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• Explanation of the engineering principles your team used in your design
• Discussion of the design iterations your team went through
• Experimental plan to test your designs

Project Design

Pheromones

The first part of our trap is based on a pheromone cocktail to attract bed bugs into our trap.

DMDS DMTS

Some studies have shown that DMDS (dimethyl disulfide) and DMTS (dimethyl trisulfide) are the most attractive pheromones for bed bugs [1].

These two volatile molecules are present in bed bugs dejections, and they allow the bed bugs to find their way back to the colonies when they go out at night. In order to make the bed bugs come to our trap, we want to create an engineered E. coli, that will produce these 2 pheromones.

The DMDS and DMTS biosynthesis pathway degrades sulfur-containing amino acids to thiol, ammonia and alpha keto-acids by using methionine gamma-lyase (MGL). The methyl sulphide produced is then naturally oxidated to DMDS and DMTS as shown below:

A similar design was already used by the Wageningen UR iGEM team in 2014. In order to test our part, to ensure that there was production of the protein and that the produced protein was active. We decided to modify the part they used in their project BBa_K1493300 in two ways. First, by adding a histidine tag, to make part BBa_K2718005, this is to allow us to purify the protein, and also to test production using an antibody. This part was the coupled to an inducible promoter BBa_R0011 to make the composite part BBa_K2718006.

E.coli bacteria is a good model organism to do such a construction, as it produces L-methionine by itself [3].

Methionine gamma lyase (MGL)

For this project we used a MGL enzyme from Brevibacterium linens organism. This MGL protein is an enzyme that belongs to the family of trans-sulfuration enzymes family. It is made up of 425 amino acids and catalyses the chemical carbon-sulfur lyase reaction [4][5][6]. The part we have created with the mgl gene can be found in our "parts" tab [7].

Benzyl alcohol

The project goal is to eliminate bed bugs by using enginered E. coli bacteria that can produce DMDS and DMTS pheromones, and by creating a contact between Beauveria Bassiana and the bed bug that will come into the trap. It will come back then to its colony and infect other bed bugs with our fungus when it will die. This infection will hapenned by fungus sporulation, and also by the contact with the infected cuticule.

The problem we met through our research is that DMDS and DMTS have growth inhibitors effect on filamentous fungus [8].

That is why we’ve decided to creat an other enginered E.coli strain, that could produce benzyl alcohol.
Benzyl alcohol is also a pheromone that can attract bed bugs[9]
In association with DMDS and DMTS pheromones we will creat a great attractive pole into the trap.

To do so, we’ve decided to insert a no-natural pathway in an E. coli strain, that derived of an other natural pathway, which is the glucose degradation in phenylalanine. This process aims to use the phenylpyruvate (one of this natural pathway’s substrats) in order to start the benzyl alcohol biosynthesis pathway. It needs 3 differents enzymes: 4-hydroxyphenylpyruvate dioxygenase, (S)-mandelate dehydrogenase and benzoylformate decarboxylase, which are respectivly encoded by hmaS, mdlB and mdlC genes.

We worked on those 3 differents genes, coming from different organism : Pseudomonas fluorescens (mdlB and mdlC) and Amycolatopsis orientalis (hmaS), because of there similitudes with E.coli [11].
Those genes will be united as an operon with an RBS and strong promoteur construction, and that operon will be insert into a plasmid that will be introduced into an E.coli enginered strain.
But the first tests were done on 3 differents E.coli strain, each one transformed with an insert-plasmid construction (hmaS, mdlB or mdlC). The 3 sequences were optimized to be correctly expressed in the host organism and ordered with an RBS sequence upstream, as well as a preffix and suffix sequence flanking the gene sequence.

hmaS

4-hydroxyphenylpyruvate synthase, or 4-hydroxyphenylpyruvate dioxygenase 2 is an enzyme that catalyzes the chemical reaction bellow, by acting as a decarboxylase and a hydroxylase:

It belongs to oxidoreductases family and is involved in the vancomycin biosynthesis pathway. This enzyme is encoded by hmaS gene, that is present in Amycolatopsis orientalis. It is the first benzyl alcohol pathway step for its biosynthesis[12][13][14]. The parts we have created with the hmaS gene can be found in our "parts" tab[15].

mdlB

(S)-mandelate dehydrogenase is the second enzyme involved in the biosynthetic pathway of benzyl alcohol. It catalyzes the (S) -mandelate reduction chemical reaction, that is provided by the 4-hydroxyphenylpyruvate synthase catalyzed reaction. The product of such a reaction is the phenylglyoxylate:

This enzyme is encoded by mdlB and contains 393 amino acids. We worked on the mdlB gene from Pseudomonas putida[16][17].

mdlC

mdlC is a gene that expresses the benzoylformate decarboxylase enzyme, which is made up to 528 amino acids. It can decarboxylate the phenyglyoxylate (see above) to produce benzaldehyde:

This product then becomes benzyl alcohol during an E.coli endogenous reaction[18][19].

Beauveria Bassiana

Entomopathogenic fungi are natural biopesticides acting through contact. Beauveria Bassiana is capable of infecting a broad range of insect hosts like bed bugs. Our goal is fighting bed bugs worldwide invasive pests, that are invading homes and biting humans for their blood.

Beauveria bassiana was first used as a way of fight against insects in agriculture in Canada, and later on, all over the world. The fungus can easily penetrate bed bug's exoskeleton: the cuticle. Once inside the insect's hemolymph, the fungus feeds and multiplies, causing its death [20].

In the wolrd, entomopathogenic fungi are common and widespread in almost all classes of insects. Only a few of fungi are currently being developed as pathogens against inset pests. Indeed, entomopathogenic fungi are effective against eggs, larvae, intermediate stages and adults.
Beauveria bassiana is a white muscardine fungus, originally known as Tritirachium shiotae, grows naturally in soils throughout the world and acts as a pathogen on various insect species causing white muscardine disease [22] [23].

So, our goal is to use this fungus to fight bed bugs. Indeed, the fungus can easily penetrate bed bug's cuticle like we said before. Our objective is to enhance the fungus so that it is highly virulent for bed bugs. We want to reduce the lethal doses and break the bugs. Especially since, the excessive use of insecticides thickened the bed bug's cuticle. As a result, pest control companies are obliged to increase the insecticides doses.

We started with a first test with our Beauveria bassiana sample on garden bugs. Then, we tested the ability of this fungus to grow on different culture media. Now, we tested Beauveria bassiana on real conditions with bed bugs.

Chitinase and lipase

Chitinase was a essential biobrick to design to improve a big part of our project which is enhanced the entomopathogenic fungus Beauveria bassiana. After bibliographic researches to design chitinase biobrick, chit1 gene from Beauveria bassiana was selected because a paper demonstrated that an overexpression of this gene has been a success for increased fungus virulence [24].
This overexpression was yet realized in laboratory, and we would reproduce this experiments and test the overexpression of an important lipase from Beauveria bassiana essential to kill bed bugs. This lipase was important to allow the fungus to cross the first layer of bed bug's cuticle.

For chitinase design, a research of the secretion signal peptide sequence localisation and the inert region between it and catalytic domain was realize. We study in detail the sequence to locate these different domains. The plan was to separate the chit1 peptide signal sequence from catalytic domain to conserve only the catalytic domain, and optionally be able to add its peptide signal sequence paired to a promoter-RBS. This plan was thank to have the opportunity to create a new biobrick with a secretion peptide signal funnctional. That is one of the principles of the standardization by biobrick concept. This promoter-RBS-secretion peptide signal biobrick will be useful for others futur projects. The INTERPRO plateform was used to identify the catalytic domain of chit1 sequence. The final chitinase biobrick was composed only by catalytic domain of chit1. This sequence was optimizated to be the most compatible with E.Coli codon expression.

The same procedure was applicated for lipase biobrick but the order by IDT wasn't available, the synthesis of our sequence failed. The lipase sequence was secondly ordered and cut for the middle to increase chances to be synthetize by IDT. But this order failed once more. We decided to amplificate directly this lipase from fungus genome designing primers and ordering them. But experiments failed too. We didn't have the choice to drop this project.

The RFC25 sufix and prefix were used for design chitinase biobrick to be compatible with promoter-RBS- chit1 secretion peptide signal biobrick, to be able to remove the Start codon from the biobrick containing only the catalytic domain of chit1, to peptide signal expression paired with chit1 expression for chitinase secretion.

The decision to use E.coli for chitinase production was to produce easily chitinase to test the functionality of the protein by activity tests. E.coli is a easy chassis to use for quick results, and we anticipated the short time in laboratory to be finally capable to transforme Beauveria bassiana, for test a potential increased virulence for future tests on bed bugs. But without time, the project to transform the fungus wasn't realized.

For chitinase expression by E.coli, we decided to used an inducible promoter to avoid toxicity problems from bacteria, a constitutive expression are generally problematical for bacteria. We planned use pLac promoter to can easily induce expression thanks to IPTG induction.