Team:UT Dallas/Project1

UT Dallas iGEM 2012

Project: Toggle Switch

Introduction:

Our project is centered around creating a working toggle switch that flips between two different states when presented with certain chemicals. We started with a simple switch that utilizes two inhibitor proteins, LacI and TetR, which bind to sites on the pLac and pTet promoters, respectively. When bound to these promoters transcription is not able to proceed; any genes downstream of the promoter are effectively off. However, certain chemicals (IPTG in the case of LacI) will prevent these inhibitor proteins from binding to their respective promoters, allowing transcription of genes to continue constituively. Our design places a fluorescent and inhibitor gene downstream of one of these promoters, as shown in the diagram below. If these parts work as intended, then side 1, when running, should turn off side 2, and vice versa. By adding the chemicals IPTG or ATc, we can turn off the inhibitor proteins of one side, allowing the other side to become dominant. INSERT DIAGRAM HERE Results of Single Toggle Mechanism: Our testing consisted mostly of microscopy experiments. Cells with our designed plasmid were inoculated into a tube of broth along with varying amounts of inducer chemicals. Although our first series of experiments were for the most part unsuccessful, we were able to find concentrations of chemicals that allowed for optimal performance of our mechanism. 1-2 mM of IPTG and 8000ng/microliter of ATc were used in our most successful experiments. Some of our test results are given below. Although noise was found to be quite high, our toggling mechanism was found to be successful to an extent. Microscopy photos do show higher levels of red fluorescence when the cells are induced with IPTG and green when induced with ATc. INSERT PICTURES HERE We also used flow readings to create graphs of fluorescence levels. INSERT GRAPHS HERE Introduction to Dual Toggle: Our secondary project involved creating another toggle switch similar in nature to our first mechanism that would serve as a relationship between two different populations of bacteria. Our dual population toggle would be equivalent to our single population toggle in its use, but split up into two populations of cells. That is, the activation of one population would mean the deactivation of the other population. We hoped to achieve this by utilizing basic components as used in our single population toggle strain along with other parts that allowed the two strains to communicate with each other via quorum signalling. When one population was “deactivated” it would secrete quorum signalling molecules that would activate the creation of fluorescence proteins in the other population. The fluorescing population was considered to be in the “activated” state. In this state, inhibitor proteins would be created alongside the flueorescence proteins in order to turn off the production of signalling molecules.Similar to our single strain toggle switch, adding either IPTG or ATc would flip the switch from one state to the other by preventing the targeted inhibitor proteins from binding to their respective promoters. Future Prospects: The ultimate goal of the dual strain toggle switch was to show that we could lock a single population into either an activated or deactivated state based on the activation or deactivation of another strain. Working on the dual strain toggle gave us experience and a better understanding of quorum signalling interactions. Also, our project allowed us to develop procedures for combining two different populations. More generally, we would show that populations of different function could coordinate in an effective manner. That is, we would cause multiple populations with different functions to interact, forming one complex system that would otherwise be impossible or very difficult to generate in a single population. For example, we had in mind a counting system that would utilize a flip flop switch mechanism as well as signalling and coordination between several population of E. coli. In essence, each population would contain a similar flip flop switch mechanism. The only differences between the mechanisms of different populations would be the input and output. That is, the output of one population would serve as the input of another population. The activation or deactivation of each population could serve as a sort of binary code from which data - such as number of chemical stimulations- could be read.