UCSF
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* '''Strategy''' | * '''Strategy''' | ||
| - | 1. Determine the transfer function of a set of heat and cold shock promoters, where the input is temperature and the output is a steady-state (!) expression of green fluorescent protein (gfp). | + | 1. Determine the transfer function of a set of heat and cold shock promoters, where the input is temperature and the output is a steady-state (!) expression of green fluorescent protein (gfp). In essence, this will provide new parts, in the form of promoters, that respond to different temperatures. |
| - | 2. | + | 2. Use existing transcriptional inverters to process the signal from the T-sensitive promoters (obtained from goal 1). For example, if we discover a cold shock promoter with a suitable transfer function, this can be 'inverted' into a heat-shock promoter. |
| + | |||
| + | 3. Build new genetic amplifiers to increase the signal from promoters with weak or adaptive responses. This is a particular problem with heat-shock promoters, which classically produce a pulse-like response when unfolded proteins are present. Even those that have been identified as reaching a T-dependent steady-state produce a very weak signal. To boost this signal, we will construct a series of new genetic amplifiers. | ||
| + | |||
| + | 4. Link genetic devices that respond to high and low temperatures using an OR-gate. This will result in an output that is on at both extreme high and low temperatures. Further, this output can be inverted such that it responds in a narrow range of temperatures. | ||
| + | |||
| + | 5. Determine if the T-dependent genetic device can function on plates to record an applied temperature gradient. | ||
* '''Progress''' | * '''Progress''' | ||
| + | |||
| + | As it turns out, identifying suitable temperature-dependent promoters was a non-trivial task. This was particularly surprising because of the number of known heat and cold shock promoters that are available in the literature. In the end, over 40 promoters were tested. These promoters were identified using micrarray data and the known architecture of the bacterial networks that respond to heat and cold. | ||
| + | |||
| + | Each promoter was screened to determine its transfer function. The input of the transfer function is temperature and the output is the steady-state activity of the promoter. The promoters were put into a screening vector, which has a high-copy (pUC) origin and gfpmut3 as an output. It took some tweaking to obtain growth conditions that produced accurate, robust, and reproducible transfer functions. The following protocol is the one that we settled on. First, the strains are grown overnight in 5ml LB with the appropriate antibiotics. The overnights are diluted first 1:1000 into 5ml fresh LB. After reaching OD ~0.4, they are rediluted 1:100 and grown to OD 0.8. Then, aliquotes are placed at different temperature (no dilutions) and grown at different temperatuers for 4 hours. The samples are then assayed for fluorescence using flow cytometry. In practice, this protocol works because it | ||
* '''New Parts''' | * '''New Parts''' | ||
| + | |||
| + | |||
* '''Team Members''' | * '''Team Members''' | ||
Revision as of 16:46, 31 October 2005
UCSF iGEM 2005: Bacterial Thermometer
- Goal
Our goal is to build a genetic circuit that enables a lawn of bacteria to respond to a temperature gradient. Inspiration came in the form of those early 1990's T-shirts with the thermosensitive dye that change color that can record palm prints.
- Strategy
1. Determine the transfer function of a set of heat and cold shock promoters, where the input is temperature and the output is a steady-state (!) expression of green fluorescent protein (gfp). In essence, this will provide new parts, in the form of promoters, that respond to different temperatures.
2. Use existing transcriptional inverters to process the signal from the T-sensitive promoters (obtained from goal 1). For example, if we discover a cold shock promoter with a suitable transfer function, this can be 'inverted' into a heat-shock promoter.
3. Build new genetic amplifiers to increase the signal from promoters with weak or adaptive responses. This is a particular problem with heat-shock promoters, which classically produce a pulse-like response when unfolded proteins are present. Even those that have been identified as reaching a T-dependent steady-state produce a very weak signal. To boost this signal, we will construct a series of new genetic amplifiers.
4. Link genetic devices that respond to high and low temperatures using an OR-gate. This will result in an output that is on at both extreme high and low temperatures. Further, this output can be inverted such that it responds in a narrow range of temperatures.
5. Determine if the T-dependent genetic device can function on plates to record an applied temperature gradient.
- Progress
As it turns out, identifying suitable temperature-dependent promoters was a non-trivial task. This was particularly surprising because of the number of known heat and cold shock promoters that are available in the literature. In the end, over 40 promoters were tested. These promoters were identified using micrarray data and the known architecture of the bacterial networks that respond to heat and cold.
Each promoter was screened to determine its transfer function. The input of the transfer function is temperature and the output is the steady-state activity of the promoter. The promoters were put into a screening vector, which has a high-copy (pUC) origin and gfpmut3 as an output. It took some tweaking to obtain growth conditions that produced accurate, robust, and reproducible transfer functions. The following protocol is the one that we settled on. First, the strains are grown overnight in 5ml LB with the appropriate antibiotics. The overnights are diluted first 1:1000 into 5ml fresh LB. After reaching OD ~0.4, they are rediluted 1:100 and grown to OD 0.8. Then, aliquotes are placed at different temperature (no dilutions) and grown at different temperatuers for 4 hours. The samples are then assayed for fluorescence using flow cytometry. In practice, this protocol works because it
- New Parts
- Team Members
Faculty Advisors
Chris Voigt
Tanja Kortemme
Graduate Students'
Liz Clarke (Voigt Lab)
Matt Eames (Kortemme Lab)
High School Students
Apple Liu (SFUSD Mission High School)
Nessa Ramos (SFUSD Mission High School)
