Long Term Goals
miRNA Profile
- Read literature
- Choose profile
Building the Cell Detector Circuit
- Build Choose profile
- Verify sensors
- Create mathematical model
- Make test bed
- Controlled miRNA expression
- Constructed miRNA sensors
Mini experiments
- Single miRNA detector circuit
Experiments
- L7ae repression
- TAL14 repression
- TAL21 repression
- Single input sensors
- Multiple input sensors
Alzheimer's Model
- Agree on a team wide, full Alzheimer's model
Today's Agenda
How To Please Ron/Brain
- Keep updating the parts tree (include experiments)
miRNA Sensors (Theory)
- Troubleshoot
- Why didn't the oligos nanodrop??
- Why didn't the primers bind to LS_miR_144?
- Learn how to acquire MATLAB from Brian
- Find natural concentrations of relevant miRNAs.
miRNA Sensors (Lab Work)
- Golden Gate
- Low Sensor miR-144-miR-181c
- Transform
- High Sensor miR-30d
- High Sensor miR-146a
- High Sensor let-7f
- High Sensor miR-125b
- Low Sensor miR-144
- Low Sensor miR-181c
- Pick colonies to miniprep and midiprep
- pEXPR: MAV1212-TRE-L7ae (LR)
- pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
- pEXPR: MAV1212-TRE-GAL4VP16 (LR)
- Prepare and run a gel (Hyperladder 2 and 4% agaros gel w/ annealed target sites)
- High Sensor miR-30d TS
- High Sensor miR-146a TS
- High Sensor let-7f TS
- High Sensor miR-125b TS
- Low Sensor miR-144 TS
- Low Sensor miR-181c TS
- FACS cell prep
- Transfect L7ae Experiment
- Analyze FACS data
NOTES
- Midi-prep hEF1a:rtTA and hEF1a: Gal4VP16 2A rtTA3
- Mini-prep MAV1212 hEF1A Tal14
- Digest and Map MAV1212 hEF1A Tal14
- Transform third LR trial if not verified
- Pick/Streak hEF1a: Gal4VP16 2A rtTA3
NOTES:
hEF1a: Gal4VP16 2A rtTA3 culture did not grow so We have a new primer to sequence with (again, from Jeremy)