Team:The Tech Museum/Notebook

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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
Conditions
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
Maturation times
Chromogenic pools ~ 5 days 37 degrees C
Fluoroescent pools ~ 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.