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.:: educatETH E.coli - Biology Perspective ::.

On this page, you can find an analysis of the function of our system, its biological design, and a list of the parts that make up the system. Are you interested in constructing educatETH E.coli in your lab? Then under Lab Notes, you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested in how educatETH E.coli was modeled and simulated outside the lab, please visit the modeling and simulations web pages.


educatETH E.coli is a system which can distinguish between anhydrotetracycline (aTc) and Isopropyl-beta-D-thiogalactopyranoside (IPTG) based on a previous training phase conducted with the same chemicals and the help of AHL. It is composed of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (LacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified and extended version of the Elowitz toggle switch found in [1]. The main difference is reflected in the use of double promoters, so that the toggle switch only changes its state when both, one of the two chemicals (aTc/IPTG), and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. AHL can therefore be seen as a training- or learning substance. In the reporting subsystem, four reporters (fluorescent proteins) allow supervision of both the chemical the system was trained with and if the system recognizes the chemical it is being exposed to in the testing phase as one it has been previously trained with or not.

The Complete System

The biological design of educatETH E.coli is presented in Fig. 1

Fig. 1: Gene interaction network of educatETH E.coli
and below, we clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modeled? Then visit the System Modeling!)

Constitutive Subsystem

The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). For now, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and LacI parts behave similarly: more specifically, the TetR protein in the absence of aTc inhibits the production of P22 cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the P22 cII production is no longer inhibited (and thus p22 cII is produced). Correspondingly, cI is produced when IPTG is present.

Learning Subsystem

The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and P22 cII (which has in turn been induced by aTc). The LuxR-AHL complex induces the cI production, whereas P22 cII inhibits it. The lower part of the toggle (P22 cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the P22 cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of P22 cII and cI into the system, which in return was caused through the exposure of the system to aTc and IPTG.

Reporting Subsystem

There are four reporters in the system. CFP (more precisely: enhanced CFP, that is eCFP) and YFP (more precisely: enhanced YFP, that is eYFP) are active during the training phase of the system and show which chemical the system is exposed to during training, whereas all four reporters (the latter and GFP and RFP) are active during the testing phase and show if the system is exposed to the same chemical as in training or not. More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. The GFP production is regulated with help of two operator sites controlled by lacI and .

System Phases

The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical and reports with the same XFP as in the training phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli.

System phases
aTc IPTG AHL p22cII cI Reporting
no input no no no no no no
Trained with aTc yes no no yes no YFP
Trained with IPTG no yes no no yes CFP
Trained with aTc yes no yes yes no YFP
Trained with IPTG no yes yes no yes CFP
Trained with aTc
Tested with aTc
yes no yes yes no YFP
Trained with aTc
Tested with IPTG
no yes yes yes no GFP
Trained with IPTG
Tested with IPTG
no yes yes no yes CFP
Trained with IPTG
Tested with aTc
yes no yes no yes RFP

System Parts

educatETH E.coli consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our In the Lab page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids. A detailed list of the used parts can be found here (due to many pictures, it takes some time to load).


[1] Elowitz MB and Leibler S "A synthetic oscillatory network of transcriptional regulators", Nature 403:335-338, 2000

To Do


  • Update and correct parts in parts list. Write better in a table
  • Update and correct full system scheme
  • What is the proof of concept mentioned?
  • Are you sure about the reporters? This is not what I understood. I though YFP and CFP were also during testing. Yes! I'm still updating the text :-)
  • I don't think it is wise to make p22cII in P22 cII, since we have in the first way all over the place. Stefan: The problem is that the correct notation is P22 cII and I already used it in all the part describtions. But I could also change it back, no worries!
  • Try to improve table with system phases. It doesn't look so nice...
  • In the memorizing phase, is there color or not? - Nope, there is no color - There are colors, however they will disappear within 2-3 hours.
  • We need to put the complete spaghetti system as well.
  • Check my terminology (operator sites etc)
  • Put Stefan's updated part on epigenetics
  • Fill in table completely, make it more reading-friendly