Team:Nanjing-China/project

Our project is to construct a system to perform tasks like amplifying expression and quantitatively detection. The system is consisted of several sections such as the QUANTITIVE DETECTOR, the accelerating expression section, the SUICIDE SYSTEM and the TIMER. Our design is modularized, replaceable, and efficient. The whole system can solve problems such as detection and degradation of toxins, enhanced expression of specific protein, timing of experiments and quantitative measurement of specific molecule.

The modularity means sections of our system can work separately as well as together. So we can carry on rather different jobs by combination of our sections. The convertibility means by changing the promoter of the detection object and the protein gene that needs to be amplified, we can detect different pollutants and amplify expression of different proteins. With the introduction of positive feedback in accelerating expression section we can work more efficiently by producing much more protein.

Here we will demonstrate our project by applying our design to detection and handling of toxins. The project is based on three kinds of escherichia coli and five plasmids. They constitute two major sections, the detector section and dispose section. We also have some auxiliary sections such as suicide section and the timer.

There are plenty of highlight in our project. First of all, we figure out different means to detect toxins. We not only find toxin promoters to detect them, but also find the method of designing riboswiches for toxins without corresponding promoters to sense them. Secondly, in the processing section, we use positive feedback circuit, which can enhance the expression of our processing proteins. What is more, to process toxins, we also think of two ways, one of them is using degrading enzymes; the other is combining it with proteins for gathering. We give the detection and disposing of microcystic and gold ion as two examples of this section. Thirdly, since we really care about the safety of this non-natural E. coli, we design a plasmid to let them commit suicide after mission accomplished. In addition, we also find a new way to stop the horizontal gene transfer of these recombinant plasmids. Last but not the least, we also designed a E. coli timer to facility our quantitative detection.

Quantitative measurement of specific toxins depends on principles of quorum sensing [1]. We construct this section by locating sender cells in the center of the plate and covering the rest of the plate with receptor cells. The sender cells will give out AHL according to concentrations of toxins. At the specific concentration of AHL, the receptor cells will send out red fluorescence.

A toxin will induce specific sensor to start the expression of downstream gene. As a result, luxI will be produced and will catalyze the generation of AHL. AHL, a signal molecule in quorum sensing, will begin to diffuse, forming a gradient of it through the whole plate [2].

AHL will be obtained by receptor cells and different cells will feel different concentrations of AHL, and then give different results. The receptor cells produce luxR in a constant level, which means that the coming AHL will combine to luxR immediately when it comes in receptor cells. The combination will induce Prlux to start to produce cI and lacI. cI is an inhibitor of PcI, which will help to control the level of lacI. At last, different lacI quantities, which will repress the activity of Plac at different levels are generated when the quantities of AHL felt by reporter cells are different.

As AHL is sufficient near the sender cells, Prlux will promote the production of lacI dramatically, almost completely blocking the expression of rfp through Plac.

Outer places with low concentration of AHL are nothing better. The lack of cI will leave PcI in an activation state, producing a huge amount of lacI in result, blocking production of rfp as well.

Only receptor cells in the middle will get suitable concentrationof AHL, producing less lacI than enough to repress Plac, and enough cI to inhabit PcI to produce lacI. As a result, receptor cells on the plate in the middle position will turn red after cultivation and the distance between the red circle and the circle center will represent the concentration of toxin quantitatively.

Using these designs, we will be able to measure concentrate of toxin in solutions, and see the results clearly.

Our system will produce specific enzyme or other binding protein to cut down on the pollutant on seeing it. To make the process faster and stronger, we introduce a circuit of positive feedback, which will magnify the effect dramatically.

This section will continuously produce luxR through Pcons2, accumulating an enough amount. When the toxin appears, it will activate specific sensor to produce luxI, which can produce a lot of AHL. AHL will combine with luxR, start the work of Prlux, producing both the aim protein which can deal with the toxin and luxI. luxI plays a major role in positive feedback, making AHL and enhancing the activation of Prlux. There may be different constructions of positive feedback, but the results of modeling and experiments prove that the feedback of luxI is the best one.

One of the specific toxins we used as example is microcystin, and to dispose of it, we introduce proteins related to the degradation of it, such as MlrD and MlrA. The former one can transport microcystin into cells and the later one serve as the degrading enzyme of it.

Our system can also be used to deal with metal ions, and we choose the gold ion as an example here. To accomplish such an aim we use PgolTS-golS-PgolB and OMPA-golB. PgolTS works as a constant promoter, producing golS which inhibit PgolB. If gold ions combine with golS, PgolB will not be stopped. So, PgolTS-golS-PgolB will work as a sensor of gold ions. We can put it in the position of P to complete our detector section. OMPA is a protein that locates on the membrane and golB is a gold ions binding protein. OMPA-golB can work as a gold disposing protein in our handling section.

To see functions of different device, click Parts

For the consideration of biosafety, we introduce suicide section in all of our cells. The section will kill our cells if there is no toxin or lactose, making them hard to survive in outdoor environment.

If there is some toxin, tetR will be produced by the promoter, inhibiting the production of lysozyme by PtetO. When there is enough IPTG but no toxin, IPTG will also start the Plac to produce tetR, blocking accumulating of lysozyme, which can facilitate the culture of our engineering bacteria. In situations without both toxin and IPTG, the engineering bacterial will die. When in use, the cells will be put in environment lacking IPTG, which means they will die after finishing their work, leaving no potential threats on the environment. The bacteria will be cultivated in environment with IPTG for storage and propagation.

To detect and dispose of more kinds of toxin, we produced some riboswitches from aptamers in the papers [3]. Using ways of modeling and experiment, we select some potential microcystin riboswitches to work as our sensors for MC.

The riboswitch is composed of a hammerhead ribozyme and an aptamer. In the picture above, the brown division is the aptamer while the remainder is the hammerhead ribozyme. In some formation the hammerhead ribozyme may degrade the RNA. However, the aptamer may combine with corresponding toxin and change the structure of the ribozyme, and then block its cleavage. So, with the toxin, the degradation rate of mRNA is lower, and more proteins will be produced. The following pictures are two formation of one of our riboswitches. The former is the formation combining with ligands, while the latter is without ligands.

The timer is designed to tell the time of observance, which can not only make the quantitative detection more acute, but also make it easier for modeling. However, because of the time limit, we only finished the modeling work of the timer. Even so, the timer can be a helpful auxiliary section for our project, since by waiting for specific amount of rfp, we can estimate when is the time to see the distance between the red circle and the center. To see more details, click Modeling.