Team:KU Leuven/Project/Oscillator/wetlab

We will shortly describe the functionality of the model as this is necessary to understand the resulting genetic network. Our proposed model is a network consisting of different transcription factors (TF), as is displayed in the figure. When there is a high level of TF A, this induces the production of both TF X and TF C, of which the latter will repress TF A and induce TF B. The production of TF C will not stop, even after TF A has disappeared below its threshold, since there is still TF X present that induces TF C. This effect creates an extended repression of TF A, in order to make sure the level of TF A drops sufficiently, so there is no production of TF X and TF C while the other halve of the system is active. This extended period in which TF C is present, also induces the production of TF B and the same story can be told for this halve. This means there are sequential peaks in the different components, which means this is an oscillating system.

Since we want a colony-wide synchronization, TF A has to be a quorum-sensing molecule (as well as TF B). We chose for the autoinducer synthetase (BBa_C0076) that leads to the production of the quorum-sensing molecule 3OH,C14:1-HSL. Before this gene we placed a TetR repressible promoter (BBa_R0040), so the quorum-sensing molecule is only produced if there is no TetR present. Because this is a proof of concept, we want to be able to control the production of the quorum-sensing molecule. Therefore we placed the gene coding for TetR (BBa_P0340) behind a T7 promotor (BBa_I719005). When we put the construct in a Rosetta strain, we can control the activity of the T7 promotor. This strain has a chromosomal copy of the T7 RNA polymerase gene behind a lacUV5 promotor, which can be induced by IPTG. By adding IPTG, T7 RNA polymerase will be formed, which leads to the transcription of the TetR gene so no quorum-sensing molecules will be produced.

To be active, 3OH,C14:1-HSL has to form a complex with the CinR activator. This complex can bind the promotor BBa_C0078 and activate transcription. The gene for the CinR activator (BBa_C0077) is also placed under control of a TetR repressible promotor. So if no TetR is present, CinR and 3OH,C14:1-HSL are formed and they combine to activate the promotor BBa_C0078 which is placed before the gene coding for AraC (BBa_C0080; representing TF X) and before a gene for GFP (BBa_K082003; representing TF C). So the complex makes sure that TF X and TF C are formed. In the real model TF C would be the gene for EBF, but in our proof of concept model we use GFP, which allows us to easily detect if the system works.

Our control mechanism does not only consist of TetR, but we also inserted a gene in our model that codes for the autoinducer inactivation enzyme AiiA (BBa_C0060). This enzyme can hydrolyze the quorum-sensing molecule. The gene is also placed behind a T7 promotor. If IPTG is added, no quorum-sensing molecule and receptor are formed anymore and the remaining quorum-sensing molecules are degraded by AiiA.

AraC also leads to the transcription of the GFP-gene by binding to an AraC regulated promotor (BBa_R0080). This means that not only TF A leads to formation of TF C but TF X as well. We want to prove in the lab that if TF X also activates production of TF C, the cell will fluoresce for a longer period than if TF X is excluded from the network.

As you can see on figure 2, the proof of concept of the oscillator exists of a lot of different genes. We started with creating TF C, which in our case exists of the GFP gene coupled to a promoter and a terminator. We ligated the GFP-reporter gene (BBa_K082003) to a double terminator (BBa_B0015). These form the new intermediate brick BBa_K1060006. On figure 3 you can see the insert and backbone after digestion of this new brick. Afterwards we focused on TF A, which consists of the genes coding for the auto-inducer synthetase and a receptor that binds the quorum-sensing molecule. We coupled the cinI gene which codes for the autoinducer synthetase (BBa_C0076) to the double terminator (BBa_B0015). These form the new intermediate brick BBa_K1060007.