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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
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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.
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