What should this page contain?

• 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

The project goal is to eliminate bed bugs by using engineered E. coli bacteria that produce pheromones to lure the bed bugs into a trap. In the trap, the bed bugs will be infected with Beauveria Bassiana. They will then return to their colony, or nest, and infect all the other bed bugs, and their eggs, with our fungus so killing the nest. This epidemic infection will be ensured by fungal sporulation. The spores should also provide lasting protection in the nest site against reinfection.

Pheromones

The first part of our trap is based on a pheromone cocktail to attract bed bugs into our trap. Bed bugs use a complex collection of pheromones and we aimed, in our project to produce 4: DMDS, DMTS, Benzaldehyde and Benzyl alcohol.

DMDS and 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 is a result of the degradation of sulfur-containing amino acids to thiol, ammonia and alpha keto-acids by using methionine gamma-lyase (MGL). The methyl sulfide produced is then naturally oxidized 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 a 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, so making part BBa_K2718005, this is to allow us to purify the protein, and also to test production using an antibody. This part was then coupled to an inducible promoter BBa_R0011 to make the composite part BBa_K2718006, allowing the inducible production in E. coli.

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

In our project, we used the methionine gamma-lyase enzyme from Brevibacterium linens. This MGL protein is an enzyme that belongs to the trans-sulfuration enzyme family. It is made up of 425 amino acids and catalyzes 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 as BBa_K2718006.

Benzaldehyde and Benzyl alcohol

We decided to create another engineered E.coli strain, that could produce benzaldehyde and benzyl alcohol. Benzyl alcohol is also a pheromone that can attract bed bugs[7].

To do so, we’ve decided to engineer a new metabolic pathway into E. coli, this pathway is derived from another natural pathway, found in Pseudomonas fluorescens. In this pathway, phenylpyruvate (a substrate in phenylalanine biosynthesis) is used as the starting material for benzyl alcohol and benzaldehyde biosynthesis. Engineering this pathway requires the introduction of 3 different enzymes: 4-hydroxyphenylpyruvate dioxygenase, (S)-mandelate dehydrogenase and benzoyl formate decarboxylase, which are respectively encoded by the 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 similarity with E.coli and previous experiments in the litterature [9].

In our design, the 3 genes will be put in a single operon with an RBS and strong promoter construction, and the operon will be inserted into a plasmid to produce an engineered E.coli strain.

But the initial tests were done on 3 differents E.coli strains, each with a separate gene from the pathway (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 biobrick prefix and biobrick suffix flanking the gene sequence.

hmaS

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

It belongs to oxidoreductases family of enzymes and is also involved in the vancomycin biosynthesis pathway. This enzyme is encoded by hmaS gene, that is present in Amycolatopsis orientalis. It is the first step in the biosynthesis of benzyl alcohol[10][11][12]. The parts we have created with the hmaS gene are BBa_K2718010 with the coding sequence and a ribosome binding site; and BBa_K2718011 with the promoter BBa_R0011.

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[13][14].

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[15][16].

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 [17].

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 [19] [20].

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 [21].
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.