Team:Johns Hopkins/Notebook

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Experiments

Crz1-GFP Experiment

Reason for doing the experiment: To establish whether shocking the cells induces calcium influx leading to CRZ1 nuclear localization.

Date: 21/7/10

Location: Imaging Center

Name of S. cerevisiae strain: Delta VCX1 Delta PMC1 with Crz1 tagged with GFP

Pictures and description of experimental apparatus:Our Device consisted of a 8 well plate with a cover slip bottom. In this we inserted two alluminium electrodes with brass backing, at a distance of 1mm from each other. The imaging was done on a Ziess confocal microscope.
Time interval between successive images: 2 seconds
Magnification: 100X.

Procedure:

The cells were cultured in suspension and then 100ul of this suspension was pipetted into the device between the electrodes and allowed to trickle to the bottom of the cell. A voltage of 3V was applied across the cell solution for a period of 5 minutes. The cells were being imaged with a picture taken every 2 seconds as they were being shocked. After 5 minutes a large majority of the cells have the Crz1 transcription factor concentrated in the nucleus in the cells near the live electrodes.

Representative images:

Conclusions and discussion:

From this experiment it is clear that there is a calcium influx into the cell on application of a voltage across the cell membrane. We have also established that there is a significant influx of Crz1 into the nucleus associated with this calcium influx as we expected. We hypothesis that the reason we saw Crz1 nuclear localisation only in cells very close to the live electrode because the voltage provided by the function generator was too low to reach across the gap, and so a hypothetical ground gets formed near the live electrode. Another reason may be that as our device has the electrodes at a large distance from each other, comparable x.

CDRE Optimization Experiment

Aim:

To hone in on the CDRE systems region of activation with respect to time of shocking. Also to compare yeast without PMC1 and VCX1 knocked out versus yeast with it knocked out to determine effect of the knockout on the CDRE systems expression levels.

Observations:

From the imaging of the shocked knock out yeast cells we find that the cells start expressing RFP after 90 seconds of shocking. We saw expression in all the samples between 90 and 130 seconds with a definite trend of increase. The overall expression of RFP was pretty low, however, and only a few of the total cells expressed. It was also difficult to establish cytosol localization of RFP due to clumping of cells. We saw no expression of RFP in the 0 second shock trial.

From imaging the non knock out yeast samples we found very high expression levels overall. There was constitutive expression in the negative control i.e. the 0 second shock trial, and the expression of RFP increased rapidly with increased shocking time, with maximum saturation at 50 seconds. After this its was not possible to visually distinguish weather there was any increase in expression levels. There was definite localization of RFP in the cytoplasm. In the non-knocked out samples, most of the cells expressed RFP. Another observation made during imaging was that the non-knock out cells were a lot healthier looking and bigger than the knock out cells.

Conclusions:

From this experiment we can conclude that the CDRE –RFP plasmid was successfully inserted into the PMC1, VCX1 knockout yeast strain. We can also establish that the activation of region of the CDRE –RFP system is between 90 and 130 seconds of shocking, with a definite increase in expression levels with an increase in time shocked. We can also establish that there is no constitutive expression of CDRE – RFP from the fact that there is no RFP seen in the 0 second control. The overall expression of the system is weak however and it is only after 110 seconds that more than half of the cells in the frame start to express. We also observed that the knock out cells are smaller and less healthy looking than the non knock out cells, which is probably because they are less able to deal with calcium shock induced by shocking and so more strained and unhealthy.

From the images of the non knocked out yeast we can see that there is significantly higher expression levels with constitutive expression. We also can see that the rate of increase in expression with time is much higher and the region where the CDRE gets activated is much earlier than with knocked out yeast. We theorize that the reason for this increased expression in non knocked out yeast could be because of a combination of two reasons. First, because they do not have PMC1 and VCX1 knocked they have the ability to pump calcium out of the cytosol and into vacuoles making them more resistant to calcium shock and hence more healthy and better able to express the RFP. The other possible reason is that PMC1 and/or VCX1 may have feedback loops, and as PMC1 does have constitutive expression the non knockout cells may be having their RFP expression rates amplified by this effect. We believe that a combination of these two effects causes the increased expression rates.

Results

CDRE Optimization

Control: no voltage, no RPF 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

Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. |}

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