The characterization of BBa_C0060 was carried out, first, by creating a composite part (BBa_K2471000) capable of expressing the biopart of interest; this was done by the addition of a promoter and RBS and terminator to the basic part. After corroborating, by agarose gel electrophoresis, that this part had been correctly synthesized using the 3A assembly method, the bacteria Erwinia amylovora was transformed, by electroporation, with this new BioBrick®. Electrophoresis in polyacrylamide gel (12%) was performed in order to corroborate if the protein of interest was indeed expressed, as this is what was theoretically proposed based on the biographical revision previously done. The general methodology (to see specifications visit protocols section ) involved a growth curve of the bacteria, then, also, a sample was taken every hour (0 - 8 hours) and stored in refrigeration to later perform total protein extraction.

In order to calculate the molecular weight of the protein, the sequence provided by the iGEM Parts Registry for BBa_C0060 was taken and introduced in ExPASy - Translate tool, which generated the open reading frame in amino acids. This peptidic sequence was then analyzed in ExPASy - ProtParam tool, where a molecular weight of 30.32026 kDa was calculated.

Figure 1.SDS-PAGE (12%) of Erwinia amylovora transformed with BBa_K2471000.

As it can be seen in figure 1, there is an appreciable band present at the approximate weight of 30 kDa from the fourth hour onwards. As a point of comparison, SDS-PAGE of wild Erwinia amylovora was also carried out, however, no banding was seen whatsoever. We think that this happened because the protocol used is not specifically designed for this bacteria, and since Erwinia amylovora forms a very complex biofilm, the cell lysis was not carried out correctly; and thus, neither did the protein extraction. In like manner, we propose that the SDS-PAGE shown in figure 1, where the protein of interest is expressed, was done correctly because of the protein´s function, which is quorum quenching; this affects the production of important components of its biofilm, which leads to its deficient production. When reviewing literature on this, not much was found, Erwinia amylovora is a poorly studied bacteria.

Nonetheless, a comparison was made with an SDS-PAGE that was carried out using the same methodology but utilizing Erwinia amylovora transformed with BBa_K2471003. As BBa_C0060 is registered as being a putative N-Acyl homoserine lactonase aiiA, this new BioBrick contains the genetic circuitry to correctly express our protein, which´s sequence (that has small changes compared to BBa_C0060, with molecular weight of 30.84286 kDa) is confirmed to indeed codify for a N-Acyl homoserine lactonase aiiA. This part is a new addition to the Parts Registry, more information about it can be found in the silver portion of our characterization.

Figure 2.SDS-PAGE (12%) of Erwinia amylovora transformed with BBa_K2471003.

In the same way as figure 1, figure 2 shows an appreciable band present at the approximate weight of 30 kDa, starting from the fourth hour onwards; meaning that this band is indeed the the enzyme aiiA. With this, we establish that we characterized BBa_C0060 by proving that Erwinia amylovora can indeed express it.