Team:Tec-Chihuahua/Design

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Design Overview

Considerations

1. In order to obtain the antimicrobial peptides of interest (apidaecin, abaecin, defensin 1 and defensin 2) and to be able to use them against both pathogens (Paenibacillus larvae and Melissococcus plutonius) an appropriate design is required; specialized in expressing heterologous proteins that are meant to be purified.


2. Given that the peptides will be exposed to the hive's environment, it is essential to provide them protection which can increase their half-life.

Genetic Design

The need for an efficient peptide production led to the use of a strong promoter. The answer was the T7 promoter which possesses a high specificity to the T7 RNA polymerase; only DNA cloned downstream from T7 promoter can serve as a template for T7 RNA Polymerase-directed RNA synthesis¹. The choice of this promoter was the first step to create the genetic design. The iGEM registry already had a T7 promoter, which also included an RBS (BBa_K525998) and this part was elected for the experimentation.

E. coli BL21(DE3), a chassis specialized in high-level expression of recombinant proteins, harbors a prophage DE3 derived from a bacteriophage λ, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter.² Given that expression control was sought out, and that the peptide expression depended on the presence of T7 RNA polymerase, this strain was chosen. When IPTG is added, RNA polymerase is produced.

Looking for better expression of proteins, further evaluation of reported parts was carried; the pelB leader sequence found in the iGEM registry (BBa_J32015) was selected. It directs the protein to the bacterial periplasmic membrane and reduces or even eliminates inclusion body formation³.

In order to be able to purify the peptides of interest, a 6X his-tag downstream the antimicrobial peptide sequence was considered, in addition an efficient terminator of transcription from the iGEM registry was selected (BBa_B0010).

How could we express antimicrobial peptides in a bacteria without killing it?

Before even thinking of transforming any E. coli, we got to make sure we have a structured plan on what to do for it to work. The basic idea around the genetic device was having a strong promoter, such as the T7 phage one, plus an RBS, our genes of interest, and finally an efficient terminator, such as the rrnB T1 terminator. Later on, we started thinking ahead, realizing that a secretion signal and a way in which it could be purified might be needed.

Figure 1: First design created based on just expressing the proteins inside the bacteria.

Final Product

Even if the genes worked as they should and the proteins were expressed as we wanted them to, we still needed a way to eliminate inclusion bodies, get the proteins to the periplasmic space for an easier purification and adding a tag to assure the proteins could be purified. The solutions we came up with were 2 main additions. The first one consists of adding a secretion leader sequence, the pelB leader sequence. This secretion signal helps our proteins of interest get to the periplasmic space once they are synthesized by the bacteria, but it has another amazing feature, it prevents the inclusion bodies from forming. The second addition was to add a 6xHis-tag that allows us to purify the proteins easily. Information related to these additions is found in the PARTS section of our wiki.

After solving these problems we finally got to a final version of the plasmids we would use to transform our E. coli´s (Figure 2). This final version contained all the parts previously described for the proteins to be produced as planned.

Figure 2: Finished genetic design considering AMP extraction and purification.

Prototype Design

Once we have pure peptides we need to find out a way for them to reach safely to the larva without denaturalizing or losing its contemplated effect. The way we would do this is by microencapsulation protocols. In simple terms, the process would go as follows.

The materials we will use are PLGA, a biodegradable copolymer whose degradation is pH dependent, and Dichloromethane, (DCM) used as a solvent for the PLGA. The solution both of these substances create is used as the oil phase in the microencapsulation process. The next step is getting our peptides in the form of a powder. Using a centrifuge we’ll combine both substances, the DCM/PLGA solution and the powdered peptides. This last combination is then passed through a membrane using pressurized nitrogen, thanks to its inert properties. After passing through the membrane tiny PLGA capsules containing our peptides are created. After letting the microcapsules dry out, they are ready to use and to be given to the bees.

Now the beekeeper has the microcapsules available, now what? They will be able to add them to the diet they commonly feed them with. It can either be added to a solid diet, which consists of powdered sugar or in a liquid form, which consists of a water and sugar mixture. In either case, these microcapsules won´t be affected nor will the peptides inside them, they won´t lose they antimicrobial activity, plus the microcapsule will keep them viable longer.