Team:Johns Hopkins/Project

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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 binding sites of both naturally-occurring and synthetic varieties that can be integrated into promoters. Genes downstream of these promoters are thus voltage-regulated in media containing calcium.

The Plan, Visualized: In this image, the pathway we are utilizing is schematically represented in an extremely accurate fashion by blobs and arrows. The calcium channels (mid1 and cch1) are shown in blue. With electrostimulation, they admit calcium into the cell. This calcium activates the the orange protein phosphatase calcineurin which dephosphorylates the purple transcription factor Crz1p. The dephosphorylated version of Crz1p, Crz1, is rapidly transported into the nucleus where it acts on the purple and blue promoters, transcribing GFP and/or RFP. Ultimately, our system did not get as far as having two coexisting promoters, but that was the plan.

Aims

• Show that voltage can be used to stimulate a transcriptional response in S. cerevisiae.
• Develop a library of voltage-inducible elements 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 or absence of vesicular calcium pumps, 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 wanted to visualize the behavior of Crz1 under electrostimulation, and confirm localization to the nucleus upon stimulation. The cells were induced, then mounted glass plate where they were imaged with a laser-scanning confocal microscope. GFP was observed to move in and out of the nucleus just 5 minutes after a voltage stimulus. In a given image, some cells displayed Crz1-GFP densely packed in the nucleus while other cells displayed Crz1-GFP in the cytoplasm, but excluded from the nucleus. Here we see a confirmation of the oscillating behavior described by Cai et al.

In the above video we can see the oscillatory behavior of Crz1-GFP after electrostimulation. Note that Crz1-GFP's oscillating movement into and out of the nucleus is not in phase throughout the cell population. Only a few cells actually exhibit a change in Crz1 localization during the course of the video.

Optimizing Parameters for the CDRE from the FKS2 Promoter

We want to find an optimum voltage amplitude and electrostimulation time for yeast containing the CDRE-RFP plasmid. Shocking at 10V caused widespread cell death, so it was necessary to find a balance between transcription and cellular damage. 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 that optimal transcription occurrs when electrostimulation voltage is 8V. We also needed at least 40 seconds of electrostimulation to see expression.

Further Honing FKS2 CDRE

We want to hone in on the FSK2-CDRE systems region of activation in the domain of time of shocking. Also to compare yeast without PMC1 and VCX1 knocked out versus yeast with it not knocked out to determine effect of the knockout on the CDRE systems expression levels. From here we see a through characterization of the system that shows increase in expression of our reporter RFP with increase in electro stimulation time. We also see that there is much higher expression of the cells without vacuoles knocked out than in cells with vacuoles knocked out. This graph to the left shows a very clear increasing, linear relationship between electrostimulation time and expression of the RFP tagged FSK2-CDRE expression.

FSK2 CDRE with vesicular pumps, 0 seconds shocking

FSK2 CDRE with vesicular pumps, 90 seconds shocking

FSK2 CDRE with vesicular pumps, 110 seconds shocking

From the images above we can see that there is some constitutive expression of RFP in the FSK2 CDRE with vesicular pumps, but there is a very clear increase in expression of RFP with increase of electro stimulating time to 90sec. In the picture taken of the cells that were stimulated for 110 seconds, we see a large vesicle, which makes sense as the was a huge calcium influx which was relocated to the vesicle. This also explain why the cells are a lot healthier looking than the cells in which the vesicular pumps were knocked out.

FSK2 CDRE without vesicular pumps, 80 seconds shocking

FSK2 CDRE without vesicular pumps, 90 seconds shocking

FSK2 CDRE without vesicular pumps, 110 seconds shocking

FSK2 CDRE without vesicular pumps, 130 seconds shocking

From the above images we can see that there is a significant increase in the expression of RFP, which is liked to our FSK2-CDRE UAS, with the increase of time of electrostimulation, with little to no expression in the 80 second case, medium expression in the 90 second case, and high expression in the 130 second case.

FKS2 CDRE and PMC1 CDRE in Vacuole Positive and Negative Yeast

Many cells were dying from the stress of voltage shocking, so FKS2 CDRE-mCherry was inserted into yeast that still had their calcium vacuoles. These cells 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. We wanted to characterize the relationship between electrostimulation time and expression of florescent protein of the synthetic CDRE promoter sequence and the PMC1 promoter sequence. We also want to demonstrate the effect of knocking out the PMC1 and VCX1 genes in yeast. The data for both vacuole positive and vaculoe negative cells are shown below. All results are normalized against OD.

This plot shows the fluorescence intensity normalized by OD 8 hours after induction by electrostimulation. The promoter was used FKS2 UAS, and the cells had vesicular calcium pumps.

This plot shows the fluorescence intensity normalized by OD 8 hours after induction by electrostimulation. The promoter was used FKS2 UAS, and the cells were null for vesicular calcium pumps.

This plot shows the fluorescence intensity normalized by OD 8 hours after induction by electrostimulation. The promoter was used PMC1 UAS, and the cells had vesicular calcium pumps.

This plot shows the fluorescence intensity normalized by OD 8 hours after induction by electrostimulation. The promoter was used PMC1 UAS, and the cells were null for vesicular calcium pumps.

From these graphs we can see a clear linear relationship between the period of stimulation and the expression of RFP or GFP in the domain of stimulation time in which we tested. We also observed that there were significantly higher percentages of live cells in the cells in which the vacuoles were not knocked out, and the presence of the vacuoles did not have significant impact on the expression rates, aside from some constitutive expression being seen in the vacuole plus samples. The controls on all our experiments seem to indicate very high constitutive expression, but that is due to a combination of cell death due to stimulation, only a small fraction of cells being stimulated and background fluorescence and so it can be discounted.

Instrumentation

We tried several approaches to electrostimulate our cells with both accuracy and precision, but without widespread cell death. Our approaches are detailed below.

A Microfluidic Chip:

This is a schematic representation of the mask used for the fabrication of our ill-fated microfluidic device.

Consists of 300 micron-wide channels with titanium and gold electrodes to electrically stimulate the cells. This approach would allow for great precision and accuracy in electrostimulation, because each cell could be treated with exactly the same electric field. This approach would also allow for high throughput quantitation without high cell volumes (and the associated reagent, media, and time concerns), by virtue of its own low volume. We completed the fabrication of this device and are currently perfecting the fabrication technique to improve device quality and performance. Microfluidic fabrication is extremely challenging and costly and, for those reasons, we were forced to abandon this approach. For further information about our construction and techniques, see our fabrication protocol.

A homemade, aluminum foil-based electrode:
Suffice it to say that this device was sufficient for initial, qualitative tests but did not provide the consistency or usability for effective long-term use. Essentially, the device was 8 well plate with aluminum electrodes with a brass backing. This design was quickly abandoned.

An 8 well, gold-plated electrode:

This is a quick photo of our delightfully multidiciplinary final setup. As noted in the text to the left, it includes the electroporator, a 96 well plate, the function generator, and our oscilloscope. Click for a high resolution version.

Finally, we have started using a gold-plated coaxial 8 well electroporater to electrically stimulate the cells. It has shown excellent results and allows us to do large scale experimentation using 96 well plates. In the image to the left, you can see our complete and final setup. In the bottom left, the 96 well plates where we kept our cells. These 96 well plates also fit into flourometer and spectrophotometer we used to quantify our cells' response. Also in the bottom left, our 8 well gold-plated coaxial electroporator. The electroporator fits neatly into a vertical column of our 96 well plate, allowing us to easily subject multiple conditions (i.e. with and without vesicles) to identical conditions, or to run a particular condition in up to 8-fold multiplicity. On the lower right, the oscilloscope that shows the alternating current produced by the function generator In the middle right, the function generator we used to produce the voltage that induced our cells. In this image, it is set to 19 Hz alternating current.

Future Research Plans

• Finish categorizing the synthetic CDREs using our high throughput electrostimulation device.
• We also plan to apply our optimized high throughput experimentation technique to a library of synthetic CDRE sequences we have developed to characterize their voltage activation domains. We hope this library might allow others to have a more fine-grained control over the voltage response in their cells.
• We hope to develop a genetic switch utilizing the repressible operator already present on FKS2.
• We noticed a lack of iGEM compatibility with the officially supported yeast chassis. We plan to develop an iGEM-compatible plasmid for yeast with a yeast-optimized origin of replication and markers. A shuttle vector would also be useful, enabling DNA to be stored or copied in bacteria and quickly transfered for testing in yeast. Our project would not be possible in a prokaryotic chassis. We feel that iGEM's apparent focus on E. coli may hinder the development of parts and systems that require eukaryotic organisms.

References

• Cyert MS. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress. Biochem Biophys Res Commun (2003)
• Long Cai, Chiraj K. Dalal & Michael B. Elowitz. Frequency-modulated nuclear localization bursts coordinate gene regulation. Nature (2008).
• Stathopoulos-Gerontides, et al. Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. Genes & Development (1999).
• Yoshimoto et al. Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae. J Biol. Chem. (2002).