It is very important to us that future iGEM teams can use information we have gathered during this year's competition and we can only hope that our contribution will prove useful.
For this year's competition we have registered several new parts, including basic and composite parts.
| Kind | Registry Part ID | Type | Description | Length |
|---|---|---|---|---|
| Basic | BBa_K4695010 | CDS | This sequence encodes an esterase (PnbA) | 1467 bp |
| Basic | BBa_K4695011 | CDS | This sequence encodes an esterase (EstJ6) | 894 bp |
| Basic | BBa_K4695020 | CDS | This sequence encodes a decarboxylase (Dcx1) | 513 bp |
| Basic | BBa_K4695021 | CDS | This sequence encodes a decarboxylase (Dcx2) | 1482 bp |
| Basic | BBa_K4695022 | CDS | This sequence encodes a decarboxylase (Dcx3) | 1659 bp |
| Basic | BBa_K4695023 | CDS | This sequence encodes a decarboxylase (Dcx4) | 789 bp |
| Basic | BBa_K4695030 | CDS | This sequence encodes a scaffoldin protein consisting of 3 kohesins and CBM (ScfL) | 2184 bp |
| Basic | BBa_K4695040 | CDS | This sequence encodes a dockerin (DocXylY) | 177 bp |
| Basic | BBa_K4695041 | CDS | This sequence encodes a dockerin (DocScaB) | 225 bp |
| Basic | BBa_K4695042 | CDS | This sequence encodes a dockerin (DocCel48A) | 210 bp |
| Composite | BBa_K4695110 | CDS | This sequence encodes a fusion protein consisting of an esterase and dockerin, divided by TEV cut site and C-terminal HisTag (PnbA-DocScaB) | 1770 bp |
| Composite | BBa_K4695111 | CDS | This sequence encodes a fusion protein consisting of an esterase and dockerin, divided by TEV cut site and C-terminal HisTag (EstJ6-DocScaB) | 1197 bp |
| Composite | BBa_K4695120 | CDS | This sequence encodes a fusion protein consisting of a decarboxylase and dockerin, divided by TEV cut site and C-terminal HisTag (Dcx1-DocXylY) | 768 bp |
| Composite | BBa_K4695121 | CDS | This sequence encodes a fusion protein consisting of a decarboxylase and dockerin, divided by TEV cut site and C-terminal HisTag (Dcx2-DocXylY) | 1737 bp |
| Composite | BBa_K4695122 | CDS | This sequence encodes a fusion protein consisting of a decarboxylase and dockerin, divided by TEV cut site and C-terminal HisTag (Dcx3-DocXylY) | 1914 bp |
| Composite | BBa_K4695123 | CDS | This sequence encodes a fusion protein consisting of a decarboxylase and dockerin, divided by TEV cut site and C-terminal HisTag (Dcx4-DocXylY) | 1044 bp |
| Composite | BBa_K4695130 | CDS | This sequence encodes a scaffoldin protein with C-terminal HisTag (ScfL-His) | 2292 bp |
| Composite | BBa_K4695210 | Device | This sequence grants an expression of fusion protein, after IPTG induction (PnbA-DocScaB) | 2031 bp |
| Composite | BBa_K4695211 | Device | This sequence grants an expression of fusion protein, after IPTG induction (EstJ6-DocScaB) | 1458 bp |
| Composite | BBa_K4695220 | Device | This sequence grants an expression of fusion protein, after IPTG induction (Dcx1-DocXylY) | 1029 bp |
| Composite | BBa_K4695221 | Device | This sequence grants an expression of fusion protein, after IPTG induction (Dcx2-DocXylY) | 1998 bp |
| Composite | BBa_K4695222 | Device | This sequence grants an expression of fusion protein, after IPTG induction (Dcx3-DocXylY) | 2175 bp |
| Composite | BBa_K4695223 | Device | This sequence grants an expression of fusion protein, after IPTG induction (Dcx4-DocXylY) | 1305 bp |
| Composite | BBa_K4695230 | Device | This sequence grants an expression of fusion protein, after IPTG induction (ScfL-His) | 2553 bp |
We were the first to take an interest in the topic of phthalates degradation through enzymatic reactions. Inspired by previous iGEM teams we decided to design an enzymatic complex based on cellulosome. In order to do so, we needed genes enabling the synthesis of:
Using three different dockerins allows designing multi-step enzymatic reactions and Biobrick® format enables exchanging sequences encoding enzymes and dockerins which results in many combinations of fusion proteins depending on needs. Scaffoldin protein consisting of three different cohesins that bind their respective dockerins allows designing protein complexes that enhances enzymatic activity due to closeness of proteins. We hope that future iGEM teams will benefit from using our parts.
We also hope that our project will inspire next iGEM teams in the terms of phthalate pollution. Our parts encoding enzymes, fusion proteins and entire devices can be used to design many phthalate-degrading enzymatic complexes. Thanks to that, iGEM teams in the future can contribute to getting rid of phthalates pollution.
Unfortunately, we were not able to use all of our parts, so their functionality is yet to be proven, but this leaves much room for testing and improvement by future iGEM teams. They can take on where we finished and make great accomplishments of their own.
In order to design our enzymatic complex we needed to choose enzymes which would enable us to perform degradation of phthalates. First part of that complex is a para-nitrobenzyl esterase, called by us esterase for short. It allows the hydrolysis of ester bonds, converting dibutyl phthalate into monobutyl phthalate and phthalic acid – the main substrates for our next enzyme. We have chosen an esterase PnbA (GTW28_17760) from Bacillus subtilis BJQ0005 identified by Xu et al. The team in their study used bacterial lysate to test enzyme’s activity. Out team took it one step further and we have obtained purified esterase and tested its activity. Future iGEM teams now have a ready-to-use part that enables them to synthesize their own esterase and further test its activity and properties.
Second part of our enzymatic complex is a decarboxylase that allows the detachment of the carboxyl group, converting monobutyl phthalate into butyl benzoate and phthalic acid into benzoic acid. We have chosen four decarboxylases from bacteria called Pantoea dispersa, genes encoding these proteins were identified by Xu et al. The team proposed a metabolic pathway of phthalate degradation but the enzymes have never been synthesized before. Our team managed to synthesize one of four mentioned decarboxylases - Dcx1 (GAY20_11210). But due to time restrictions we were unable to test its activity. However, this is the opportunity for future iGEM teams to take part in synthesis of these proteins and testing their activity, therefore contributing to purifying wastewater from phthalates.
Our enzymatic complex is a part of water filter of our design. We are positive that this filter will be applicable in both industry and households. But we are open for suggestions from other teams and we will be more than happy to share with you the blueprints of our filter, so you can make one yourselves and give us feedback on whether it works and your thoughts or suggestions on how we can improve it.
Previous iGEM teams have been tackling the challenge of obtaining reliable and repeatable measurements in different labs through the International InterLaboratory (Interlab) Measurement Studies. This year we have signed up for the 7th Interlab study. We have conducted the third experiment, you can check the results on our Experiments Page. We hope that our effort will help determine the unit of fluorescence and therefore allow scientists as well as future iGEM teams to compare their fluorescence data.
During the competition, our team was very involved in educational activities and we have prepared a lot of materials that can be used by future iGEM teams. We are happy to share our presentations – the one prepared for high school students and the one prepared for a guest lecture at the Pomeranian Science and Technology Park in Gdynia. We also prepared a description of our SynBio workshops from Project: High School, with detailed information on how to prepare and use our genetic puzzles. We also had the opportunity to participate in the INSA ENS Lyon 1 project on antibiotic resistance – it resulted in a nearly 10-minute video in which several teams from around the world share their thoughts and knowledge about this growing problem. In addition, we also prepared a series of educational posts about great Polish scientists and some basic issues connected to biotechnology and synthetic biology. We also share more than 20 questions that we prepared for our biological pub quiz which was a resounding success among science enthusiasts in Gdańsk. Finally, we provide some ideas we did not implement, but which may be an inspiration for future iGEM teams: the International Day of Synthetic Biology on September 9 (on the birthday of Prof. Wacław Szybalski, Polish scientist, founder of our Faculty and one of the fathers of synthetic biology), our own mini-newspaper on synthetic biology, an art competition for primary schools, an online course on the basics of synthetic biology and a workshop inspired by the Montessori method, combining movement with education.
