Results

We are proud of our results obtained during our past ten months in iGEM 2023 competition lab work. We were fortunate to have learned more then we could expect. We managed to perform multiple experiments (Golden Gate assemblies, Restriction analyses, PCRs, transformations, overproductions, purifications and HPLC runs) and each of them brung us closer to the reaching the finished project. All of the experiments performed and all of our results are meticulously described in the experiments subpage of our 2023 iGEM Wiki.

On this page we want to briefly show our most important conclusions, highlighting positive and negative outcomes of our efforts. We also present our future plans, as the project is far from being over and we want to continue developing our ideas and gathering data to confirm our theories.

WHAT WE ARE PROUD OF

• It was our first time working with the iGEM 2023 DNA Distribution Kit Plate. We learned how to design functioning devices with its contents and how to use the deposited parts in wet lab.
• We aqcuired a library of E. coli clones harbouring parts from iGEM DNA Distribution Plate Kit that we needed to assembly our constructs.
• We learned how to use the RFC1000 system to construct desired parts and devices.
• We learned and tested different cloning methods (Golden Gate assembly, Blunt End ligation) which allowed us to obtain desired constructs.
• We performed dozens of restriction analyses and some PCR analyses to verify the correctness of our cloning experiments.
• We tried different conditions for the overproduction of PnbA-DocScaB and chose the optimal one for the large scale overproduction.
• We overproduced and purified the PnbA-DocScaB fusion protein.
• We determined the stability of PnbA-DocScaB.
• We proved that PnbA-DocScaB catalyses ester bond hydrolysis in dibutyl phthalate (DBP) and butyl benzoate (BB).

WHAT WE REGRET

• We failed to achieve protein expression from constructs assembled using separate parts via Golden Gate assembly. We optimised the method for scaffoldin construct and right now the PCR results are ambigous - we can confirm presence of the coding sequence but we are still unsure of the proper assembly. Unfortunately, still no protein expression was observed.
• Despite promising results about obtaining EstJ6-DocScaB, Dcx1-DocXylY and ScfL-His we did not manage to fully confirm the correct assembly of those constructs. Fortunately, we have the tools to achieve that in the future.
• We did not have time to fully characterize the PnbA-DocScaB enzymatic activity.
• We managed to overproduce and describe only 1 out of 7 designed constructs. Although we are unhappy because of this result, we are also hungry for more and cannot wait to continue our project after the competition.

Although we have worked hard and produced plenty of data, we did not manage to complete all of our ambitious plans. Here, we have described the experiments we want to conduct in the future, as we did not have enough time to complete them during our iGEM competition participation. They are listed in order that we will probably follow, given a chance to continue our endeavors:

• We want to repeat the PnbA-DocScaB activity tests using different method, that would allow as to reliably measure the changes in the reagents concentration and also gather information about reaction kinetics. Currently we suspect that stopping the reaction with a strong acid and then extracting the reagents with a organic solvent, such as ethyl acetate.
• We want to test PnbA-DocScaB activity against as the only available substrate MBP, as we tested this activity only indirectly, in the analysis where it was the product of DBP hydrolysis.
• We want to compare the activity of PnbA-DocScaB and native form PnbA obtained by TEV protease cleavage.
• We want to test PnbA-DocScaB activity in different conditions of pH, temperature, ionic strength and acetonitrile content.
• Prof. Dr. Jörn Kalinowski Head of Technology Platform Genomics, Center for Biotechnology from Bielefeld University have offered us the service of nanopore sequencing, so we want to sequence pJUMP23-1A-pnbA-docscaB vector and pJUMP23-1A-scfL-docscaB vector to finally confirm that they were assembield correctly.
• We want to purify Dcx1-DocXylY from the pellet after cell lysis as it seems to be insoluble.
• We want to test the activity of Dcx1-DocXylY against monobutyl phthalate (MBP) and phthalic acid PA.
• If sequencing indicates that our pJUMP23-1A-scfL-docscaB vector was not assembled correctly, we will modify the sequence of ScfL-His amplicon with PCR to add appropriate enzyme restriction sites in primer overhangs that would allow us to clone this sequence to pCOLADuet-1. We want to test if the assembly and overexpression of our device would be easier in videly recognised expression vector might. Appropriate primers for this procedure have already been synthetised.
• An alternative way of obtaining functional expression vector for scfL gene would be synthetising the complete scfL device with all the regulatory sequences and clone it to pJUMP23-1A vector as one insert.
• We want to obtain the other decarboxylases: Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylY and together with Dcx-DocXylY test if they have the desired decarboxylase activity against MBP and PA. If more than one if them catalyse this reaction we will compare their activity to find the best one.
• If we find functional decarboxylase we will obtain their native forms by TEV protease cleavage and compare their activity to their conterparts with dockerin domains.
• We want to test the affinity of ScfL to the cellulose by packing fine cellulose beads into a column and load a sample containing ScfL onto the column. We would compare the initial sample and the flow through in SDS-PAGE. We could try eluting the protein from the column by applying a gradient of increasing ionic strength to test the stability of the immobilisation.
• We want to test the if the enzymes bind to the ScfL. We could do that by first immobolising the ScfL on the cellulose bed in the column in the column and then loading the samples with enzymes onto the column. Comparison of input and output samples on SDS-PAGE would tell us if the binding occured. The results would be compared with the control with no ScfL on the column.
• The interaction of enzymes containing dockerin domain with the ScfL could also be tested by microscale thermoforesis it.
• We want to test the activity of enzymes immobilised to the ScfL and then compare the results with the free enzymes.
• We want to test the activity of our protein complexes in the filter by filling the filter with a cellulose medium and filling the filter with water containing DBP and then measuring the changes of DBP and its degradation products concentration on HPLC.
• We want to test the efficiency of the filter by filling it with cellulose medium with enzymes immobilised and pass a certain volume of water with known concentration of DBP and then measure the concentration of DBP and its degradation products in the effluent.

These ideas constitute a path that we envision before us, leading us to our goal - water safe from phthalates pollution, both for the people and for the wild life. We hope that our effort will make a contribution in solving the global problem of endocrine disrupting chemicals. We are aware that down the road there will be many unexpected complications to resolve and difficult decisions to make. Nevertheless we are determinated to press forward and excited to explore all the vast possibilites to make our region a safe place to live and play a part in ensuring the sustainability of our civilization.