Team:Minnesota/Project
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The Project
We studied the potential of a synthetic, single promoter AND gate. This helps us understand at a fundamental level what is happening in a network with multiple regulators and opens further research paths. The device consists of parts of the Tet (tetracycline) and Lac (lactose) and responds to commonly used inducers IPTG (Isopropyl β-D-1-thiogalactopyranoside) and aTc. Three or fewer of these operators can be combined to form a promoter and the order and quantity of each operator site result in different constructs, that is, given three blank spots for operators on a promoter and the choice of either -, T or L for each spot, there are a variety of possible promoter designs. We chose to focus on four constructs:
- T T L
- T T -
- T - -
We also examined 4 mutations in the Tet operator site for each construct.
The Experiments
Results
Protocols
These are the standard techniques that we used in the wet lab:
Bacterial Culture
Sterile Technique
- Always work around a flame or in the hood
- Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping
- Sterilize metal instruments between uses by dipping in 100% ethanol and flaming
Bacterial Culture Maintenance
Culture cells:
- At 36 degrees Celsius
- Shaking at 220 rpm
- At 10% total flask/tube volume
- In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x108cell/ml)
Bacterial Culture For Gene Expression Experiments
- Pick and individual colony from a plate and inoculate 2ml LB + amp media
- Incubate overnight at 37 C, shaking at 220 rpm
- Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture
- Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)
- Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture
- Continue cultures as described above in "bacterial culture maintenance" for 9 hrs
- Isolate cell samples from cultures at 3, 6, and 9 hour time points
- Remove 100ul sample aliquots from cultures
- Pellet samples at 5K rpm for 5 minutes
- Remove supernatant
- Wash cells with 1 ml chilled 1xPBS, pH 7.6
- Resuspend cells by vortexing
- Re-pellet cells at 5K rpm for 5 minutes
- Remove supernatant
- Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)
- Incubate at RT for 30 minutes
- Pellet cells at 5K rpm for 5 minutes
- Remove supernatant
- Resuspend cells in 1 ml 1xPBS
- Store samples at 4 C until analysis by flow cytometry