Team:Aachen/Notebook/Engineering/ODF

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= OD/F device =
 
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Measuring Optical Density (OD) is a '''central element in microbiological work and synthetic biology'''. One question that has to be answered often is how many cells are in a suspension. Here, the OD can give you a hint. Unfortunately, commercially available OD meters '''cost several hundred dollars''' ([http://www.laboratory-equipment.com/laboratory-equipment/cell-density-meter.php OD meter]), and can '''limit''' the spread of synthetic biology.
 
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Therefore, we wanted to devenlop an alternative for measuring OD, '''specifically designed for BioHack-Spaces, DIY and community laboratories and schools'''. With our OD/F device, we want to enable many people to do good, precise and inexpensive science research.
 
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Especially for the [[Interlab Study]] fluorescence, too, has been of importance. One aim of this study was to measure the correlation between OD and fluorescence. Since the taks of measuring OD and fluorescence are often performed at the same time, we want to present a device that can '''measure both fluorescence and OD''' with just some easy adjustments. This way, we can measure how much fluorescence there is per amount of cells.
 
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In fact, you can find some DIY posts for turbidity meters such as [http://www.thingiverse.com/thing:74415 turbidity sensors]. However, a proper assessment of their linearity as well as a calculated OD-value are missing.
 
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Regarding fluorescence, we are of course not re-inventing the wheel (well, not totally). The [http://2010.igem.org/Team:Cambridge 2010 iGEM Cambridge] team actually built a very similar device, the [http://2010.igem.org/Team:Cambridge/Tools/Eglometer E.glometer]. However, there's no data available showing an actual comparison of the data from their device and some proven commercial system to, for example, assess linearity of the measurement.
 
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== Development ==
== Development ==

Revision as of 20:12, 13 October 2014

Development

Building the OD/F device has been an interesting task. On the one hand, this device has been developed mainly by the IT division of our team. On the other hand, we got assistance from biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs. For the next year, you really have to select carefully who's going to help with which task!

Cuvette Holder

The essential part of this device is the cuvette holder which has also been the most tricky thing to design. In short, we had to overcome a dilemma created by the need for an optimal height for the sensor:

  • A too low sensor position bears problems with sedimentation as well as light diffraction from the bottom of the cuvette.
  • The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.

As a compromise, we place the sensor at a height of 0.75 cm, which, as it turned out later, is very close to one of the standard heights (0.2 cm, 0.8 cm, 1.2 cm) of OD meters. It is important to note that despite the official minimal fill height of 1.2 mL of the 1.5 mL cuvettes we used, our device also works with filling volumens of just 1 mL which in fact comes closer to reality in the lab.

The final cuvette holder design was rendered in a stl-file shown below:

Light filters

Once the cuvette holder was finished, finding good filters was a tough challenge. A main goal throughout our project has been to choose easily available parts which are also inexpensive. Thus choosing Schott glasses as filters unfortunately could not be considered. Instead, filters used for illumination of theaters seemed to be ideal solution.

Especially for the fluorescence measurements of GFP finding the right filter has been a big problem. GFPmut3b has a peak excitation at 501 nm and a peak emission at 511 nm - too close together for our low-cost filters to block the excitation light but transmit the emitted light. Thus, we chose to excite at around 485 nm reduce false positive results below 500 nm. However, no adequate filter for these settings could be found. Eventually, using the dark greenish Twickenham Green filter only little amounts of light shorter than 500 nm got through, reducing any bias from excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, 20 % only, for the target emission wavelength of 511 nm.

For the OD measurement, too, we had similar problems. The solution to this problem is presented in the F device section.

1. Quite a good random number generator from a computer-scientific perspective!

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Setup of the measuring unit
On the left, the setup of a classical spectrophotometer is depicted. The setup around the cuvette holder of our device is shown on the right.

Combined Device

Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing. All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino. Right at the bottom we present you the differences in wiring things up.

Linearity

As for any scientifc device it is crucial to question the results one gets from the device. To ensure that our device actually works, we performed a set of measurements which are presented below.

It is crucial that the selected hardware is mapping reality into the digital world of our $\mu$-Controller. In order to sense reality our setup uses a light to frequency sensor, TSL235R-LF. The light to frequency sensor resembles the most to a photo transistor and thus is less sensible to temperature than a light dependant resistor. Additionally counting a frequency using interrupts seems to be easier and more accurate than using the analog to digital converter.

Using a dilution series of purified [ iLOV] we could determine the characteristic curve for the light sensor. Finally we can conclude that the sensor is linear as expected and shown in the datasheet.

Interlabstudy overview wiki.png
Interlab Study Results
Our measurements of fluorescence and optical density of the three genetic devices and a negative control.

OD device

Aachen 14-10-09 Flowsheet OD-device ipo.png
How to use our OD/F device
XXX


From Transmittance to True Optical Density

At very low levels, uncorrected photometric determinations of cell densities show a decreasing proportionaility to actual cell density.

This can also be observed using our OD measurement device.

In general, photometric determination of bacterial concentrations depends primarily on light scattering, rather than light absorption. Therefore. often not absorption is measured, but transmittance. For this, the relationship between optical density (OD) and transmitted light $\frac{I_0}{I}$ exists as:

$$ OD = \frac{I_0}{I} = \kappa \cdot c$$

However, this equation is linear only in a certain range. While one can tackle this non-linearity by using dilutions of the culture, correcting the error systematically is another way to overcome this limitation.

For our OD device we needed to correlate the transmittance measured by our sensor to an optical density anyway. Our team members from the deterministic sciences emphasized on the correction method, which was conducted according to Lawrence and Maier [1]:

  • The relative density ($RD$) of each sample in a dilution series is calculated using $\frac{min(dilution)}{dilution}$.
  • The uncorrected optical density is derived from the transmission T [%]: $OD = 2 - \log T$
  • Finally, the unit optical density is calculated as $\frac{OD}{TD}$.
  • The average of the stable unit optical densities is used to calculate the true optical density $ OD_{unit} \cdot RD $.

This way, the correlation between transmission and true optical density can be computed. The derived function allows the conversion from transmission to optical density on our device and therefore calibrates our device.

Lawrence and Maier could show that correcting transmittance this way, the corrected optical density shows a linear relationship between true optical density and dry weight in cell suspensions.

In our experiments, we find that different cell types have a different correction function. While this at first sight looks disappointing, it should be expected: Transmittance is the fraction of light coming through some medium relative to the cell-free and clear medium. However, the transmittance is not only depedent on the amount of cells in the way of the light beam but also on the cells' shape, their size and possibly also their cell membranes.

[1] Correction for the Inherent Error in Optical Density Readings, Lawrence, J.V. and Maier, S., Applied and Environmental Microbiology, 1977, p. 482-484

DIY: How to Build Your Own OD Device

Aachen ODdevice Steckplatine.png
Breadboard of our OD device
To build your own OD device, connect the parts as shown in this diagram.

If you want to build our OD device, make sure to use the following secret ingredients:

F device

Similarly to the OD measurement, the fluorescence is measured using the same cuvette holder. In fact, if one does not build a combined device, the only thing one is supposed to change is the cuvette holder. However, as for optical density measurement, a filter needs to be placed between led, sample and the light sensor. Selecting the filter has been troublesome. Either the tried filters had a good transmittance but did not screen for the correct wavelength, or they screened for the correct wavelength but showed bad transmittance. Finally we chose the [ Twickenham green] filter with bad transmittance, and raised the sampling interval from 1 s to 4 s to allow a distinct signal. This is by far not optimal, but delivers stable and reliable results.

For fluorescence measurement we luckily are not that much relying on the optical density of the cell culture to measure (if the sample contains cells at all). We compared the values of our device against the [Team:Aachen/LabDevices#platereader platereader].

Evaluation

Figure 1 shows the absolute measurements for both the platereader and our OD/F device. The abrupt jump at 50% concentration can be explained by a second dilution step and is prevalent in both devices. It can be seen that the platereader show a much higher difference between the GFP and non-GFP cell culture at a higher standard deviation. Another interesting metric is the difference between the GFP and non-GFP, which can be seen as the normalized fluorescence measure.

If one compares the results there, as in Figure 2, interesting observations can be made. First, both platereader and OD/F device show very similar results. The regression curves differ only in a linear factor. Most interestingly general fit of the OD/F device to a linear function seems to be better than with the platereader. Overall the linearity which has been observed earlier (in testing the general setup) could be verified. Therefore our do-it-yourself OD/F device can be used to determine fluorescence.


Hint: Building it

Aachen Fdevice Steckplatine.png
Our novel biosensor approach
Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.

If you want to build the OD device, make sure to use the following secret ingredients:

Economical View

TODO:

  • what does the market offer, what does the market not offer
  • what is the closest available device to ours and what does it cost? where is it possibly better? where is ours better?
  • how easy is it to get the parts?


Table 2: Needed number of pieces, components and prices for creating your own OD or F device

number of pieces components costs [$]
1 arduino UNO R311.65
1 light to frequency converter TSL 235R5.71
1 display 2x163.28
1 LCD display to I2C 1.99
1LED (600 nm for OD or 480 nm for F (but any LED should do))~0.20
1pushbutton5.23
1filter slide2
20 jumper-wire-cable2.28
1 small breadboard4.00
1power supply5.00
1 case20.24
1 cuvettes-holder7.99
-odds and ends like header sockt/pins2.52
-total85.16


Building your own OD/F device

While the casing and the cuvette holder are custom made, most of the parts are pre-made and only need to be bought. The previous section Economical View lists all needed parts.

Please find our custom parts for download below[1]. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F device a test :) !

You will need a special library for the display, which can not be uploaded for legal reasons.

Build you own device

Aachen ODF 9.JPG First we want to assemble the casing. Once you have all the cut parts, you can start to assemble them. For cutting, we really recommend using a laser cutter.
Aachen ODF 8.JPG Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.
Aachen ODF 4.JPG Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.
Aachen ODF 3.JPG Your lid finally should look like this.
Aachen ODF 11.JPGAachen ODF 10.JPG Next we want to assemble the cuvette holders. On the side with the square hole attach the light-to-frequency sensor with glue. For the OD case place the orange LED opposite, or for fluorescence, the LED in the hole in the bottom. Make sure to close any remaining open hole!
Aachen ODF 12.JPG Your final assembly should then look like this. Now place the correct filter into the cuvette holder, directly in front of the sensor. Make sure that the filter does not degrade due to the glue!
Aachen ODF 14.JPG As the case can be used for both, fluorescence and OD measurement, we use a combined plug. Just three header rows (7 pins) and connect them as we did.
Aachen ODF 1.JPG Now we're doing the wiring. Connect the Arduino 5V and GND such that you have one 5V and one GND line on your breadboard.
Aachen ODF 2.JPG Then connect the button to 5V on the one side, and to GND via a resistor on the other side. Connect this side also to port __ on your Arduino. This will sense the blank. Next connect the display to the Arduino and our connector. See the Fritzing diagram at the bottom for a detailed information.
Aachen ODF 13.JPG Now put everything into the case and ...
Aachen ODF 6.JPG ... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.
Aachen ODF 7.JPG Congratulations! You have finished constructing your own OD/F device!

1. iGEM really does not make it easy to distribute non-common files!

Building the combined device

Aachen ODF combined Steckplatine.png
Our novel biosensor approach
Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.

Table 1: Needed number of pieces, components and costs for building your own OD/F device

number of pieces components costs [$]
1 arduino UNO R311.65
2 light to frequency converter TSL 235R10.42
1 display 2x163.28
2LEDs 600nm and 480 nm0.39
1taster5.23
1 filter slide5.17
20 jumper-wire-cable2.28
1 small breadboard4.00
1power supply5.00
1 case20.24
2 cuvettes-holder15.98
-odds and ends like header sockt/pins2.52
-total86.16