Team:Aachen/OD/F device

From 2014.igem.org

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(Building your own OD/F device)
(Building your own OD/F device)
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* cuvette holder [http://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]
* cuvette holder [http://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]
* casing [ SVG file]
* casing [ SVG file]
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* Arduino Code
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{| class="wikitable centered"

Revision as of 12:00, 11 October 2014

OD/F device

Measuring Optical Density (OD) is a central element in microbiological work and synthetic biology in general. Often the qeustion is, how many cells are in a suspension. The OD can give you a hint.

Commercial OD meters cost several hundred dollars (OD meter), and can limit the spread of synthetic biology. Especially for BioHack-Spaces, DIY laboratories and schools we wanted to develop an alternative.

With our OD/F device we want to enable many to people for good, precise and cheap science.

Especially for the Interlab Study also fluorescence has been of importance. Here the correlation between OD and fluorescence should be measured. 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 with easy changes. Finally we can tell you, 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 is missing.

Also regarding fluorescence, we're not re-inventing the wheel (well, not totally). The 2010 iGEM Cambridge team actually build 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, e.g. to assess linearity of the measurement.

Development

Developing the OD/F device has been an interesting task. On the one hand, this device has been developed mainly by the computer guys. On the other hand, we got assistance from the 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!

The essential part of this deivce is the cuvette holder and has been developed in an incremental development approach. First, 3 ml have been consideres, and an optimal height of 1.5 cm from ground has been determined as optimal. Indeed, this works quite well for the large cuvettes, but unfortunately delivers random[1] results on semi-micro cuvettes. We could determine that the cause of this problem originates from refractions on the sample surface in the cuvette, as this coincedences with the measurement height.

Being naive, the computer guys first attached the sensor to the couvette holder at approximately 1.5cm, which was perfectly suited for 3ml cuvettes. However, those are not widely used anymore, and we migrated to 1.6ml cuvettes. As it turns out, if the sensor sits at 1.5cm, it just hits the position where the cuvette enlarges again - and most importantly, where the edge of the sample solution is. This results in diffraction on the surface of the sample, and finally renders any measurement results into - from a computer scientific point of view - a perfect random number generator.

However, placing the sensor very low brings problems with sedimentation as well as diffractions from the bottom. Finally we place the sensor in 0.75cm height, which, as it turns out later, is very close to one of the standard heights (0.2cm, 0.8cm, 1.2cm) of OD meters. The main problem here was also to have the sensor as close to the bottom, such that enough lights shines through for the fluorescence measurement, and to have it closer to the top, such that effects from sedimentation are reduced.

It is important to note, that despite the minimal fill heights of the 1.6ml cuvettes of 1.2ml, our device also works with fills of only 1ml, which comes closer to reality in the lab.

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

Once the cuvette holder has been finished, finding good filters was a tough challenge. The overall goal has been to choose easily available parts, which are also inexpensive. So choosing Schott glasses as filters unfortunately could not be considered. Filters, as used for illumination of theatres, seemed to be ideal. However, despite testing a lot of filters, no ideal filter could be found. The selection of filters here represents our best efforts to choose the most suitable filter. Especially for the fluorescence measurements of GFP this has been a big problem.

GFPmut3b has a peak excitation at 501nm and a peak emission at 511nm - too close for our low-cost filters. Instead we choose to excite at around 485nm, try to avoid and response lower than 500nm. However, no filter with these strict properties could be found. Finally, using the dark greenish Twickenham Green filter only little amounts of sub-500nm-light gets through, reducing any bias from this significantly. Unfortunately the transmission rate of this filter is still quite bad at 20% for the target emission wavelength. Also for the OD measurement 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!

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

  • linearity
  • true od
  • transmission -> true od

Hint: Building it

Aachen ODdevice 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:

F device

  • filter selection
  • led selection
  • resolution
  • correlation to platereader


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:

  • check prices
  • 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 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

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
1LED (600 nm for OD or 480 nm for F)~0.20
1taster5.23
1filter slide5.17
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 :) !

  • cuvette holder STL file
  • casing [ SVG file]
  • Arduino Code
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