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)
- Obtain Plasmids
- pEXPR: hEF1a-GAL4VP16
- pENTR: TRE (promoter)
- pEXPR: TAL14-mKate
- Troubleshoot
- So where are the siRNA though... (keep looking around, they were ordered)
- Why didn't the GH golden gate work on try #3?
- Retransform products at higher concentration
- Fill out the Genewiz Order Form to sequence Low Sensor miR-144
- Design primers to sequence high sensors
- Ask Jeremy about sequencing primers
- Look into siRNA transfection
- Re-analyze all of our data in MATLAB (Gary)
- Learn how to acquire MATLAB from Brian
- Find natural concentrations of relevant miRNAs.
miRNA Sensors (Lab Work)
- TOP PRIORITY: SEQUENCE LOW SENSORS (can't be done till primers arrive)
- Pick colonies
- pEXPR: TAL14-mKate
- LR
- pEXPR: MAV1212-TRE-L7ae
- Transfection
- L7ae experiment
- Golden Gate
- High Sensor miR-30d (I13 & J13)
- High Sensor miR-146a (I5 & J5)
- High Sensor let-7f (1.13 & 1.14)
- High Sensor miR-125b (A3 & B3)
- Combined Low Sensor miR-144-miR-181c
- Get ultramers from Brian
- Anneal target sites
- All high sensors
- Transformation
- Re-do Low Sensor miR-181c
- Midiprep
- pEXPR: TAL14-mKate
- 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 NOTES