Team:Aachen/OD/F device

From 2014.igem.org

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= Building your own OD/F device =
 
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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 [http://2014.igem.org/Team:Aachen/OD/F_device#economicalview Economical View] lists all needed parts.
 
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Please find our custom parts for download below<sup>[[http://2014.igem.org/wiki/index.php?title=Team:Aachen/OD/F_device#fn2 1]]</sup>. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F device a test :) ! <span class="anchor" id="ref2"></span>
 
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* cuvette holder [http://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]
 
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* casing single device [ SVG file]
 
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* casing combined device [ SVG file]
 
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* Arduino Code for single device [http://2014.igem.org/Template:Team:Aachen/arduino_single.ino?action=raw Sketchbook (.ino)]
 
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* Arduino Code for combined device [http://2014.igem.org/Template:Team:Aachen/arduino_combined.ino?action=raw Sketchbook (.ino)]
 
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You will need a special [http://www.exp-tech.de/images/product_images/description%20images/YWRobot/1602/LiquidCrystal_I2C1602V2.rar library] for the display, which can not be uploaded for legal reasons.
 
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== Build you own device ==
 
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{| class="wikitable centered"
 
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|-
 
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| [[File:Aachen_ODF_9.JPG|300px]] || 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.
 
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|-
 
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| [[File:Aachen_ODF_8.JPG|300px]] || Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.
 
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| [[File:Aachen_ODF_4.JPG|300px]] || Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.
 
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| [[File:Aachen_ODF_3.JPG|300px]] || Your lid finally should look like this.
 
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| [[File:Aachen_ODF_11.JPG|150px]][[File:Aachen_ODF_10.JPG|150px]] || 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!
 
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| [[File:Aachen_ODF_12.JPG|300px]] || 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!
 
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| [[File:Aachen_ODF_14.JPG|300px]] || 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.
 
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| [[File:Aachen_ODF_1.JPG|300px]] || 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.
 
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| [[File:Aachen_ODF_2.JPG|300px]] || 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.
 
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| [[File:Aachen_ODF_13.JPG|300px]] || Now put everything into the case and ...
 
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| [[File:Aachen_ODF_6.JPG|300px]] || ... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.
 
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| [[File:Aachen_ODF_7.JPG|300px]] || Congratulations! You have finished constructing your own OD/F device!
 
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|}
 
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<html>
 
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<sup><span class="anchor" id="fn2"></span>1. iGEM really does not make it easy to distribute non-common files!<a href="#ref2" title="">↩</a></sup>
 
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</html>
 
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== Building the combined device ==
 
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{{Team:Aachen/Figure|Aachen_ODF_combined_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}
 
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Table 1: Needed number of pieces, components and costs for building your own OD/F device
 
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{| class="wikitable"
 
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! number of pieces !! components !! style="text-align:right" | costs [$]
 
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| 1|| [http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600#.VDfbWhY2KBk arduino UNO R3]||style="text-align:right" |11.65
 
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|-
 
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| 2|| [http://www.newark.com/ams/tsl235r-lf/light-to-frequency-converter/dp/06M3670 light to frequency converter TSL 235R]||style="text-align:right" |10.42
 
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| 1|| [http://www.dx.com/p/16-x-2-character-lcd-display-module-with-blue-backlight-121356#.VDfhkxY2KBk display 2x16]||style="text-align:right" |3.28
 
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| 2||LEDs [http://www.newark.com/avago-technologies/hlmp-c423/led-orange-t-1-3-4-5mm-170mcd/dp/06B2750 600nm] and [http://www.newark.com/cree/c503b-bcs-cv0z0461/led-blue-t-1-3-4-5mm-4-1cd-480nm/dp/04R6675 480 nm]||style="text-align:right" |0.39
 
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|-
 
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| 1||taster||style="text-align:right" |5.23
 
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|-
 
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| 1|| [http://www.prolighting.de/Sprache/en/Englisch.htm filter slide]||style="text-align:right" |5.17
 
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|-
 
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| 20|| [http://www.dx.com/p/diy-male-to-female-dupont-breadboard-jumper-wires-black-multi-color-40-pcs-10cm-339078#.VDgHqBY2KBk jumper-wire-cable]||style="text-align:right" |2.28
 
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|-
 
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| 1|| [http://www.newark.com/adafruit-industries/65/tiny-breadboard-prototype-electronics/dp/52W9088 small breadboard]||style="text-align:right" |4.00
 
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|-
 
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| 1||power supply||style="text-align:right" |5.00
 
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|-
 
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| 1 ||case||style="text-align:right" |20.24
 
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|-
 
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| 2|| [http://www.trinckle.com/en/index.php cuvettes-holder]||style="text-align:right" |15.98
 
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|-
 
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| -||odds and ends like header sockt/pins||style="text-align:right" |2.52
 
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|-
 
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| -||total||style="text-align:right" |86.16
 
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|}
 
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{{Team:Aachen/Footer}}
 

Revision as of 09:09, 14 October 2014

OD/F device

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 (OD meter), and can limit the spread of synthetic biology.

Therefore, we wanted to devenlop an alternative for measuring OD, specifically designed for Biohackspaces, 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.

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.

In fact, you can find some DIY posts for turbidity meters such as turbidity sensors. However, a proper assessment of their linearity as well as a calculated OD-value are missing.

Regarding fluorescence, we are of course not re-inventing the wheel (well, not totally). The 2010 iGEM Cambridge team actually built a very similar device, the 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.

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.

OD device

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



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: