ug/ml Amp<br>
 +
<LI>0.3 ul unamplified plasmid pool in 100ul CaCl2<br>
 +
<LI>20 ul competent bacteria<br>
 +
<LI>Current visitor wetlab transformation protocol: 30 sec on ice, 40 sec heat shot at 42 degrees C<br>
 +
<LI>Chromogenic pool maturation time ~ 5 days 37 degrees C<br>
 +
<LI>Fluoroescent pool maturation time ~ 3 days 37 degrees C<br>
 +
<LI>Low copy fluorescent plasmid pool gave more reliable results with most color variety </UL></p><br>
 +
<p><b>Safety:</b><br></p>
 +
<p>We did not use any dangerous organisms or reagents. Transformation of bacteria was done in the musuem’s existing wetlab setup. </p>
 +
 
 +
 
</td>
</td>

Latest revision as of 21:14, 17 October 2014

Home Team Project Notebook Community Engagement Attributions

Notebook

Timeline of major events:

  • May: Basic concept research and design
  • June: Software development initiated; plasmid design started and promoter-RBS’s picked
  • July: Wetlab testing of possible color protein combinations
  • August 10: Design of complete tri-color plasmids finalized
  • August 24: Assembly of tri-color plasmid pools completed (DNA2.0)
  • September 10: Tri-color plasmids optimized on museum wetlab setup
  • September 29: Software fully integrated with hardware on mobile exhibit
  • September 30 - October 3: Prototyping of mobile exhibit with museum staff and visitors
  • October 6 - October 17: Data collection on the museum floor with visitor!


Biology Details:

Design of tri-color plasmid pool

  • 9 promoter-rbs pairs of varying strength from Kosuri et al paper (2013):

Promoter - RBS Pairs Sequence
1 BBa_J23117 - BBa_J61112 TTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGCCAATCTCTAGAGAAAGAGGTGACATAC
2 BBa_J23104 - BBa_J61107 TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCTTACGTCTAGAGAAAGAAGAGACTCAC
3 apFAB78 - apFAB954 TTGACATTTATCCCTTGCGGCGATAGATTTAACGTATGACGGATCTTAATCTAGCTCAGGACAATTT
4 apFAB76 - apFAB927 TTGACATTTATCCCTTGCGGCGATATAATAGATATCTTAATCTAGCCCGGGAGTTTTTTCATTCCGGATCTTAATCTAGCTGGGGACTGTTT
5 apFAB70 - apFAB844 TTGACATCGCATCTTTTTGTACCTATAATGTGTGGATAGAGTATCTTAATCTAGCAGGGGACACTTT
6 BBa_J23104 - apFAB909 TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCTTACGATCTTAATCTAGCGAAGGATAGTTT
7 apFAB45 - apFAB901 AAAAAGAGTATTGACTTCGCATCTTTTTGTACCTATAATGTGTGGATAGCGG
8 apFAB92 - apFAB863 AAAAAATTTATTTGCTTTCAGGAAAATTTTTCTGCATAATTATTTCATGGAGCATCTTAATCTAGCGGGGGAGCGTTT
9 apFAB71 - salis-4-10 TTGACATCGCATCTTTTTGTACCTATAATAGATTCATGATGAAATCTCTTTTATCAAATATAAGCAGGAT

Selection of colored reporter proteins

  • Co-transformation testing of multiple color combinations of proteins from DNA2.0
  • Transformations on Kanamycin and IPTG





Final tri-color plasmid designs, assembled by DNA2.0:

Chromogenic plasmid pool

  • Blitzen (blue)
  • Kringle (yellow)
  • Paprika (red)

Fluorescent plasmid pool

  • CindyLouCFP (400/495)
  • KringleYFP (520/542)
  • PaprikaRFP (554/590)

Optimization of plasmid pools with visitor-accessible museum wetlab set up. Transformation condition:

  • 6cm plates
  • 400 ug/ml Amp
  • 0.3 ul unamplified plasmid pool in 100ul CaCl2
  • 20 ul competent bacteria
  • Current visitor wetlab transformation protocol: 30 sec on ice, 40 sec heat shot at 42 degrees C
  • Chromogenic pool maturation time ~ 5 days 37 degrees C
  • Fluoroescent pool maturation time ~ 3 days 37 degrees C
  • Low copy fluorescent plasmid pool gave more reliable results with most color variety


Safety:

We did not use any dangerous organisms or reagents. Transformation of bacteria was done in the musuem’s existing wetlab setup.

Retrieved from "http://2014.igem.org/Team:The_Tech_Museum/Notebook"