iGEM 2024 — Team anatolia-college

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{% block title %}Notebook{% endblock %}
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In order to walk you through our progresses and lab adventures we’ve created a concise pdf file for you. This includes chronological notes and brief descriptions of what your team did, along with pictures showcasing our progress.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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# Let us walk you through the thinking process behind our bioengineering design.

From the degradation of PET plastic, using an enzyme which specializes in breaking down exclusively PETase, three products result: BHET (Bis(2-Hydroxyethyl) terephthalate) in small amounts, MHET (Mono-(2-hydroxyethyl)terephthalic acid) as the main product, and EG (Ethylene glycol).
From the degradation of PET plastic using the enzyme PETase, which specializes in breaking down PET, three products are produced: MHET (Mono-(2-hydroxyethyl) terephthalic acid) as the main product, BHET (Bis(2-hydroxyethyl) terephthalate) in smaller amounts, and EG (Ethylene glycol).
TPA and EG are then normally produced by the action of MHET-ase. Hydrolysis of polyethylene terephthalate (PET) is an efficient strategy to depolymerize PET waste into terephthalic acid (TPA), which can be used as a fundamental building block for repolymerization of PET or for the synthesis of biodegradable plastics and metal-organic frameworks and does not pose a risk to the environment.
The enzyme cutinase derived from the fungus Humicola insolens, hydrolyzes ester bonds found in natural and synthetic polyesters, like PET . Its versatility allows it to act on both hydrophobic and hydrophilic ester bonds, effectively targeting the surface of PET. The enzyme degrades amorphous regions of PET, which are less crystalline and more accessible, increasing the surface area for hydrolysis. This process breaks down PET into its monomers: TPA and EG. Cutinase can also hydrolyze other aliphatic polyesters, such as polylactic acid (PLA) and polycaprolactone (PCL).
The reason we chose this specific enzyme is because it is an enzyme that can be found to have very similar abilities with PETase, same optimum conditions and they have been used together in previous studies.
The potential benefits of creating the hybrid enzyme are:

1. The biodegradation of products made up of more than one type of plastic as mentioned above (PLA and PCL)

2. Acceleration of biodegradtion rate: PETase in optimal conditions requires a minimum period of 6 days to biodegrade the PET material, so the synergestic action of the two enzymes will reduce this time period.

3. PETase and cutinase may target different parts of PET molecules, increasing the efficiency of biodegradation as PETase can initially cleave PET into intermediates such as MHET, which can be further degraded by cutinase, facilitating faster degradation.

4. PETase and cutinase can work sequentially, with PETase initiating degradation and cutinase accelerating the breakdown of intermediates, leading to faster overall degradation.

5. The fused protein is suitable for industrial applications that require enzymatic activity under relatively mild environmental conditions, due to the fused enzyme’s optimal operating conditions, namely a pH of 8.0 and temperature of 37℃. This reduces the need for extreme conditions (high temperatures or strong acids) that are typically used in chemical plastic recycling processes.

We chose to create a fused enzyme using a protein linker. In bioengineering, a protein linker is a short peptide or protein sequence used to connect two or more proteins or protein domains. We found out that linkers serve various purposes, including: flexible connection, spatial orientation stability, functionality, modularity.
When designing a linker, factors like length, amino acid composition, and flexibility are considered to ensure optimal performance of the linked proteins. Existing protein linker types are the following:

\* Flexible

\* Rigid

\* Cleavable

However after a series of meetings with both George Kalliolias and the iGEM team MetaThess (overgrad prize for best diagnosis 2022) , as well as a lot of research, we decided that the most appropriate linker type was definitely a flexible linker, as it enables the modular assembly of proteins, making it easier to design and build complex protein constructs with desired properties. After exploring multiple options we decided that the ideal flexible linker was the (GGGGS)3 linker, commonly used in iGEM and bioengineering generally and also previously used on fused enzymes with both PETase and cutinase enzymes.
This is due to its small length( 15 amino acids- 45 bases), chemical properties (Rich in small or hydrophilic amino acids) and geometric features (minimize steric hindrance when linking two proteins or domains while still providing enough separation for functional interactions)
The (GGGGS)3 linker is typically a synthetic sequence of amino acids used to physically connect polypeptide domains, as seen in fusion proteins like green fluorescent protein (GFP) constructs.  While some linkers, like GS, are chemically synthesized , the (GGGGS)3 linker can also be chemically synthesized or transcribed from a gene. This flexibility allows for various applications in protein engineering, where the linker's sequence diversity is explored to optimize fusion protein functionality.
So in order to provide flexibility between protein domains, allowing them to move independently and maintain their functional conformations and help position proteins at specific distances or orientations, which are crucial for their activity, especially in multi-domain proteins or in creating fusion proteins.

We transcribed the linker from the gene (GGGGS)3

Linker mRNA sequence:
GGU-GGU-GGU-GGU- UCU- GGU-GGU-GGU-GGU-UCU-GGU-GGU-GGU-GGU-UCU

Linker dna used
GGT GGA GGC GGT AGC GGG GGA GGC GGT AGC GGA GGG GGC GGT AGC

Linker protein sequence
NH2-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-COOH

## The base sequence for PETase:

ATG AAC TTC CCC CGT GCA TCG CGC CTA ATG CAG GCT GCT GTG CTG GGC GGA CTT ATG GCA GTT TCC GCA GCT GCC ACG GCG CAG ACC AAT CCG TAT GCG CGC GGA CCC AAT CCT ACC GCC GCC TCG TTG GAA GCC AGC GCT GGA CCA TTT ACT GTT CGA AGC TTC ACC GTT AGC CGT CCG TCC GGA TAT GGT GCA GGG ACC GTC TAT TAC CCA ACC AAC GCA GGC GGC ACA GTT GGC GCC ATT GCT ATC GTC CCC GGT TAC ACC GCA CGA CAA AGC AGT ATT AAG TGG TGG GGT CCA CGC TTA GCT AGC CAT GGC TTT GTG GTT ATT ACC ATC GAT ACG AAC AGT ACT CTA GAC CAG CCC AGT AGC CGA AGT TCG CAA CAG ATG GCC GCT CTT CGT CAA GTT GCA AGC TTA AAC GGA ACC AGT AGT AGC CCG ATT TAC GGA AAG GTC GAT ACT GCC CGC ATG GGT GTG ATG GGC TGG TCA ATG GGA GGC GGA GGT TCA CTT ATT AGC GCC GCA AAC AAC CCG AGT TTA AAA GCA GCG GCA CCG CAG GCG CCA TGG GAC TCT TCA ACG AAT TTC AGC AGT GTT ACC GTG CCG ACG CTG ATT TTC GCG TGC GAG AAT GAT AGC ATT GCA CCG GTG AAC AGT AGC GCA CTG CCG ATT TAT GAT AGC ATG TCC CGC AAC GCT AAA CAG TTT CTG GAA ATT AAC GGC GGT AGC CAC TCT TGT GCC AAC TCT GGG AAC AGC AAC CAG GCA CTG ATC GGA AAA AAA GGG GTT GCA TGG ATG AAA CGA TTC ATG GAT AAT GAC ACC CGT TAC TCA ACC TTC GCC TGT GAG AAT CCC AAC AGT ACA CGC GTG TCG GAT TTC CGC ACC GCG AAC TGT TCC CTC GAG CAC CAC CAT CAC CAC CAC TGA

## The base sequence for Cutinase:

ATG GGC AGT AGC CAC CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGT CAT ATG CTG CCG ACC AGC AAC CCG GCG CAA GAG CTG GAA GCG CGC CAA CTG GGC CGT ACC ACC CGT GAC GAT CTG ATC AAC GGT AAC AGT GCC AGC TGC GCG GAC GTG ATC TTC ATT TAC GCG CGT GGC AGC ACC GAA ACC GGT AAC CTG GGC ACC CTG GGT CCT AGC ATT GCA AGC AAC CTG GAG AGC GCA TTT GGC AAG GAC GGT GTG TGG ATT CAG GGT GTT GGT GGC GCG TAT CGT GCG ACC CTG GGT GAT AAC GCG CTG CCG CGT GGC ACC AGT AGT GCT GCG ATC CGT GAA ATG CTG GGC CTG TTC CAG CAA GCG AAC ACC AAA TGC CCG GAT GCG ACC CTG ATT GCG GGT GGC TAC AGC CAG GGT GCG GCG CTG GCG GCG GCG AGC ATT GAG GAC CTG GAT AGC GCG ATC CGT GAT AAG ATT GCG GGC ACC GTG CTG TTC GGT TAT ACC AAA AAC CTG CAA AAC CGT GGT CGT ATT CCG AAC TAT CCG GCG GAC CGT ACC AAG GTG TTC TGC AAC ACC GGC GAT CTG GTG TGC ACC GGT AGC CTG ATT GTT GCG GCG CCG CAC CTG GCG TAT GGC CCG GAT GCG CGT GGT CCG GCG CCG GAA TTT CTG ATT GAG AAA GTG CGT GCG GTT CGT GGT AGC GCG GGT GGC GGT GGC AGC GGT GGC GGT GGC AGC CAG CAA CGT TTC GAG TGG GAA TTT GAG CAG CAA TAA

## The sequence for the final used enzyme that we designed and expressed:

AAC TTC CCC CGT GCA TCG CGC CTA ATG CAG GCT GCT GTG CTG GGC GGA CTT ATG GCA GTT TCC GCA GCT GCC ACG GCG CAG ACC AAT CCG TAT GCG CGC GGA CCC AAT CCT ACC GCC GCC TCG TTG GAA GCC AGC GCT GGA CCA TTT ACT GTT CGA AGC TTC ACC GTT AGC CGT CCG TCC GGA TAT GGT GCA GGG ACC GTC TAT TAC CCA ACC AAC GCA GGC GGC ACA GTT GGC GCC ATT GCT ATC GTC CCC GGT TAC ACC GCA CGA CAA AGC AGT ATT AAG TGG TGG GGT CCA CGC TTA GCT AGC CAT GGC TTT GTG GTT ATT ACC ATC GAT ACG AAC AGT ACT CTA GAC CAG CCC AGT AGC CGA AGT TCG CAA CAG ATG GCC GCT CTT CGT CAA GTT GCA AGC TTA AAC GGA ACC AGT AGT AGC CCG ATT TAC GGA AAG GTC GAT ACT GCC CGC ATG GGT GTG ATG GGC TGG TCA ATG GGA GGC GGA GGT TCA CTT ATT AGC GCC GCA AAC AAC CCG AGT TTA AAA GCA GCG GCA CCG CAG GCG CCA TGG GAC TCT TCA ACG AAT TTC AGC AGT GTT ACC GTG CCG ACG CTG ATT TTC GCG TGC GAG AAT GAT AGC ATT GCA CCG GTG AAC AGT AGC GCA CTG CCG ATT TAT GAT AGC ATG TCC CGC AAC GCT AAA CAG TTT CTG GAA ATT AAC GGC GGT AGC CAC TCT TGT GCC AAC TCT GGG AAC AGC AAC CAG GCA CTG ATC GGA AAA AAA GGG GTT GCA TGG ATG AAA CGA TTC ATG GAT AAT GAC ACC CGT TAC TCA ACC TTC GCC TGT GAG AAT CCC AAC AGT ACA CGC GTG TCG GAT TTC CGC ACC GCG AAC TGT TCC CTC GAG CAC CAC CAT CAC CAC CAC TGA GGT GGA GGC GGT AGC GGG GGA GGC GGT AGC GGA GGG GGC GGT AGC ATG GGC AGT AGC CAC CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGT CAT ATG CTG CCG ACC AGC AAC CCG GCG CAA GAG CTG GAA GCG CGC CAA CTG GGC CGT ACC ACC CGT GAC GAT CTG ATC AAC GGT AAC AGT GCC AGC TGC GCG GAC GTG ATC TTC ATT TAC GCG CGT GGC AGC ACC GAA ACC GGT AAC CTG GGC ACC CTG GGT CCT AGC ATT GCA AGC AAC CTG GAG AGC GCA TTT GGC AAG GAC GGT GTG TGG ATT CAG GGT GTT GGT GGC GCG TAT CGT GCG ACC CTG GGT GAT AAC GCG CTG CCG CGT GGC ACC AGT AGT GCT GCG ATC CGT GAA ATG CTG GGC CTG TTC CAG CAA GCG AAC ACC AAA TGC CCG GAT GCG ACC CTG ATT GCG GGT GGC TAC AGC CAG GGT GCG GCG CTG GCG GCG GCG AGC ATT GAG GAC CTG GAT AGC GCG ATC CGT GAT AAG ATT GCG GGC ACC GTG CTG TTC GGT TAT ACC AAA AAC CTG CAA AAC CGT GGT CGT ATT CCG AAC TAT CCG GCG GAC CGT ACC AAG GTG TTC TGC AAC ACC GGC GAT CTG GTG TGC ACC GGT AGC CTG ATT GTT GCG GCG CCG CAC CTG GCG TAT GGC CCG GAT GCG CGT GGT CCG GCG CCG GAA TTT CTG ATT GAG AAA GTG CGT GCG GTT CGT GGT AGC GCG GGT GGC GGT GGC AGC GGT GGC GGT GGC AGC CAG CAA CGT TTC GAG TGG GAA TTT GAG CAG CAA TAA

We chose to express the fused enzyme in Escherichia coli, as it has the ability to reproduce every few minutes (high replication rate), increasing the speed of decomposition. Thus, it multiplies many times, reproducing many times the copy of its new piece, which turns into a protein. E. coli is also the safest for laboratory processing due to its known properties.

## Reaching out

In the process of researching PETase, its properties, the right enzyme to choose for the fusion process and well as the experimental procedure, we had online meetings with George Kalliolias MD PhD, a physician-scientist trained in Internal Medicine and Rheumatology who works as a Clinical Sciences Director in Early Clinical Development at Regeneron Pharmaceuticals, Inc in New York, as well as the members of the overgrad team MetaThess from Aristotle University of Thessaloniki who participated in the IGEM competition in 2022 and won the award of the Best Diagnostics Project (Overgrad). They helped us to choose the most suitable and efficient method for the creation of the enzyme, so that we could implement our idea despite our limited knowledge in the procedures and terms of biology (due to our team being undergrad) and the lack of equipment and knowledge in the lab, They also explained and clarified difficult and sophisticated biological terms and generally promoted our research.

## References

\* https://www.sciencedirect.com/science/article/abs/pii/S1369703X22003783

\* https://bioresourcesbioprocessing.springeropen.com/articles/10.1186/s40643-022-00532-4

\* https://www.sciencedirect.com/science/article/abs/pii/S0141391016303299

\* https://www.mdpi.com/2076-2607/11/2/328

\* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10577388/

\* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8669999/

\* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10577388/

\* https://link.springer.com/article/10.1007/s10924-022-02435-z#:~:text=It%20has%20been%20reported%20that,been%20used%20as%20mulching%2

\* https://hal.inrae.fr/hal-02545880v1/document

\* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8715580/pdf/12934\_2021\_Article\_1725.pdf

\* Fusion Protein Linkers: Property, Design and Functionality - PubMed

\* How do I design a fusion protein linker? - ResearchGate

\* Inducible Protein Expression Using GeneSwitch Technology - Thermo Fisher Scientific

\* https://www.sciencedirect.com/science/article/pii/S0734975022001124#:~:text=Synthetic%20fusion%20enzymes%20are%20formed,et%20al.%2C%201989.

\* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/

\* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/table/T3/

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## Take a look at the protocols we used in our iGEM project

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