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Team:Johns Hopkins/Project
From 2010.igem.org
Contents |
Abstract
If the goal of iGEM and the Parts Registry is to take the messy world of genetic engineering and transform it into something like the standardized world of electrical engineering, it may be useful if electronic systems could directly interface with biological systems. Past iGEM projects have used chemical or optical stimuli to actuate transcriptional responses. Our project, however, seeks to add voltage sensitivity to Saccharomyces cerevisiae. Baker’s yeast was chosen because in some sense yeast have a system that responds to voltage input. With a voltage stimulus one can open the voltage-gated calcium channels of yeast, causing calcium ions to rush into the cytoplasm. This causes calcineurin to dephosphorylate Crz1, which enters the nucleus and binds various promoters. Our group presents a library of characterized Crz1-sensitive promoters of both naturally-occurring and synthetic varieties. Genes downstream of these promoters are thus voltage-regulated in media containing calcium.
Aims
- Show that voltage can be used to stimulate a transcriptional response in S. cerevisiae.
- Develop a library of voltage-inducible promoters with differing voltage response curves.
- Determine the functional range of and optimized values for our system with respect to the following variables:
- Voltage applied
- Duration of voltage application
- Presence of vacuoles, yeast’s natural mode of intracellular Calcium control
- Develop an effective experimental apparatus to apply voltage and measure response.
Methods
Visualizing the Crz1 Transcription Factor
We want to characterize the electro stimulation voltage domain in which we see movement of the GFP tagged CRZ1 transcription factor into the nucleus. Crz1-GFP was grown and passed into two rows of a 96 well plate. The cells were then shocked every two hours at 10 Volts. The cells were passed to a glass plate where they were fixed and then imaged. GFP was observed to move in and out of the nucleus just 5 minutes after voltage stimulus. In a given image, some cells displayed GFP densely packed in the nucleus while other cells displayed GFP in the cytoplasm, but excluded from the nucleus. Here we see a confirmation of the oscillating behavior described by Elowitz et al (Elowitz 2008).
Optimizing Parameters for the CDRE from the FKS2 Promoter
We want to find an optimum voltage amplitude and electro stimulation time for yeast containing the CDRE-RFP plasmid. Shocking at 10 Volts caused large amount of cell death, as such, it was necessary to find an optimal voltage to cause transcription, but not to damage our cells. CDRE-mCherry were grown and passed into 96 well plate. The electroporator was used to shock the cell with voltages from 2-10 Volts with an exposure time from 0-80 seconds. We find optimum electro stimulation voltage is 8V and we need at least 40 seconds of electrostimulation to see expression.
| 2s | 5s | 10s | 20s | 40s | 80s | |
|---|---|---|---|---|---|---|
| 10V | None | None | None | None | None | N/A |
| 8V | None | None | None | None | High | High |
| 6V | None | None | None | None | Moderate | High |
| 4V | None | None | None | None | Low | Low |
Further Honing FKS2 CDRE
FKS2 CDRE in Vacuole Positive Yeast
A large amount of cells were dying from the stresses of voltage shocking, as such, a FKS2 CDRE-mCherry was inserted into yeast that still had their calcium vacuoles. Yeast were grown and passed into two rows of a 96 well plate and shocked at 2-10 volts with an exposure time of 0-80 seconds.
Results
Visualizing the Crz1 Transcription Factor
GFP was observed to move in and out of the nucleus just 5 minutes after voltage stimulus. In a given image, some cells displayed GFP densely packed in the nucleus while other cells displayed GFP in the cytoplasm, but excluded from the nucleus. Here we see a confirmation of the oscillating behavior described by Elowitz et al (Elowitz 2008).
Optimizing Parameters for the CDRE from the FKS2 Promoter
We found that exposure to a stimulus of 8V for over 40 seconds produced the strongest response. Our results are summarized below.
| 2s | 5s | 10s | 20s | 40s | 80s | |
|---|---|---|---|---|---|---|
| 10V | None | None | None | None | None | N/A |
| 8V | None | None | None | None | High | High |
| 6V | None | None | None | None | Moderate | High |
| 4V | None | None | None | None | Low | Low |
First, observe that the channels are inactive for the lowest stimulus durations. Some of these durations are on cellular diffusive timescales, so not enough calcium can enter the cells. Also, cells can quickly move to extrude the excess calcium.
Second, consider the channel mechanics. Calcium channel gates alternate between open and closed states based on first order kinetics, where the rate constants are exponential functions of voltage. The open probability curve increases rather steeply, made even steeper since having n channels raises the probability curve to the nth power. This curve was found to rise rapidly between 4V and 8V to a maximum around 8V. At 10V, however, shorter stimulus durations are insufficient to trigger calcineurin, but longer durations kill the cells.
Further Honing the FKS2 CDRE parameters
Conclusions
Future Research Plans
References
- Long Cai, Chiraj K. Dalal & Michael B. Elowitz. Frequency-modulated nuclear localization bursts coordinate gene regulation. Nature (2008).
