To conclude our project, we have been aiming to alter organisms’ behaviors by cloned bacteria. And we successfully designed 2 parts and produced 1 of the two to meet our goal.

Where did our idea come from?

We started up just from observing the ability of controlling other organism of horsehair worms in a YouTube video. Horsehair worm is parasitic. These worms can go inside another insect’s body and make them go into water, which the insect originally won’t ever do that. So that the horsehair worm can reproduce in water later when it crawls back out from the insect’s body, leaving the insect drowned. We thought we might be able to change the behaviors of some organisms too, just like what the horsehair worms do but using transformed bacteria. That’s how we started this idea.

How we chose our target organism and what is it?

We aim organisms that have highly observable behaviors, easy to handle and low maintenance cost. Also, the organism is better to be well studied and had used by other iGEM teams before. That will make sure we have sufficient information to support our project. Finally, we figured out C.elegans, a transparent tiny nematode, which has a nickname called the round worms. Unlike horsehair worms, C.elegans is free-living and non-parasitic. C.elegans is approximately 1mm in length when they are in mature stage. We can keep them alive on agar and feed them with bacteria. The main reason that we chose C.elegans is because they have the ability to detect volatile compounds and chemical substances, which is essential for our project. Plus, C.elegans is used by Tongji University in iGEM 2015.

What are our chemical attractant and repellent?

We would like to engineer some bacteria to produce some chemicals that might attract C.elegans or make them go away. So we need to find suitable attractants and repellents of C.elegans. And choose among these chemicals which attractant and repellent to study and produce in our project. We looked through the online resources - the Wormbook, that is a glossary about C.elegans. Meanwhile, we tried to find substances which have been studied in the iGEM community. There we discovered a potential attractive chemical called cinnamaldehyde. The pathway to producing cinnamaldehyde is studied by the British Colombia team in iGEM 2013. From further online research, we figured out that C.elegans is attracted by volatile organic compounds, VOC. And that’s what cinnamaldehyde was, volatile and organic. Searching through other useful studies, we found another substance called the phenylpyruvic acid that might be a repellent to C.elegans. The far-fetched reason was the word ‘acid’, where C.elegans is repelled by acids. Yet phenylpyruvic acid is new to iGEM community. However, what interesting was, later our assay results completely overthrown our deduction.

What was our plan?

At the beginning of the story, we aimed three things. First, to produce an attractant with transformed bacteria. Second, to produce a repellent. Third, to produce attractant under control of a repellent-sensitive promoter. We planned to design three devices to achieve these aims. We design the orientations of parts of the three devices. Unfortunately, when we went on testing the attractiveness of the chemicals, we are surprised by the results that were completely opposite of our deductions. Considering this, we were forced to give up the third device. The design of producing attractant under control of a repellent-sensitive promoter is no long sensible. So we carried on with the first two devices, producing attractant and repellents.

What have we done?

Cloning - Cinnamaldehyde

We found a synthesis pathway of cinnamaldehyde with genes in the iGEM parts library, given by the British Colombia team in iGEM 2013. The pathway is constructed with 3 genes, where the starting substrate is phenylalanine, an amino acid. With the sequence of the three genes provided, we designed and ordered the 3 oligo DNA from IDT. Each ordered oligo DNA sequence encloses an iGEM promoter, RBS, a gene in the cinnamaldehyde synthesis pathway and a terminator, with iGEM prefix and suffix for a more convenient use in 3A assembly cloning steps. We were supposed to sub-cloned these genes into 3 separate plasmid backbones then clone the 3 parts into one plasmid backbone. And lastly transform the plasmid into bacteria. Unfortunately, we were not successful in doing it. Take a look on our part Kxxxxxx.

Cloning - Phenylpyruvic acid

Phenylpyruvic acid is an attractant. However, we had difficulties find a protein sequence that enables the production of phenylpyruvic acid in the iGEM community. As such, we decided to synthesize it ourselves. We discovered a protein sequence for producing phenylpyruvic acid on NCBI website. We again ordered the oligo DNA from IDT. The ordered sequence also encloses an iGEM promoter, RBS, a phenylpyruvic acid producing gene and a terminator, with iGEM prefix and suffix. The gene is an amino acid deaminase gene. Take a look on our part Kxxxxxx.

Assay

We have done assays on pure chemicals and bacteria synthesized chemicals, testing whether the substances are attractants or repellents to C.elegans. And we used response index to describe whether the chemicals are attractants or repellents to C.elegans. basically, a positive index indicates an attractant and a negative index implies a repellent. For more detailed explanation and calculations, please refer to experiment page.

Assay - Pure chemicals

We tested on pure chemicals of cinnamaldehyde and phenylpyruvic acid to check if our assumptions about the attractiveness were correct. The results turned out to be not meeting our expectations. Cinnamaldehyde is a repellent and phenylpyruvic acid is an attractant! For more detailed assay results, please refer to result page.

Assay - Bacteria synthesized chemicals

After successful cloning bacteria with phenylpyruvic acid producing gene, we used this composite part to perform our assays. We did two assay concerning phenylpyruvic acid, Bacteria synthesized phenylpyruvic acid verses water and backbone-only plasmid (empty vector) verses water. The response index taken in the former assay is higher significantly higher than that of the latter. The result has proved our success in cloning the gene and is effective in terms of our aim of producing an attractant. For cinnamaldehyde, we were not able to clone the genes into a plasmid and do transformation and thus we did not conduct any assay on bacteria synthesized cinnamaldehyde.

How can our idea and products be used for?

C.elegans is a pest that would attack the root of grapevines. We can tell that cinnamaldehyde, the organic compound giving cinnamon its flavor and odor is indeed a repellent to C.elegans. On the other hand, Phenylpyruvic acid is an attractant to C.elegans. Our project can be used as biochemical trap and fence. By using phenylpyruvic acid, known as the attractant, we can gather C.elegans in a designated place. Vice versa, by applying cinnamaldehyde solution around the crops, we can avoid C.elegans from them. This method can replace the use of traditional pesticide, which kills pests directly, or meanwhile damages plants. Additionally, it is harmful to surroundings and human being. On the contrary, our method is beneficial to plants. Having low toxicity, cinnamaldehyde is an effective agrichemical, being used as fungicide, insecticide and mosquito larvae. Furthermore, it is not harmful to surroundings thus human being. With such safe and beneficial method, we all concur the use of our method is more preferable for the agriculture.

What are things to be followed up?

First, as we mentioned above, we have not successfully cloned or transformed the bacteria that produces cinnamaldehyde. We barely have the sequence of the plasmid, excluding the plasmid backbone. We have to identify the problem in cloning the plasmid and transformation efficiency in order to make improvements. For phenylpyruvic acid, although we are able to make the design come true, we still need to work on the actual effects on insects. Since we don’t have enough resource, we are not sure about the actual effect when this method is being applied. In our project, we only did assays on C.elegans. we hope our idea can be widely used to control other pests. Secondly, we hoped this project is the beginning of a brand new way to cure pest problems. Further investigation on the relationship between the behavior of pests and their olfactory system can also be carried out.

Reference

[1]: iGEM. (2016). Safety/Risk Groups. Available: https://2016.igem.org/Safety/Risk_Groups. Last accessed 26th Aug 2016.

[2]: iGEM. (2016). Safety/White List. Available: https://2016.igem.org/Safety/White_List. Last accessed 26th Aug 2016.