Requirements Specification

Overview:

Many physiological processes have an endogenous daily periodic variation that arises from oscillations of the transcription of specific, identified master “clock” genes. To further study these circadian rhythms, a reporter construct has been made in which the sequence for the enzyme lucifease has been spliced to the Period2 gene. By measuring light generated upon cleavage of luciferin substrate by tissues of this transgenic mouse, the operation of the circadian oscillator can be assessed and its entrainment properties can be determined.

Statement of Problem:

It is necessary to obtain sensitive measurements of luciferase activity in multiple tissue samples at sufficient sampling frequencies for assessment of the phase and period of the biological oscillator. The tissue must be bathed in oxygenated medium containing nutrients and the luciferin substrate. It must be kept at the appropriate temperature and held in otherwise constant conditions. We must be able to add physiological (chemical) stimuli to the flow-through medium in order to examine effects of these drugs or hormones on the phase and period of the luciferase expression rhythm. In order to examine effects of the stimuli on the secretory functions of the tissue, we also wish to collect the output of the column at hourly intervals. Here, we need a light proof box that will fit into an incubator in order to contain the experiment apparatus. This box must contain equipment that measures the light output of each biological sample, and allow the experiment to be modified via addition of a liquid without letting light into the box. As these experiments take at least 10 days, theses devices must be extremely reliable.

Operational Description:

Inside this box, there will be two circles of vials arranged such that if one were to view it from the top down, one would see the circles. Each circle will have an angled mirror in the center to reflect the light emitted from a single mirror up to a photo-multiplier tube, or PMT.

The mirror will rotate in order to change which sample the PMT is collecting light from. Each sample will be contained in a small cylindrical column These vials will have a tube that goes in from the outside of the device, allowing them to be fed with chemicals or enzymes, as well as a tube near the bottom that allows collection of used liquid. The PMTs will be connected to a computer that will control the whole system, both setting the mirror position and reading light data. What one would have to do to use this device, is to set up the experimental matter inside standard vials, place vials in the device, connect both feed and output tubing to each vial, close device door, and then set up experiment on the computer. The computer will then run the experiment for a pre-set amount of time, summing light emission in bins of durations that could be adjusted by the programmer.

Requirement Specification:

-capability to monitor 24 samples
-ability to run at constant temperature (set at a fixed value between 35-38°C)
-must not let outside light in
-must connect to existing lab equipment for sample modification
-must collect accurate data from PMT
-variable experiment length set in software -must collect data from each sample at least once every 10 minutes
-must interface with appropriate software for analysis of period and phase of the luminescence rhythm
-must be able to collect column effluent at hourly intervals without disrupting light input or luminescence data collection

Design Deliverables:

We will deliver a working sample monitoring device along with a control computer.

Preliminary System Test Plan:

The design of one rotating mirror that can be precisely set to one of 12 positions is the first main step. This can be tested by sending commands from a computer to the microcontroller controlling the mirror, and measuring the movement. As for the light portion, we cannot test the PMTs easily, as if they are exposed to room light, they will break. We will probably have to build a simple test apparatus that consists of a cheap light detecting diode as well as some LEDs to make sure our mirror is calibrated correctly. Once that is set, our final test will most likely be an actual experiment, as there is no other good way to test the PMTs.

Implement Considerations:

The system must be able to run at 38°C, so all internal components must not have problems at that temperature. [Sometimes these experiments seem to work better at a slightly cooler temperature and at some point we may want to examine effects of varying the temperature, but it definitely won’t be cooler than room temp or hotter than 38C). Also, there must be some ventilation in the box, so the internal temperature matches the outside temperature, where “outside” refers to the temperature in the incubator. Another consideration is that each PMT retails for $1200, and our maximum budget set by our customer is $5000, so we must use the minimum number of PMTs possible. Also, the commercial application uses four PMTs, and since we want to re-use the commercial software, we may have to modify our design to do that.