iGEM Bielefeld 2015

All-in-One Performance

It LITERALLY works on paper!

Project aim: Achieved!

Device box
Our measurement prototype for easy and fast evaluation of fluorescence emission

We aimed at developing a cell-free test strip for the parallel detection of various substances at once. We started with many different subprojects. A lot of cloning needed to be performed, the ideal design of the teststrip had to be developed, a cell-free detection approach had to be established and the processing of the output signal had to be taken care of. So in our last weeks our main focus was to bring all this different aspects together to one working approach. And we did it successfully!

We achieved a lot during this project, just take a look at this (far from complete) list to get an idea:

  1. Highly efficient and robust cell-free synthesis of sfGFP on simple paper that can be stored. By the way, you just have to spend 16 ¢ for each reaction
  2. Cloning and characterization of multiple heavy metal sensors
  3. Cloning and characterization of a date rape drug sensor
  4. Establishment of a smartphone based fluorescence detection method
  5. Implementing of an app to easily evaluate the measured fluorescence signals.

So this is, how they actually work together!

  1. At first, you have to combine all reagents that are necessary to conduct cell-free protein synthesis.
  2. When all compounds are combined, the reaction is applied on paper and freeze-dried. This procedure prevents the reaction from premature starting. Additionally, all remaining cells are killed.
  3. The paper with the reaction is storable and transportable. For examination of water quality, you can simply add a water drop to the paper.
  4. In no time you can take a picture of the paper with your smartphone. A filter combination for your smartphone enables you to capture the fluorescence that is emitted by the reporter protein.
  5. Our app evaluates the data and tells you if your water sample is contaminated.

Heavy Metal Detection in Detail

We made it possible to detect various heavy metals with our sensor. The sensor is based on standard ligand-transcription factor interaction. We tested our sensor by using mercury and copper contaminated water. The water sample was applied to the cell-free paper teststrip, which contained the freeze dried cell-free protein synthesis (CFPS) reaction. Our sensor was able to discriminate between clean and contaminated water. This result was obtained by taking a picture of the strip with a common smartphone. The only thing one needs is our black case, filters in front of your flash and camera as well as our self-programmed app. The app quantifies the fluorescence in real-time and calculates whether a contamination is present in the sample or not. A typical workflow is depicted in this video:

Exemplary wet lab detection of Mercury

At molecular level, all our biosensors work on basis of the simple transcription factor-ligand interaction concept: The expression of a gene is regulated by a transcription factor protein. In case of a transcriptional repressor, the reporter protein (sfGFP) is not expressed unless a particular ligand is present and changes the conformation of the transcription factor. In case of a transcriptional activator, expression of the reporter protein is enhanced in presence of the ligand.

This regulation is a central process in nature. Because our system uses this concept, it is highly extensible and can be used to tackle various real world problems.

Cell free Hg sensor
Paper discs containing the cell-free standards and Hg sensor, pictured without filters.
Cell free Hg sensor
Cell-free biosensor for mercury detection in water samples. While the standard setup is strongly inhibited by Hg2+, the sensor setup strongly reacts when the metal is present.

We consider our cell-free biosensor an outstanding instrument for the detection of harmful substances in liquids.

We see great need for a storable, transportable and easy-to-use water quality test strip all around the world.

Our development can help to improve the quality of living by revealing the quality of water.

During the reaction we had measured the fluorescence in a plate reader, in order to verify the results obtained by the app. In the nearby figures you can see: The standard CFPS setup performed not sufficent when mercury was present, HOWEVER, when using our mercury detection setup, cell-free synthesis of our reporter protein sfGFP was greatly enhanced!

Water containing 6 µg/L Hg2+ strongly inhibits sfGFP expression in our standard cell-free setup. Data were acquired with Tecan plate reader. Error bars represent standard deviation of triplicates.
bar chart hg biosensor
Water containing 6 µg/L Hg2+ strongly enhances sfGFP expression in our cell-free mercury detection setup. High fluorescence signal can be measured after 60 min. Data were acquired with Tecan plate reader. Error bars represent standard deviation of triplicates.

Detection of Date Rape Drugs

With our developed biosensor we introduce an easy and unprecedented date rape drug ingredient biosensor! To detect the date rape drug ingredient γ-hydroxy butyrate (GHB), we had to adjust only two components of our setup: The repressor protein and the DNA sequence that the protein recognizes.

GHB in standard reaction
Water containing 1% (v/v) of date rape drug ingredient GHB inhibits sfGFP expression in our standard cell-free setup. Data were acquired with Tecan plate reader. Error bars represent standard deviation of triplicates.
GHB detection
Detection of date rape drug ingredient GHB with our cell-free biosensor. Data were acquired with Tecan plate reader and normalized (for details see date rape drug result page). Error bars represent standard deviation of triplicates.