My Proposal for How We Tackle These Projects
My proposal for how we tackle these projects
Function / Part / Location / WellTerminator / B0015 / 1 / 23L
Ribosome binding site / B0034 / 1 / 2M
Light sensor generator / M30109
ECFP+LVA / E0022 / 1 / 6C
mCherry / J06504 / 1 / 13F
pAra / I0500 / 1 / 14N
pLac / R0010 / 1 / 1D
p λcI / R0051 / 1 / 6K
Ribolock RBS / J23032 / 1 / 3N
Ribokey / J23066 / 2 / 2C
λcI+LVA / C0051 / 1 / 4E
Constituitive promoter1 / J23118 / 1 / 22A
Constituitive promoter 2 (stronger) / J23102 / 1 / 18G
Hin / J31000 / Order / Order
HixC / 1 / 1B
Cre / J61047 / 1 / 5D
Lox / J61046 / 1 / 24B
luxR / C0062 / 1 / 4O
pLux / R0062 / 1 / 6O
LuxI (AHL synthase) / C0061 / 1 / 4M
Ho1 / I15008 / 2 / 13J
PcyA / I15009 / 1 / 20F
Cph8 / I15010 / Order 2008 1022-6E / Order
pOmpR / R0082 / 1 / 16K
Dual regulated light sensor generator / M30109 / Order 2008 1022-8C / Order
Aspartate synthase CDS / C0083 / 2 / 17A
Function / Part# / Plate / Well / Made of…
λcI Tripart / P0151 / 1 / 10C / B0031-C0051-B0012-B0010
TetR tripart / P0140 / 2 / 1F
ECFP tripart / E0422 / 1 / 8M
GFP tripart / K091012 / 3 / 12M / B0030-E0040-B1006
RFP tripart / K081014 / 3 / 12O / B0030-E1010-B1006
LuxI (AHL) generator / K081015 / 3 / 14A / B0030-C0061-B1006
Constituitive GFP generator rith Ribolock RBS / K145303 / 2 / 6H / J23109-J23032-E0040-B0010-B0012
Ho1-PcyA generator / K098010 / 3 / 5F / Basic part. Polysictronic. Includes promoter and terminator
(A) / P1010(A) / 1 / 3K
(c) / P1010(c) / 1 / 5E
(T) / P1010(T) / 1 / 9E
E1010 / 1 / 18F
Step one
- Start work on the NAND gate. (9 initial rehydrations/transformations) During assembly, ensure the following two part composites are intermediates.
- cI lambda-stop
- mCherry-stop
- cI promoter-RBS
- Characterize light sensor device and OmpC promoter. If the part doesn’t work, try the plasmid from Calgary. If that doesn’t work rebuild the device.
- Test the device with an OmpC-RBS-GFP-Stop fusion (2 additional rehydrations) keep the following intermediates
- OmpC-RBS
- GFP-Stop
- Rehydrate and transform additional parts required for Derek and layne’s projects (4 additional rehydrations/transformations)
- Characterize cI inverter for leakiness
If NAND gate, Light sensor generator (any version) work and inverter isn’t inappropriately leaky
- Move on to Layne’s project ensuring the following composites are created as intermediates, and using the composite parts from the NAND gate where appropriate and the OmpC-RBS-GFP-Stop (saves 6 fusions)
- OmpRC-RBS-cI lambda-stop
If NAND gate doesn’t work, but everything else runs smoothly (i.e., problem isn’t in the cI inverter)
- Same protocol as if the NAND gate worked. We put that project aside and focus on light sensor based projects
If the inverter is too leaky
- Characterize some other inverters that don’t cross talk with anything in our projects (tetR if the light sensor needs reengineering?)
If the Light sensor doesn’t work (any version) or no suitable inverter is developed
- Move on to the thermometer project
Step 2 (assuming we haven’t moved to the thermometer)
- Move to Derek’s project. Only 2 additional rehydrations/transformations are required to get the parts ready, and enough composite parts are prepared and tested that around 6 fusions would be required to assemble the circuit.
- See if the cI inverter is sufficient to control the Cre recombinase expression.