Team:Aachen/Notebook/Protocols/detection

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(Measurement of Fluorescence)
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Revision as of 00:11, 17 October 2014

2D Detection of IPTG and HSL

The protocol for the chip production was developed by this year's iGEM Team Aachen with Anna as driving force and person in charge. A lot of parameters were tested and the final protocol for the production of the sensor chips the way there were used for detection in the project is published below.

Chip Production

Cell Preparation

  1. over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
  2. centrifuge all 50 mL by 3000 g for 10 min at RT (21 °C).
  3. discard the supernatant
  4. re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium.


Agar Preparation

  1. autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
  2. cool it down to 45 °C in a water bath.


Chip Preparation

  1. mix the cooled medium with the cells by inverting gently.
  2. pour it in the chip form, avoiding bubble formation (!).
  3. wait for approximately 20 min until the agar has solidified.
  4. cut out the chips with a scalpel.
  5. put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
  6. incubate two chips for 1 h at 37 °C prior to induction.
Aachen 14-10-09 flowsheet chip manufacturingV8 ipo.png
Sensor-chip manufacturing
Scheme illustrating the work flow during chip production.

Measurement of Fluorescence

For the measurement of fluorescence in our sensor chips we used three different methods. The first was done by using the Gel DocTM XR+ from BIO-RAD. There UV light was used with a exposure time of 1 s. Secondly we used our own device WatsOn. We use blue light (450 or 480 nm) and special filter. You can read even more about building your own WatsOn here. Thirdly we used the Platereader Synergy Mx from BioTek. We put the sensor chips into the lid of a normal well plate and then measured GFP with an excitation of 496 ± 9 nm and an emission of 516 ± 9 nm and iLOV with an excitation of 450 ± 9 nm and emission of 495 ± 9 nm.