Team:CIDEB-UANL Mexico/Project-Parts

The agriculture is very important because from this activity we get our food and alimentation; but also, there exist the, normally of insects, and they can affect the agricultural products. An answer for this problem is the use of pesticides but there are negative consequences for their use in the ground that is supplied and to the nature. The potato crop is the culture that we are interested to work with and the plague that affects it is the worm white. The reason is because this worm is a harmful pest that can affect the roots of the plant and even it can destroy it. We want to eliminate this problem in secure conditions with a system that can destroy the plague without harm the crop and only affect the insect.

In our system, we are contemplating to avoid this problem with a pesticide that it is going to be produced in specific conditions of temperature. It is going to be a modified bacterium, in this case we will be working on E. coli, that it is going to be releasing the pesticide (Vip3Ca3) regulated by temperature related to the optimum temperature in which the worm grows. We constructed a model of a circuit that will be finally inserted into the bacteria. In order to do so we divided it in two main parts:

The first part is about a regulable promoter the one that is repressed by the cI lambda (in the parts registry this promoter is named as R0051), linked to a rbs and followed by the Vip3ca3, ending with another rbs. This part did not exist prior to the registration of parts, for this reason we have considered to insert it as a new part, by converting it into a bioBrick with the standard cutting sites. That’s why it was requested to be synthesized for a more practical use, and not found marketed directly. After the rbs we will assembly manually a reporter to notice that the production of the Vip3Ca3 is actually occurring. In this case we will use the GFP reporter with degradation tag LVA in order to see if the Vip3Ca3 production is actually occurring. Later we are going to explain a little more about the degradation tag.

The second part has of a constitutive promoter and a riboswitch that will regulate, by temperature, the Vip3ca3 production. After the riboswitch the gene that will be expressed is the Vip3Ca3, then a RBS in order to add a repressor after this. It was decided to synthesize these parts for the assembly because it was more practical. After the synthesized parts, we are going to assembly manually adding the repressor lamba cI (In the parts registry is located as the C0051).

Two important aspects of our circuit are the riboswitch and the Vip3Ca3 protein.
Riboswtich: this functions as a RBS but it activates transcription at higher temperatures of 20ºC having its better production at 37ºC. As we needed the inverse of this function we use a set of promoter repressor to stop the production at 20ºC.
Vip3Ca3: this is a new protein. It is not found in the partsregistry so we asked it synthesized. To know more information about this protein you can see an article in the safety section.

One system is going to be repressing the other one with the riboswitch when the specific range of temperature is from 20oC to 37oC, but when the temperature is lower to this interval the first system is going to be off and then the second system is going to be active with the production of the pesticide (Vip3Ca3) and the GFP. When the temperature is low, the promoter (J23100) is going to inhibited and then the first system it’s going to shut down. Then the second system is activate it and the production of the pesticide (Vip3Ca3) is going to be active and also the GFP, that it is a mode to notice if it is producing the pesticide. As we know, the part that has the GFP reporter will be repressed after the 37ºC so it needs a degradation tag to reduce the fluorescence. Our circuit will be inserted into E. Coli. We had the idea of placing the bacteria in a gellan bead, like the team of Bristol 2010(https://2010.igem.org/Team:BCCS-Bristol), this is a way to enclose our bacteria and to maintain it safe from the environment and another things that could represent a risk for them and it help in bio safety because, the bacteria would not be in direct contact with the crops. Also this helps to extend the life period of the bacteria.

To spread our bacteria in the field, we have a mode of a robot constructed by our team, to develop this task, and make more efficient the work. This has the purpose to take advantage of the ground conditions for the spread of bacteria. You can see a little more about this model of the robot in the software section.

Our goal is to have another way to combat or eliminate the problem of the plagues that in this case is the worm white in the crop potato in a secure way which controls the system regulating the production. This is with the objective to avoid:

• Overproduction
• As the Vip3Ca3 can be broken down after certain time there is no problem of bioaccumulation in species.
• The Vip3Ca3 does not harm the environment (there is no contamination).
• The pesticide protein is not in the plant, it is about of the plant but in the ground.
• This system is regulated by temperature because it was better against the white worm life cycle but it can be regulated by different intervals of temperature (with different riboswitch). Also we can inverse the activation of the system by only using the part with the Vip3Ca3 without the promoter-reppresor set.

One of the purposes of our work is to function as a model, in what the riboswitch and the toxin can be exchanged for other kind of riboswitches that sense another thing, and other kind of pesticides that affect other insects, OR another reporter instead of GFP.