Team:The Tech Museum/Notebook

From 2014.igem.org

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<p>Final tri-color plasmid designs, assembled by DNA2.0<br>
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<p>Final tri-color plasmid designs, assembled by DNA2.0:</p>
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Chromogenic plasmid pool <br>
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<p>Chromogenic plasmid pool <br>
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Blitzen (blue) <br>
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<UL><LI>Blitzen (blue) <br>
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Kringle (yellow)<br>
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<LI>Kringle (yellow)<br>
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Paprika (red)<br>
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<LI>Paprika (red)</UL></p>
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Fluorescent plasmid pool<br>
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<p>Fluorescent plasmid pool<br>
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CindyLouCFP (400/495)<br>
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<UL><LI>CindyLouCFP (400/495)<br>
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KringleYFP (520/542)<br>
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<LI>KringleYFP (520/542)<br>
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PaprikaRFP (554/590)</p>
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<LI>PaprikaRFP (554/590)</UL></p>
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<p>Optimization of plasmid pools with visitor-accessible museum wetlab set up<br>
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<p>Optimization of plasmid pools with visitor-accessible museum wetlab set up. Transformation condition:<br>
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Conditions<br>
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<UL><LI>6cm plates<br>
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6cm plates<br>
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<LI>400 ug/ml Amp<br>
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400 ug/ml Amp<br>
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<LI>0.3 ul unamplified plasmid pool in 100ul CaCl2<br>
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0.3 ul unamplified plasmid pool in 100ul CaCl2<br>
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<LI>20 ul competent bacteria<br>
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20 ul competent bacteria<br>
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<LI>Current visitor wetlab transformation protocol: 30 sec on ice, 40 sec heat shot at 42 degrees C<br>
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Current visitor wetlab transformation protocol: <br>
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<LI>Chromogenic pool maturation time ~ 5 days 37 degrees C<br>
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30 sec on ice<br>
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<LI>Fluoroescent pool maturation time ~ 3 days 37 degrees C<br>
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40 sec heat shot at 42 degrees C<br>
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<LI>Low copy fluorescent plasmid pool gave more reliable results with most color variety </UL></p>
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Maturation times<br>
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Chromogenic pools ~ 5 days 37 degrees C<br>
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Fluoroescent pools ~ 3 days 37 degrees C<br>
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Low copy fluorescent plasmid pool gave more reliable results with most color variety </p>
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<p><b>Safety:</b><br>
<p><b>Safety:</b><br>
We did not use any dangerous organisms or reagents. Transformation of bacteria was done in the musuem’s existing wetlab setup. </p>
We did not use any dangerous organisms or reagents. Transformation of bacteria was done in the musuem’s existing wetlab setup. </p>

Revision as of 17:51, 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 (June 2014)
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.

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