Overview

As you already may know, Tec-Chihuahua’s main purpose is to inhibit Erwinia amylovora´s main virulence factors by using synthetic biology techniques to synthesize three different enzymes; the aiiA enzyme or N-Acyl homoserine lactonase, the Cyclic-di-GMP phosphodiesterase from the yhjH gene and the Putative Glycosyltransferase encoded by epsE. Nevertheless, our project can be divided into two parts: the proof of concept regarding Fire blight (characterizing and proving how the proteins work and how effectively. Also whether is it a good option to design a genetic circuit with one or more of them) and the process beyond (the whole process that goes afterwards, necessary to transform Erwinions into a commercial product).

Proof of Concept

Genetic System

In order to inhibit Erwinia’s virulence factors, we knew we first had to design four genetic systems, one for each enzyme (1) plus a polycistronic gene that includes all of them (2), for both Erwinia and BL21(DE3) as part of our proof of concept. For this, we knew that it was necessary to prove that either one enzyme or the combination of them could act effectively against the progression of the disease. That is why we established, as our proof of concept, the molecular and microbiology characterization of each of them. Nevertheless, we ran into one important question: Do we design our Biobrick as a constitutive or regulated gene?

IOne of the most important things to do when starting a research is to acknowledge your possibilities (economical, technical, etc.). Even though we were transforming E. coli and E. amylovora, the fact that we were a lacking some reagents was key for us to decide to construct a constitutive circuit. At last, what we were trying to prove at this point of the project was that both bacterias were able to express those exogenic proteins. For this purpose, a constitutive gene was perfect because, we believe, that this way we were able to characterize an exogenous gene easier. The only problem could be the creation of inclusion bodies. If you continue reading, you may notice that this definitely is not our last genetic system. We decided to use a T7 promoter because we knew for sure that it will work at the BL21(DE3) for sure, while on Erwininia we were not for sure which promoter was going to work.

Bronze

When we did our research and found out of those three enzymes, we did not know that they were already at the iGEM registry. Nevertheless, this turned out to be good news as they were registered but lacked complete characterization. It is important to highlight the fact that we practically needed to develop a new Biobrick. Because the coding sequences (BBa_C0060, BBa_K861090 and BBa_K143032) did not have a complete circuit and we lacked an expression vector, we had to develop our own vector by digesting and ligating a promoter (BBa_K525998) and a terminator (BBa_B0010) to each protein. When we were able to complete it we started characterizing both molecular and microbiologically. We did some protein kinetics with the help of SDS Page technique, colorimetry, and motility protocols. You can see the results either at our [wiki] or at the [iGEM Registry].

Silver

For our silver construct, we realized, with the help of BLAST, that most of the sequences we were using were reported as putative proteins, while there were some different sequences at the NCBI database better characterized. Therefore, we decided to construct two new Biobricks with better experimental backgrounded proteins that will help us compare the results from the existing at the iGEM registry against the those in the NCBI.

The epsE gene was the first we decided to design as it was reported that it had never been characterized in any other microorganism than Bacillus. We thought that if we have a negative expression in BL21(DE3), it could be due to the fact that the iGEM`s epsE was not the optimal. If at least we had two different sequences that might code for the same protein we could make better assumptions and conclusions.

On the other hand because the iGEM`s aiiA was reported as putative at the NCBI and the characterization at the registry was just a substrate-enzyme kinetic (that might be influenced by an isoform), we decided to construct a new gene aiiA. Plus if we were unable to characterize epsE because of a negative expression, therefore we this could be also our back up plan.

If you want to look at the complete sequence of our construct you can click here.

It is important to mention that couldn't achieve this without the help of IDT.

Final Product

For this phase of the project, the first complication that we encountered was: If we prove that our solution work, then how am I going to apply it? As mentioned on the project description tab, there were many ideas that we thought were good applications for our product. Nevertheless, it was until we started gathering information for our Human Practices that one agricultural engineer, Rafael Quevedo owner of Optihumus, told us about what ended being the best option (you can read more about this integrated human practice here): a biocontrol which the main composite is our modified Erwinions. He recommend us to develop a biocontrol under the same principle of the modified Mosquito for the Malaria.

NEW Genetic System

We knew that for us to develop this product until the commercialization stage, we needed to create a new genetic system that ensures our transformation and, therefore, our GMO as biosecure and efficient. Unfortunately, the regulations on Mexico regarding this matter are inefficient and/or inexistent. This way, as part of our Human Practices and Integrated HP, we developed Official Mexican Norms (NOM`s) that were discussed and validated by the Ministers involved; agriculture and environment. Plus, we internationally validated it according to The Cartagena Protocol on Biosafety [Check it here]. These stakeholders did give us some feedback that we take into account in the creation of this new genetic system.

According to what we developed for the governmental entities,