iGEM 2023 — Team thessaly

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

Engineering
Engineering

Introduction

Synthetic biology is an emerging engineering discipline, focused on leveraging the intricate workings of biological systems in order to address contemporary challenges, spanning from climate change to critical medical phenomena. What sets synthetic biology apart from traditional biology is its emphasis on the engineering process, encompassing the designing, building, testing, and iterative learning from these created biological systems.

Overview

During our time in the lab, we successfully completed several Design-Build-Test-Learn (DBTL) cycles. These cycles involved addressing various challenges we faced, ranging from basic troubleshooting of standard procedures like clonings to more intricate aspects related to our overall design. On this page, we will showcase our engineering process and the impact it had on our final design.

Cloning method

To build our constructs, our team followed the GoldenBraid 2.0[1](#references), an advanced and highly efficient cloning method widely used in synthetic biology and genetic engineering. This method involves several key steps, such as the domestication of the DNA parts to ensure compatibility with the GoldenBraid standard grammar and the assembly of parts into higher-level constructs, using universal domesticator plasmids (in our case pUPD2) and destination vectors (in our case pDGB3α1, pDGB3α2 and pDGB3ω1). One of its notable features is the ability to create multi-level constructs, facilitating the construction of intricate genetic circuits. The different levels of GoldenBraid correspond to different stages of construct assembly.

* Level 0: In this level, individual DNA parts are inserted into universal domesticator plasmids and prepared for higher level assembly. For that to happen a process known as domestication must occur, which involves modifying these DNA parts to remove unwanted internal restriction sites and add specific overhangs compatible with the GoldenBraid grammar. Level 0 parts are typically inserted into universal domesticator plasmids, creating what is known as "level 0 constructs." These constructs serve as the building blocks for higher-level assemblies.
* Level 1: Level 1 (alpha plasmids) involves the assembly of multiple level 0 constructs to create complete transcriptional units.
* Level 2: level 1 modules are combined to create more intricate genetic circuits or systems. Specifically, level alpha plasmids can be combined with an omega destination vector to create a construct with 2 transcriptional units.

FadR/PfadBA system

The PhaJ gene encodes the enzyme trans-enoyl-CoA hydratase, an enzyme that catalyzes the linking step between β-oxidation and the PHA producing machinery[2](#references) (see [Design](https://2023.igem.wiki/thessaly/design) page for more details). The overexpression of this enzyme in P. putida would facilitate the efficient conversion of the free fatty acids derived from OMW or E. coli, shifting them away from the β-oxidation pathway and redirecting them towards the PHA biosynthetic pathway[3](#references), making its efficient regulation essential for our system. For this reason, we decided to develop a free fatty acid (FFA)–inducible system for phaJ expression[4](#references), based on FadR, an Escherichia coli transcription factor regulated by fatty acyl-CoAs and the fadBA promoter ([PfadBA](http://parts.igem.org/Part%3ABBa_K4624625)), containing a FadR-recognition site[5](#references) ( Fig. 1.)

![](https://static.igem.wiki/teams/4624/wiki/fig-1.png)

Fig. 1. Schematic representation of the PfadBA-FadR system. In absence of free fatty acids ( FFAs), the consistently produced FadR protein inhibits the function of the  PfadBA promoter, suppressing the  phaJ expression. When FFAs are present, they are activated to acyl-CoAs by acyl-CoA synthase (encoded by fadD).  Acyl-CoAs bind to theFadR protein, allowing the  PfadBA promoter to function properly, leading to the transcription of the  phaJ gene.

Cycle 1 - Characterization of the fadBA promoter

Designing

Since we could not find enough data characterizing the fadBA promoter to an extend that could help us with our decision making, we decided to attempt a basic characterization, by firstly designing a construct with the PfadBA upstream of a reporter gene encoding the SYFP2 ([BBa\_K864100](http://parts.igem.org/Part%3ABBa_K864100)), a monomeric fluorescent protein with optimized folding, maturation and a narrow fluorescence emission spectrum[6](#references), contained in this year’s Distribution kit ( Fig. 2).

![](https://static.igem.wiki/teams/4624/wiki/fig-2.png)

Fig. 2. Schematic representation of the level 1 (alpha) construct we designed for the characterization of the  PfadBA.

Additionally, we designed a level 2 construct with two transcriptional units; the level 1 construct described before and an additional transcriptional unit of the fadR under the control of a constitutive promoter ( Fig. 3) to evaluate the level of inhibition the protein FadR exerts on the promoter.

![](https://static.igem.wiki/teams/4624/wiki/fig-3new.png)

Fig. 3. Schematic representation of the level 2 (omega) constructs we designed for the evaluation of the inhibition fadR imposes on the promoter  PfadBA .

However, due to time constraints, the whole level 2 (omega) construct ( Fig. 3), was not assembled. So, we decided to test only the reporter transcription unit ( Fig. 2), in the E. coli BL21 (DE3) strain. This decision was also based on the fact that FadR is a transcription factor found natively in Escherichia coli [7](#references), so its inhibitory effects could still be noticeable on the fluorescence output of the construct. The sequence of PfadBA was acquired and then domesticated, using the [GoldenBraid Domesticator](https://gbcloning.upv.es/do/domestication/) tool, which removes any internal restriction sites that did not comply with the GoldenBraid standard and adds the appropriate 4-nt 3’ and 5’ flanking overhangs in order for the inserts to be compatible with our level 0 pUPD2 cloning vector.

Building

After the domestication process, the part was ordered and synthesized by IDT. When received, the DNA was resuspended and the cloning procedures began. To get to the desired reporter construct, we began by cloning the PfadBA into a level 0 cloning vector, pUPD2. Through digestion-ligation reaction, DH5α transformation, plasmid isolation and restriction-digestion confirmation ( Fig. 4), we successfully constructed the level 0 construct.

![](https://static.igem.wiki/teams/4624/wiki/fig-4.png)

Fig. 4. Diagnostic digestion of pUPD2\_pfadBA with EcoRI and EcoRV, expected bands (bp): 1309 and 896. Lane 2: pUPD2 (no insert).

The level 0 construct was then combined, via a digestion-ligation reaction, with the Distribution kit’s level 0 constructs of the B0030 RBS ([BBa\_J428032](http://parts.igem.org/Part%3ABBa_J428032)), the syfp2 (BBa\_K864100) and the B0015 double terminator ([BBa\_J428092](http://parts.igem.org/Part%3ABBa_J428092)), in order to build the complete transcriptional unit that we could then test. We successfully built the level 1 (alpha) construct, which was confirmed with a restriction digestion reaction ( Fig. 5).

![](https://static.igem.wiki/teams/4624/wiki/fig-5.png)

Fig. 5. Diagnostic digestion of pDGB3α1\_pfadBA-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): 2327, 2026, 1628 and 1291. Lane 2: pDGB3α1 (no insert).

Once we had the complete construct, we transformed E. coli BL21 (DE3) chemically competent cells with the isolated plasmid. To further confirm that our insert was correct and functional, a single colony was picked, inoculated in LB medium and cultured O/N at 37οC and 210 rpm. The next morning, 5 x 200 ul of the culture were loaded into a black plate with transparent bottom (with the appropriate blanks) and then a fluorescence intensity measurement was taken at wavelengths of 511 nm (excitation) and 529 nm (emission). The tested culture showed distinct fluorescence when compared to the negative control (non-transformed E. coli BL21 (DE3)), so we knew that the construct was indeed functional.

Testing

To adequately characterize PfadBA , we included two controls: 1. a positive control construct which we designed and built, carrying the same reporter gene (syfp2) under the control of the well-documented constitutive Anderson promoter J23118, and 2. non-transformed BL21 (DE3) cells as a negative control. The detailed protocol we followed can be found on our [Experiments](https://2023.igem.wiki/thessaly/experiments) page. In short, single colonies were picked for both constructs, as well as a colony of non-transformed BL21 (DE3) cells and inoculated into 5 ml of LB with the appropriate antibiotic. The cultures were grown O/N at 37οC and 210 rpm. The next day, a dilution was performed to reach an OD600 of approximately 0.02 and a black plate with a transparent bottom was prepared, blanks included. The construct was tested in three different conditions: with the addition of non-dissolved oleic acid, addition of oleic acid dissolved in DMSO[8,9](#references) and without oleic acid. Four biological repeats were performed for each condition, the plate was placed into the plate reader and measurements of absorbance and fluorescence were taken after 6h incubation. The results we got are shown below ( Fig. 6).

![](https://static.igem.wiki/teams/4624/wiki/fig-6-eng.png)

Fig. 6. Normalized fluorescence intensity measurement for pfadBA-syfp-rrnB T1/T7TE construct on three different conditions ( No oleic acid addition, addition of non-dissolved oleic acid, addition of oleic acid dissolved in DMSO) after 6h incubation.

Learning

From the data we got, we noticed that the PfadBA resulted in low expression levels of the SYFP2 protein when compared to our positive control, the PJ23118 Anderson promoter, a result for which we were previously alerted to by Dr. Tsampika, suggesting that a promoter with higher expression rates would be better suited for the adequate transcription of the phaJ gene since the low expression levels could hamper the efficiency of our system. So, we set out to repeat the DBTL cycle in search of a better suited promoter for the phaJ.

Cycle 2 - A more suitable promoter for the phaJ gene

Designing

Going back to the designing stage, we searched for alternative promoters, regulated by the FadR protein. Utilizing the iGEM registry and after thorough literature review, we discovered two synthetic promoters,[PLR](http://parts.igem.org/Part%3ABBa_K4624602) and[PAR](http://parts.igem.org/Part%3ABBa_K4624601)[5](#references). These promoters had previously been characterized as having higher relative strength than PfadBA. Our objective was to evaluate and compare their intensity using the same reporter gene and compare them to the same positive control. To achieve this, we designed two additional reporter constructs, one for PLR and the other for PAR, both regulating the expression of syfp2 (Fig. 7). Again, the promoter sequences were first domesticated for the removal of internal restriction sites not compatible with the GoldenBraid cloning method, as well as the addition of the 3’ and 5’ overhangs.

![](https://static.igem.wiki/teams/4624/wiki/fig-7.png)

Fig. 7. Schematic representation of the standard construct for the characterization of PLR and PAR .

Building

The same cloning strategy was followed, as described above. The -1 domesticated part sequences were successfully inserted into a pUPD2 vector ( Fig. 8).

![](https://static.igem.wiki/teams/4624/wiki/fig-8.png)

Fig. 8. Diagnostic digestion of (1) pUPD2\_pLR and (2) pUPD2\_pAR with EcoRI and EcoRV, expected bands (bp): (1) 1300 and 896, and (2) 1305 and 896. Lane 3: pUPD2 (no insert).

Then, the level 0 parts were used to build the 2 new reporter constructs. Finally, both constructs were built and the inserts were confirmed by restriction-digestion reactions (Fig. 9).

![](https://static.igem.wiki/teams/4624/wiki/fig-9.png)

Fig. 9. Diagnostic digestion of (1) pDGB3α1\_pLR-syfp-rrnB T1/T7TE and (2) pDGB3α1\_pAR-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): (1) 2327, 2017, 1628, 1291 and 58, and (2) 2327, 2022, 1628, 1291 and 58. Lane 3: pDGB3α1 (no insert).

The isolated plasmids were used to transform E. coli BL21 (DE3) chemically competent cells. To further ensure that the correct functional insert was built, we performed a preliminary fluorescence intensity measurement, as in the case of PfadBA. Both constructs exhibited significant fluorescence levels, so we concluded that the clonings were indeed successful.

Testing

Having all the devices transformed into the same E. coli strain, we repeated the experiment with the same conditions. The results we got are shown below ( Fig. 10).

![](https://static.igem.wiki/teams/4624/wiki/fig-10.png)

Fig. 10. Normalized fluorescence intensity measurement for pLR-syfp-rrnB T1/T7TE and pAR-syfp-rrnB T1/T7TE constructs on three different conditions ( No oleic acid addition,  addition of non-dissolved oleic acid,  addition of oleic acid dissolved in DMSO) after 6h incubation.

Learning

Comparing the two new promoters to the PfadBA and the PJ23118 we can clearly see the elevated levels of fluorescence intensity ( Fig. 11). However, as in the first cycle, no significant difference can be seen with the addition of oleic acid. We assume that if we were to test the complete level 2 construct ( Fig. 3), the presence of the additional FadR regulator would cause greater downregulation of the system in the absence of the inducer. However, we must mention that additional experiments are required to clarify which of the two promoters is the most suitable for our system. Considering those results, we deemed the second engineering cycle as a success, since our initial goal to find a higher intensity fatty acyl-CoA-responsive promoter, was achieved.

![](https://static.igem.wiki/teams/4624/wiki/fig-11.png)

Fig. 11. The results of both engineering cycles for all fatty acyl-CoA-responsive promoters in a common chart.

Future

Having acquired these data, to decide on the appropriate promoter for our design, we would have to build two level 2 (omega) constructs, each containing one of the promoters regulating the reporter gene and another transcription unit containing the fadR under the control of the AraC/PBAD system. These two constructs would eventually be transformed into P. putida and then be tested with the addition of oleic acid and a gradient of concentrations of L-arabinose. The ideal promoter would be the one with lower levels of leakiness combined with high induced gene expression at a concentration of oleic acid that is similar to the levels found in OMW. Due to time constraints, we were not able to perform this experiment, but we hope that a future team will be able to.

Laccase secretion

The use of E. coli for the secretion of a functional laccase enzyme would take us a step further towards the holistic detoxification of the OMW[10,11](#references) ( Fig. 12). Although we performed experiments to evaluate the activity of the enzyme (see [Experiments](https://2023.igem.wiki/thessaly/experiments)), we mainly focused on tackling the secretion aspect in an engineering cycle, by reviewing the literature and actually building the constructs to test the secretion of the laccase, using the native peptide from Trametes versicolor as well as 3 different well-documented signal peptides, NSP4 (BBa\_K3606042), PelB (BBa\_K208004) and MalE (BBa\_K1012004) in search of a more efficient secretion system[11,12](#references).

![](https://static.igem.wiki/teams/4624/wiki/fig-12.png)

Fig. 12. The laccase secretion system in E. coli. The  laccase is under the regulation of the araBAD promoter and is secreted from the cell (pac-man), thanks to the fusion with a signal peptide, suitable for protein secretion in bacterial expression systems.

Cycle 1 -Testing the original secretion peptide.

Designing

Our first objective was to test the native signal peptide (Na.SP), originating from Trametes versicolor, since, belonging to different biological kingdoms, we could not be sure about its functionality in E. coli. To test its functionality, we designed a construct where the coding sequence of the laccase was fused with the sequence of the fluorescent protein SYFP2 at the 3’-terminal ( Fig. 13). This sequence was then domesticated to the GoldenBraid standards and ordered to be synthesized by IDT .

![](https://static.igem.wiki/teams/4624/wiki/fig-13.png)

Fig. 13. Schematic representation of the level 1 (alpha) construct of the laccase fused with syfp and carrying its native peptide.

Building

Once the level -1 DNA fragment arrived, we began the process of cloning the insert into the pUPD2 vector, turning it into a level 0 construct. Once this procedure was successful, with confirmation from restriction-digestion reaction and gel electrophoresis ( Fig. 14), the isolated plasmid was combined by digestion-ligation reaction with the level 0 constructs syfp2 and B0015 double terminator provided by this year’s iGEM distribution kit, and the AraC/PBAD regulatory system we had previously constructed. The complete construct was successfully built, verified by restriction-digestion reaction and gel electrophoresis. ( Fig. 15).

![](https://static.igem.wiki/teams/4624/wiki/fig-14.png)

Fig. 14. Diagnostic digestion of pUPD2\_Na.SP-laccase with EcoRV and NotI, expected bands (bp): 1562, 1143 and 903. Lane 2: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/fig-15.png)

Fig. 15. Diagnostic digestion of pDGB3α1\_pBAD-Na.SP-laccase-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): 2327, 2049, 1628, 1361, 1291, 610, 423, 185, 163, 81 and 58. Lane 2: pDGB3α1 (no insert).

The isolated plasmid was transformed into E. coli BL21 (DE3) chemically competent cells and to further ensure that the insert is functional we performed the diagnostic fluorescence measurement, as described previously. The tested samples exhibited significant fluorescence levels which further confirmed the insertion of the functional construct.

Testing

The detailed protocol we followed can be found on our [Experiments](https://2023.igem.wiki/thessaly/experiments) page. In short, a single colony carrying the level 2 (omega) construct was used to inoculate 5 ml of LB medium, with the appropriate antibiotic, and the culture was grown O/N at 30οC and 160 rpm. E. coli BL21 (DE3) cells with the construct containing the syfp2 under the control of the J23118 Anderson promoter and non-transformed E. coli BL21 (DE3) cells were used as negative controls. The next day, O/N cultures were diluted in order to reach the same OD600 and the addition of L-arabinose to a final concentration of 10 mM followed. Finally, after a 6h incubation the cultures were centrifuged in order to measure absorbance and fluorescence of the supernatant and the pellet fraction, after cell pellet resuspension[14](#references). Four biological repeats were performed for each condition, the plate was placed into the plate reader and measurements were taken. The results of the normalized fluorescence intensity measurements for each fraction are depicted in Fig. 16.

![](https://static.igem.wiki/teams/4624/wiki/fig-16.png)

Fig. 16. Normalized fluorescence intensity of the supernatant and pellet fraction for pBAD-Na.SP-laccase-syfp-rrnB T1/T7TE construct after 6h incubation with 1,5% arabinose, (-) control 1: pJ23118-syfp-rrnB T1/T7TE, (-) control 2: non-transformed cells.

Learning

From those results we can conclude that the signal peptide from Trametes versicolor is not suitable for laccase secretion from E. coli, since supernatant fluorescence intensity exhibited low values compared with the negative controls. Thus, we decided to go through another cycle, in order to find and test additional, well characterized signal peptides for protein secretion in bacterial expression systems.

 Cycle 2 -Testing signal peptides NSP4, PelB and MalE

Designing

After a thorough evaluation of the related literature, the 3 signal peptides NSP4[15](#references),  PelB[16](#references) and MalE[17](#references) were selected to test with the laccase. To evaluate the secretion efficiency of these peptides, we substituted the sequence of the native signal peptide of the laccase with each of the three peptides. These 3 coding sequences were then fused with the sequence of the syfp as illustrated in Fig. 17.

![](https://static.igem.wiki/teams/4624/wiki/fig-17.png)

Fig. 17. Schematic representation of the standard level 1 (alpha) construct of the laccase fused with the syfp and carrying one of the selected signal peptides (NSP4, PelB and MalE).

Building

Once we designed the constructs, the sequences of the 3 domesticated signal peptides with the appropriate overhangs were ordered from IDT. The cloning procedures began with the insertion of the -1 parts into the pUPD2 vector, to create the level 0 constructs. Once successfully built, with confirmation by restriction-digestion reaction ( Fig. 18 and 19), the isolated level 0 parts of the signal peptides were combined with the level 0 parts for the laccase, the syfp2, the B0015 double terminator and the AraC/PBAD , to build the complete constructs ( Fig. 17).

![](https://static.igem.wiki/teams/4624/wiki/fig-18.png)

Fig. 18. Diagnostic digestion of (1) pUPD2\_NSP4 and (2) pUPD2\_PelB with EcoRV and NotI, expected bands (bp): (1) 1143, 903 and 122 and (2) 1143, 903 and 128. Lane 3: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/fig-19-new-eng.png)

Fig. 19. Diagnostic digestion of pUPD2\_MalE with EcoRI and EcoRV, expected bands (bp): 1290 and 896. Lane 2: pUPD2 (no insert).

Following the standard protocol for digestion-ligation reaction, we were able to build the complete transcriptional units, again confirmed by restriction-digestion reaction and electrophoresis ( Fig. 20).

![](https://static.igem.wiki/teams/4624/wiki/fig-20-new-eng.png)

Fig. 20. Diagnostic digestion of (1) pDGB3α1\_pBAD-NSP4-laccase-syfp-rrnB T1/T7TE, (2) pDGB3α1\_pBAD-PelB-laccase-syfp-rrnB T1/T7TE and (3) pDGB3α1\_pBAD-MalE-laccase-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): (1) 2327, 2049, 1628, 1361, 1291, 625, 423, 185, 163, 81 and 58, (2) 2327, 2049, 1628, 1361, 1291, 613, 423, 185, 163, 81 and 58 and (3) 2327, 2049, 1628, 1361, 1291, 625, 423, 185, 163, 81 and 58. Lane 4: pDGB3α1 (no insert).

Testing

We once again began by transforming all 3 test devices into bacteria of the same E. coli strain and we repeated the same steps in order to measure absorbance and fluorescence of both the supernatant and the pellet fraction. The protocol we followed for this experiment is available in detail in [Experiments](https://2023.igem.wiki/thessaly/experiments) page. The results are depicted in Fig. 21. It can be seen clearly that NSP4 consistently gave the best results followed by the signal peptide MalE, while PelB and the native signal peptide constructs gave the lower secretion output.

![](https://static.igem.wiki/teams/4624/wiki/fig-21.png)

Fig. 21. Normalized fluorescence intensity of the supernatant and pellet fraction for pBAD-NSP4-laccase-syfp-rrnB T1/T7TE, pBAD-PelB-laccase-syfp-rrnB T1/T7TE and pBAD-MalE-laccase-syfp-rrnB T1/T7TE constructs after 6h incubation with 1,5% arabinose, (-) control 1: pJ23118-syfp-rrnB T1/T7TE, (-) control 2: non-transformed cells.

Learning

After processing the results, we concluded that NSP4 signal peptide might be the most suitable for our purposes, since both supernatant fluorescence intensity value and supernatant fluorescence intensity per pellet fluorescence intensity value were the highest among the tested signal peptides (Fig. 22).

![](https://static.igem.wiki/teams/4624/wiki/fig-22.png)

Fig. 22 . The results of both engineering cycles, for the normalized fluorescence intensity of the supernatant and the pellet fraction, in a common chart.

Conclusion

After completing two DBTL cycles, each comprising two iterations, we have gained a significantly deeper understanding of the oPHAelia system. This progress brings us closer to envisioning the promising future it may hold.

### References

1. Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez, GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology, Plant Physiology, Volume 162, Issue 3, July 2013, Pages 1618–1631, https://doi.org/10.1104/pp.113.217661
2. Tsuge T, Fukui T, Matsusaki H, Taguchi S, Kobayashi G, Ishizaki A, Doi Y. Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS Microbiol Lett. 2000 Mar 15;184(2):193-8. doi: 10.1111/j.1574-6968.2000.tb09013.x. PMID: 10713420.
3. Vo MT, Lee KW, Jung YM, Lee YH. Comparative effect of overexpressed phaJ and fabG genes supplementing (R)-3-hydroxyalkanoate monomer units on biosynthesis of mcl-polyhydroxyalkanoate in Pseudomonas putida KCTC1639. J Biosci Bioeng. 2008 Jul;106(1):95-8. doi: 10.1263/jbb.106.95. PMID: 18691538.
4. Ma Y, Zheng X, Lin Y, Zhang L, Yuan Y, Wang H, Winterburn J, Wu F, Wu Q, Ye JW, Chen GQ. Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas. Metab Eng. 2022 Jul;72:325-336. doi: 10.1016/j.ymben.2022.04.003. Epub 2022 May 2. PMID: 35513297.
5. Zhang, F., Carothers, J. & Keasling, J. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol 30, 354–359 (2012). https://doi.org/10.1038/nbt.2149
6. Kremers GJ, Goedhart J, van Munster EB, Gadella TW Jr. Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry. 2006 May 30;45(21):6570-80. doi: 10.1021/bi0516273. PMID: 16716067.
7. DiRusso CC, Heimert TL, Metzger AK. Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A. J Biol Chem. 1992 Apr 25;267(12):8685-91. Erratum in: J Biol Chem 1992 Nov 5;267(31):22693. PMID: 1569108.
8. Römer A, Rawat D, Linn T, Petry SF. Preparation of fatty acid solutions exerts significant impact on experimental outcomes in cell culture models of lipotoxicity. Biol Methods Protoc. 2021 Dec 3;7(1):bpab023. doi: 10.1093/biomethods/bpab023. PMID: 35036572; PMCID: PMC8754478.
9. B. Atef, R.A.H. Ishak, S.S. Badawy, R. Osman. Exploring the potential of oleic acid in nanotechnology-mediated dermal drug delivery: an up-to-date review. J. Drug Deliv. Sci. Technol. (2021), Article 103032, 10.1016/j.jddst.2021.103032
10. Mo Y, Lao HI, Au SW, Li IC, Hu J, Yuen HM, Cheong WM, Lo OLI, Seak LCU. Expression, secretion and functional characterization of three laccases in E. coli. Synth Syst Biotechnol. 2021 Dec 7;7(1):474-480. doi: 10.1016/j.synbio.2021.12.002. PMID: 34938906; PMCID: PMC8665402.
11. Ntougias S, Baldrian P, Ehaliotis C, Nerud F, Merhautová V, Zervakis GI. Olive mill wastewater biodegradation potential of white-rot fungi--Mode of action of fungal culture extracts and effects of ligninolytic enzymes. Bioresour Technol. 2015;189:121-130. doi: 10.1016/j.biortech.2015.03.149. Epub 2015 Apr 3. PMID: 25879179.
12. Sørensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol. 2005 Jan 26;115(2):113-28. doi: 10.1016/j.jbiotec.2004.08.004. PMID: 15607230.
13. Freudl, R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 17, 52 (2018). https://doi.org/10.1186/s12934-018-0901-3
14. Linton E, Walsh MK, Sims RC, Miller CD. Translocation of green fluorescent protein by comparative analysis with multiple signal peptides. Biotechnol J. 2012 May;7(5):667-76. doi: 10.1002/biot.201100158. Epub 2011 Sep 20. PMID: 21834133.
15. Han S, Machhi S, Berge M, Xi G, Linke T, Schoner R. Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express. 2017 Dec;7(1):93. doi: 10.1186/s13568-017-0394-1. Epub 2017 May 12. PMID: 28497288; PMCID: PMC5427057.
16. Lei SP, Lin HC, Wang SS, Callaway J, Wilcox G. Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J Bacteriol. 1987 Sep;169(9):4379-83. doi: 10.1128/jb.169.9.4379-4383.1987. PMID: 3040692; PMCID: PMC213756.
17. Bassford PJ Jr, Silhavy TJ, Beckwith JR. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19-31. doi: 10.1128/jb.139.1.19-31.1979. PMID: 110778; PMCID: PMC216822.

Experiments
Experiments

Experiments

During our time in the lab, we designed and conducted the experiments that would offer the most valuable insights at the outset of our project. Following thorough discussions, we pinpointed specific aspects of our project's design to concentrate on, aiming to provide a practical and realistic demonstration. For better comprehension, we encourage you to first read the ‘Cloning method’ section on our  [Engineering](https://2023.igem.wiki/thessaly/%20engineering%20) page.

Experiment 1: FapR/PFR1 system characterization

One of the goals we set for our experiments was to shed light on a pivotal component of the malonyl-CoA-based negative feedback system we have implemented in our design, the PFR1 promoter. (for more details, refer to our [Design](https://2023.igem.wiki/thessaly/design) page ). To comprehensively characterize this component, we devised two experiments:

1. Testing a level 1 construct featuring the PFR1 promoter controlling the syfp2 reporter (Fig. 1) to assess its relative strength.

2. Testing a level 2 construct that includes the aforementioned construct but also incorporates a transcriptional unit of the fapR , regulated by the AraC/PBAD system (illustrated in (Fig. 2).

For more details, refer to our  [New Basic Part](https://2023.igem.wiki/thessaly/new-basic-part) page.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-1.png)

Fig. 1 .Schematic representation of the level 1 (alpha) construct for the characterization of the  PFR1 (1st experiment).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-2.png)

Fig. 2.Schematic representation of the level 2 (omega) construct for the characterization of the PFR1 (2nd experiment).

Process

We obtained and domesticated the sequences of these three components using the [Domestication tool]. This process involved eliminating internal restriction sites incompatible with the GoldenBraid 2.0 grammar while also incorporating the appropriate 3' and 5' overhangs. To confirm the accuracy of this domestication, we imported the modified parts into Snapgene to simulate the restriction-insertion reaction and ensure that the correct overhangs were added to each part.
After verifying all the parts, we were ready to begin the cloning process. The initial step involved the insertion of the fragments into a pUPD2 vector to generate level 0 constructs, following our standard digestion-ligation cloning protocol. We maintained the optimal 3:1 insert:vector molar ratio, as suggested by our protocol.
Continuing, 10 ul of the digestion-ligation reaction was used to transform DH5α chemically competent cells. The following day, single colonies were picked (through blue-white screening), inoculated in 5 ml of LB medium, with the appropriate antibiotic, and finally the cultures were incubated overnight at 37°C and 210 rpm. These cultures were used to isolate the plasmids, which were subsequently subjected to restriction-digestion reactions to confirm the successful insertion of the fragment (Fig. 3, 4 and 5).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-3.png)

Fig. 3 . Diagnostic digestion of pUPD2\_pFR1 with EcoRI and EcoRV, expected bands (bp): 1305 and 896. Lane 2: pUPD2 (no insert)

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-4.png)

Fig. 4. Diagnostic digestion of pUPD2\_araC/pBAD with EcoRI and EcoRV, expected bands (bp): 2107, 896 and 466. Lane 2: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-5.png)

Fig. 5. Diagnostic digestion of pUPD2\_fapR with EcoRV, expected bands (bp): 1477 and 1196. Lane 2: pUPD2 (no insert).

The resulting level 0 constructs, along with selected parts from the distribution kit, such as syfp2 ([BBa\_K864100](http://parts.igem.org/Part%3ABBa_K864100)), B0015 double terminator ([BBa\_J428092](http://parts.igem.org/Part%3ABBa_J428092)), and B0030 RBS ([BBa\_J428032](http://parts.igem.org/Part%3ABBa_J428032)), were combined to build 3 level 1 constructs : 1. a construct carrying the PFR1 with the syfp2 downstream, 2. the PJ23118 Anderson promoter, again regulating syfp2, and 3. the fapR gene under the control of AraC/PBAD .
After a few failed attempts, we tried to optimize our digestion-ligation protocol, adjusting the insert:vector ratio depending on a few parameters. Specifically, the length of the final construct was taken into account. In cases where the final construct surpassed 2.0 kb, higher quantities of both vector and insert were used (e.g. 40-120 fmol instead of 20-80 fmol respectively ). Secondly, the length of the part itself was also considered, where the ratio of smaller parts was increased.
With those adjustments to our process, we managed to successfully build the level 1 construct we required for the 1st part of this experiment (Fig. 1), the PFR1 strength evaluation.

 PFR1 strength evaluation

Having the correct level 1 construct, we were ready to start the 1st part of this experiment. The verified plasmid was used to transform E. coli BL21 (DE3) chemically competent cells. Single colonies were picked and inoculated in 5 ml LB medium with 5 ul kanamycin and cultured O/N at 37οC and 210 rpm.
The following day, the cultures were taken to the cold room and intermediate dilutions 1:10 were prepared with the addition of 500 ul of culture in 4,5 ml of LB broth + kanamycin. Then intermediate dilutions were measured for absorbance at 600 nm and the values were inserted in the culture dilution calculator, [https://static.igem.org/websites/technology/interlab/2023/protocolsculture-dilution-calculator-template.xlsx] provided by the InterLab committee. Using those measurements, we prepared final dilutions for all three cultures (positive, negative and test construct). Fluorescence measurements at wavelengths of 511 nm (excitation) and 529 nm (emission) were taken after 6 hours of incubation and the results we got are shown below (Fig. 6). For the interpretation of the results see [New Basic Part](https://2023.igem.wiki/thessaly/new-basic-part) page.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-6.png)

Fig. 6. Normalized fluorescence intensity of the pFR1-syfp-rrnB T1/T7TE construct, compared to a positive control (pJ23118-syfp-rrnB T1/T7TE) and a negative control (non-transformed E. coli cells) after 6h incubation.

Evaluation of FapR - mediated PFR1 repression

After testing the level 1 construct, we were ready to use it to build the level 2 construct, in order to perform the second part of the experiment, the evaluation of the repression FapR poses on the promoter. To achieve this, we had to combine the two level 1 constructs containing the PFR1-driven reporter module and the PBAD-driven FapR repressor module via a new digestion-ligation reaction. The standard protocol was followed with the appropriate optimizations. Eventually, we were able to build the construct (Fig. 2).
The verified plasmid was used to transform E. coli BL21 (DE3) chemically competent cells. The next day, single colonies were picked and inoculated in 5 ml LB + 5 ul spectinomycin and cultured O/N at 37οC and 210 rpm. The O/N cultures were retrieved the next day, intermediate 1:10 dilutions were prepared and their OD600 was measured. From those intermediate dilutions we prepared final dilutions for the level 2 construct, the positive and negative controls. The appropriate amounts of arabinose were added in each sample (+ the blanks) so in the end we were left with 5 repeats of each culture, each containing increasing concentrations of arabinose. The results we got are shown below (Fig. 7). For the interpretation of the results see [New Basic Part](https://2023.igem.wiki/thessaly/new-basic-part) page.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-7.png)

Fig. 7. Normalized fluorescence intensity for the level 2 construct (pDGB3ω1\_pFR1-syfp-rrnB T1/T7TE + araC/pBAD-fapR-rrnB T1/T7TE) in different concentrations of L-arabinose, after 3h and 6h incubation.

Experiment 2: PfadBA and alternative promoters’ characterization

The objective of this experiment was to create six constructs: three level 1 constructs aimed at assessing the relative strength of the PfadBA , PLR and PAR promoters, and three level 2 constructs that included the aforementioned level 1 constructs along with fadR to evaluate the level of promoter inhibition exerted by the regulator. This would help us get closer to selecting the optimal promoter, one that would offer the highest sensitivity and efficiency in regulation. However, we were only able to construct and test the level 1 constructs and determine the strengths of the PfadBA , PLR , and PAR promoters (Fig. 8) (see [Engineering](https://2023.igem.wiki/thessaly/engineering)  for more details on the results).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-8.png)

Fig. 8. Schematic representation of the standard construct for the characterization of PLR and PAR .

 Process

The sequences for PfadBA , PLR and PAR were acquired and domesticated using the domestication tool, to be compatible with the GoldenBraid standards. The domesticated part sequences were then imported into Snapgene to simulate the restriction-insertion reaction procedure and verify the 3’ and 5’ overhang compatibility. Once all constructs were checked, all the fragments were ordered by IDT.
The DNA fragments were then resuspended and the first digestion-reaction was performed for the insertion of the parts into a pUPD2 domestication vector. For the level 0 clonings, the insert:vector ratio was kept at a 3:1. Following the standard protocol, 5 ul of the digestion-ligation reactions were used to transform E. coli DH5α chemically competent cells. Plates were streaked and 10ul of IPTG and 40 ul of X-gal were added for the blue-white screening. The following day, white colonies were picked, inoculated in 5 ml LB broth + 5 ul chloramphenicol, and finally the cultures were incubated O/N at 37οC and 210 rpm. From the O/N cultures, we performed plasmid isolation and next restriction-digestion to confirm that we had indeed isolated the right plasmids. After a few rounds of this procedure, we had successfully acquired all level 0 constructs (Fig. 9 and 10) and we continued with the level 1 clonings.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-9.png)

Fig. 9. Diagnostic digestion of pUPD2\_pfadBA with EcoRI and EcoRV, expected bands (bp): 1309 and 896. Lane 2: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-10.png)

Fig. 10. Diagnostic digestion of (1) pUPD2\_pLR and (2) pUPD2\_pAR with EcoRI and EcoRV, expected bands (bp): (1) 1300 and 896, and (2) 1305 and 896. Lane 3: pUPD2 (no insert).

The same pattern as with the previous experiment was observed where the first attempts for the construction of the level 1 parts failed. Thus, the same optimization took place, adjusting the quantities of the smaller parts, and the constructs were eventually successful (Fig. 11 and 12). Once we had successfully built our constructs, we were ready to test the devices

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-11.png)

Fig. 11. Diagnostic digestion of pDGB3α1\_pfadBA-syfp-t1te with BsaHI, expected bands (bp): 2327, 2026, 1628 and 1291. Lane 2: pDGB3α1 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-12.png)

Fig. 12. Diagnostic digestion of (1) pDGB3α1\_pLR-syfp-t1te and (2) pDGB3α1\_pAR-syfp-t1te with BsaHI, expected bands (bp): (1) 2327, 2017, 1628, 1291 and 58, and (2) 2327, 2022, 1628, 1291 and 58. Lane 3: pDGB3α1 (no insert).

To begin, once we had all the devices in our hands, we transformed E. coli BL21 (DE3) chemically competent cells. The next day, single colonies were picked for the preparation of liquid cultures, which were incubated O/N at 37οC and 210 rpm. The cultures were retrieved for the preparation of 3 final dilutions for each construct, including positive and negative controls. The constructs were tested in three different conditions: with the addition of non-dissolved oleic acid, addition of oleic acid dissolved in DMSO (final concentration 10 mM in each case) and without oleic acid. The plate was prepared with four repeats for each condition and placed into the plate reader. Measurements were taken at 0h and 6h timepoints. The final results are depicted in Fig. 13. For the interpretation of the results see [Engineering](https://2023.igem.wiki/thessaly/engineering) page.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-13.png)

Fig. 13. Normalized fluorescence intensity for pfadBA-syfp-rrnB T1/T7TE, pLR-syfp-rrnB T1/T7TE and pAR-syfp-rrnB T1/T7TE constructs on three different conditions (No oleic acid addition, addition of non-dissolved oleic acid, addition of oleic acid dissolved in DMSO) after 6h incubation. E. coli BL21 (DE3) cells with pJ23118-syfp-rrnB T1/T7TE construct and non-transformed cells were used as (+) and (-) control respectively.

Experiment 3: Laccase secretion and functional characterization

The secretion of a functional laccase from E. coli is essential for our system since it would mean that the poly-phenolic content of OMW would be degraded to the maximum. To establish that, we conducted two experiments: 1. an assay to determine the location of the enzyme inside or outside the cell and 2. an assay for the calculation of the enzyme activity, to prove functionality of the secreted laccase. Below, we describe the procedures for each of the two aspects and our results.

 Process

To test the secretion of the enzyme, we decided that the best way for us would be to fuse the genetic signal peptide-laccase entity with the sequence of SYFP2 fluorescent protein, the reporter we had chosen for the rest of our experiments. However, to evaluate the enzyme activity, we deemed that the enzyme should be tested without being fused with the fluorescent protein, since that might interfere with its functionality. So, for each peptide, including the native, 2 constructs were built: one containing the signal peptide-laccase sequence in fusion with the syfp2 (Fig. 14) and one where the coding sequence of the mature laccase carries upstream only the appropriate signal peptide (Fig. 15), making a total of 8 level 1 constructs.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-14.png)

Fig. 14. Schematic representation of the standard level 1 (alpha) construct of the laccase fused with the syfp and carrying one of the signal peptides (Na.SP, NSP4, PelB and MalE).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-15.png)

Fig. 15. Schematic representation of the standard level 1 (alpha) construct of the laccase carrying one of the signal peptides (Na.SP, NSP4, PelB and MalE).

  The sequences for the signal peptides and the laccase were domesticated to the Golden Braid standards. However, because of the Golden Braid method's characteristics, specific 3' and 5' overhangs are limited to use in a single position within the transcription unit. So, to create constructs where the SP-laccase genetic entity is fused with the syfp2 and constructs where it is not, we needed to create two distinct sets of overhangs for the laccase. These sets were designated for the B4 and the B4+B5 position respectively within the transcriptional unit. For the addition of the overhangs, we designed primer pairs specifically for each of the 2 positions we wanted to assemble laccase in. The primers we designed for this procedure have been added to the iGEM registry and can be found on our Parts page.
  Once the compatibility of the parts was confirmed, the parts were ordered to be synthesized by IDT. The DNA fragments were resuspended and we performed 2 different PCR reactions, one for each position of the laccase in our transcriptional units (B4 and B4+B5). The samples were then prepared for gel electrophoresis, and once we confirmed that the main product was indeed in the appropriate height, we followed the standard protocol for Gel Extraction. Then the 2 laccase parts, along with the rest of the domesticated sequences were inserted into pUPD2 part domestication following standard protocol. A diagnostic restriction digestion reaction and gel electrophoresis followed for each of the level 0 constructs to verify their correct insertion (Fig. 16-19).
Finally, the resulting level 0 constructs were combined with the ones of AraC/PBAD and B0015 double terminator, for the creation of the complete level 1 (alpha) constructs (Fig. 15 and 16). The inserts were once again confirmed through restriction-digestion reactions and electrophoresis (Fig. 20 and 21).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-16.png)

Fig. 16.Diagnostic digestion of (1) pUPD2\_NSP4, (2) pUPD2\_PelB and (3) pUPD2\_Na.SP with EcoRV and NotI, expected bands (bp): (1) 1143 903 and 122, (2) 1143, 903 and 128 and (3) 1143, 903 and 125. Lane 4: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-17.png)

Fig. 17. Diagnostic digestion of pUPD2\_MalE with EcoRI and EcoRV, expected bands (bp): 1290 and 896. Lane 2: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-18.png)

Fig. 18. Diagnostic digestion of pUPD2\_laccase(B4) with EcoRV and NotI, expected bands (bp): 1562 1143 and 903. Lane 2: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-19.png)

Fig. 19. Diagnostic digestion of pUPD2\_laccase(B4+B5) with EcoRV and NotI, expected bands (bp): 1563, 1143 and 903. Lane 2: pUPD2 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-20.png)

Fig. 20.Diagnostic digestion of (1) pDGB3α1\_pBAD-Na.SP-laccase-syfp-rrnB T1/T7TE (2) pDGB3α1\_pBAD-NSP4-laccase-syfp-rrnB T1/T7TE, (3) pDGB3α1\_pBAD-PelB-laccase-syfp-rrnB T1/T7TE and (3) pDGB3α1\_pBAD-MalE-laccase-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): (1) 2327, 2049, 1628, 1361, 1291, 610, 423, 185, 163, 81 and 58, (2) 2327, 2049, 1628, 1361, 1291, 625, 423, 185, 163, 81 and 58, (3) 2327, 2049, 1628, 1361, 1291, 613, 423, 185, 163, 81 and 58 and (4) 2327, 2049, 1628, 1361, 1291, 625, 423, 185, 163, 81 and 58. Lane 5: pDGB3α1 (no insert).

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-21.png)

Fig. 21. Diagnostic digestion of (1) pDGB3α1\_pBAD-Na.SP-laccase-rrnB T1/T7TE, (2) pDGB3α1\_pBAD-NSP4-laccase-rrnB T1/T7TE, (3) pDGB3α1\_pBAD-PelB-laccase-rrnB T1/T7TE and (4) pDGB3α1\_pBAD-MalE-laccase-rrnB T1/T7TE with EcoRV and BsaHI, expected bands (bp): (1) 2327, 1628, 1291, 1290, 807, 610, 554, 423, 185, 163, 81 and 58, (2) 2327, 1628, 1291, 1290, 807, 607, 554, 423, 185, 163, 81 and 58 (3) 2327, 1628, 1291, 1290, 807, 613, 554, 423, 185, 163, 81 and 58 and (4) 2327, 1628, 1291, 1290, 807, 625, 554, 423, 185, 163, 81 and 58. Lane 5: pDGB3α1 (no insert).

Having all 8 constructs ready, we were able to begin with the first experiment, the secretion evaluation assay.

Secretion evaluation

For the secretion evaluation, we decided to measure both the absorbance and fluorescence intensity of the supernatant fraction and pellet fraction after centrifugation. Single colonies of the 4 constructs were picked, inoculated in 5 ml of LB medium with the appropriate antibiotic and cultured O/N at 37oC and 210 rpm. The next day, the OD600 of the O/N cultures was measured and used to prepare dilutions with the same starting value. Arabinose was added to a final concentration of 100mM for the induction of the devices and the samples were placed for incubation at 30oC and 160 rpm. After 6 hours the cultures were retrieved and 2 ml of each was transferred in 2 ml eppendorf tubes and centrifuged at 11,000 x g for 3 minutes. The supernatant was collected and loaded onto a black plate with a transparent bottom. The cell pellets were resuspended in fresh LB broth with the appropriate antibiotic and then also loaded onto the plate. Measurements of the absorbance were taken at 600 nm and for the fluorescence at 511 nm for excitation and 529 nm for emission. Two negative controls were used, E.coli BL21 (DE3) cells with a construct containing the syfp2 under the control of the J23118 Anderson promoter and non-transformed cells. For the interpretation of our results we got (Fig. 22) please see our Engineering page.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-22.png)

Fig. 22. Normalized fluorescence intensity of the supernatant and pellet fraction for pBAD-Na.SP-laccase-syfp-rrnB T1/T7TE, pBAD-NSP4-laccase-syfp-rrnB T1/T7TE, pBAD-PelB-laccase-syfp-rrnB T1/T7TE and pBAD-MalE-laccase-syfp-rrnB T1/T7TE constructs after 6h incubation with 1,5% arabinose, (-) control 1: pJ23118-syfp-rrnB T1/T7TE, (-) control 2: non-transformed cells.

Laccase activity

  For this experiment, we followed the ABTS assay designed to assess laccase activity in the culture of the white-rot fungus Trametes Versicolor. The protocol was provided by Dr. Panagiotis Karas, a Post-Doctoral researcher in the Plant Biotechnology lab of the Department of Biochemistry and Biotechnology. The basis of this assay involves the oxidation of the non phenolic dye ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by laccase to the radical cation ABTS\*+, a soluble chromogen that is green in color and can be determined spectrophotometrically at 425 nm. After discussing the protocol with Dr. Karas, he suggested that we should optimize it by adjusting the ratio of bacterial culture to the ABTS reagent. This adjustment is necessary since E. coli is unlikely to produce the same quantity of the enzyme as fungi naturally do.
Having all the needed devices transformed into E. coli BL21 (DE3) cells, liquid cultures were prepared and incubated O/N at 37oC and 210 rpm. The next day, these cultures were retrieved and used in order to prepare the final cultures with the same starting OD600 values. Addition of L-arabinose to a final concentration of 100mM was followed and the cultures were incubated at 30oC and 160 rpm for 6h. So, after 6h incubation we began the procedure by adding 1,2 ml of sodium tartrate 0.1 M (pH of 4,5 adjusted with NaOH addition) in a cuvette. Next, 0,6 ml of the bacterial culture was added, the cuvette was placed in the spectrometer and the absorbance was set to zero. To initiate the reaction, 0,4 ml of ABTS solution 3 mM were added in the cuvette and pipetted three to four times to mix. The instrument was set back to zero again and the timer started. Absorbance measurements at 425 nm were taken every 20 seconds for 2 minutes [1](#references). The above procedure was repeated 3 times to get as reliable results as possible. The results for each construct are summarized in Fig. 23. Non-transformed E. coli BL21 (DE3) cells were used as negative control.

| Time (sec) | 20 | | | 40 | 60 | 80 | 100 | 120 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Na.SP | 0,029 | | | 0,039 | 0,043 | 0,046 | 0,048 | 0,049 |
| 0,026 | | | 0,033 | 0,037 | 0,041 | 0,044 | 0,047 |
| 0,019 | | | 0,021 | 0,022 | 0,026 | 0,028 | 0,030 |
| NSP4 | 0,067 | | | 0,078 | 0,082 | 0,084 | 0,085 | 0,086 |
| 0,016 | | | 0,067 | 0,076 | 0,081 | 0,085 | 0,087 |
| 0,02 | | | 0,025 | 0,075 | 0,083 | 0,088 | 0,09 |
| PelB | 0,043 | | | 0,049 | 0,052 | 0,056 | 0,058 | 0,06 |
| 0,03 | | | 0,042 | 0,046 | 0,048 | 0,049 | 0,05 |
| 0,038 | | | 0,047 | 0,051 | 0,054 | 0,056 | 0,056 |
| MalE | 0,012 | | | 0,045 | 0,051 | 0,056 | 0,059 | 0,062 |
| 0,003 | | | 0,034 | 0,04 | 0,044 | 0,05 | 0,051 |
| 0,007 | | | 0,037 | 0,043 | 0,047 | 0,051 | 0,054 |
| (-) control | 0,026 | | | 0,034 | 0,038 | 0,043 | 0,047 | 0,05 |
| 0,003 | | | 0,013 | 0,014 | 0,025 | 0,027 | 0,028 |
| 0,003 | | | 0,008 | 0,021 | 0,03 | 0,034 | 0,039 |

Fig. 23.Fig. 23. Absorbance measurements at 425 nm of the 4 constructs (Fig. 15) designed for laccase activity calculation. Measurements were taken every 20 sec after the addition of the ABTS reagent for 2 minutes. Non-transformed E. coli BL21 (DE3) cells were used as (-) control.

Once the measurements were collected, we calculated the enzyme activity based on the absorbance changes using the formula:
      U / L = (ΔΑ x Vt)/(Δt x ε x Vs)
U enzyme activity umol min-1 L-1, ΔΑ (final absorbance - initial absorbance) the maximum absorbance difference measured at 2 min, Vt total reaction volume (ml), Δt elapsed time between two measurements (min), ε molar extinction coefficient (L mol-1 cm-1) = 36 L mol-1 cm-1, Vs sample volume (ml). The enzyme activity values for each sample are depicted in Fig. 24.

![](https://static.igem.wiki/teams/4624/wiki/exp-fig-24.png)

Fig. 24. Laccase activity of the four different constructs estimated with ABTS assay, after 6h incubation with 1,5% arabinose. Non-transformed E. coli BL21 (DE3) cells were used as (-) control.

Comparing the above results with those of the laccase secretion, we notice that although there is no perfect measurements matching for the PelB and MalE signal peptides, the NSP4 signal peptide exhibited the best results in both aspects. Therefore, the results of both experiments indicate the selection of NSP4 as the ideal one for our system.

Lab Book

At the outset of our experiments, we recognized firsthand the critical importance of accurately and comprehensively documenting a scientist's daily work. This documentation is crucial for interpreting results and drawing credible, logical conclusions. Consequently, we promptly made the decision to maintain a digital lab notebook, which encompassed nearly allall of the experiments we conducted throughout the iGEM competition. Our goal was to present our work in as realistic a manner as possible, with the aim of providing future teams with a valuable introduction and insight into the role of a wet lab member in an iGEM team.

Standard protocols

Here you can find all the protocols our team used during our time in the lab, in great detail.

Materials

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Vectors

PUPD2 (UNIVERSAL GB PART DOMESTICATOR)

The pUPD2 vector serves as a universal domestication plasmid designed for level 0 parts. Typically used in synthetic biology and the domestication of DNA segments for GoldenBraid assembly, this vector includes a chloramphenicol resistance gene and features two cloning sites with BsmBI cutting sites. Positioned between these cloning sites is a transcriptional unit responsible for the expression of β-galactosidase.

PDGB3Α1 AND PDGB3Α2 VECTORS

The pDGB3α vectors are versatile domestication plasmids designed for level α transcription units. These vectors find common application in synthetic biology and the domestication of DNA segments for GoldenBraid assembly. Each pDGB3α vector comprises a kanamycin resistance gene and features two cloning sites with BsaI restriction sites. Positioned between these cloning sites is a transcriptional unit responsible for the expression of β-galactosidase.

PDGB3Ω VECTOR

The pDGB3ω vector serves as a universal domestication plasmid designed for level ω constructs. This plasmid is commonly employed in synthetic biology and the domestication of DNA fragments intended to be used for GoldenBraid assembly. It carries a spectinomycin resistance gene and features two cloning sites with BsmBI restriction sites. Positioned between these cloning sites is a transcriptional unit responsible for the expression of β-galactosidase.

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BACTERIAL STRAINS

ESCHERICHIA COLI DH5Α CELLS

E. coli DH5α Competent Cells were used for our cloning experiments since they are engineered to maximize transformation efficiency.

ESCHERICHIA COLI BL21 DE(3)

E. coli BL21 DE(3) were used to perform experiments were we meant to detect and measure expression of certain proteins since this strain is specifically engineered to for high yields of production.

P. putida KT2440

P. putida KT2440 was used as the chassis organism we have chosen in our design

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NUTRIENT MEDIA FOR BACTERIAL CULTURES

LB BROTH

Luria Broth (LB) Broth was used in liquid form, dissolved in ddH2O and sterilized, for liquid bacterial cultures. The desired antibiotic should also be added.

LB AGAR

Luria Broth (LB) Agar was dissolved in ddH2O, sterilized and added in Petri dishes for bacterial cultures. The desired antibiotic should also be added before its solidification.

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ANTIBIOTICS

The antibiotics used were the following: ampicillin, chloramphenicol, kanamycin, and spectinomycin.

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 ENZYMES

For the Restriction Enzyme Digestion and Ligation cloning experiments, the following enzymes were used: BsaI-HF, BsmBI, Esp3I and T4 Ligase.

For the Polymerase Chain Reaction, the Q5® High-Fidelity DNA Polymerase and Kapa Taq polymerase were used.

For the diagnostic digestion experiments, the following enzymes were used:
BamHI-HF, BsaHI,, EcoRI-HF, EcoRV-HF, HindIII-HF, NotI, KpnI.

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 DNA ELECTROPHORESIS IN AGAROSE GEL

- TAE 50X ELECTROPHORESIS BUFFER
  The DNA electrophoresis buffer with a final volume of 1L contains 100 ml EDTA (0.5M), 242g Tris Base, 57.1 liquid acetic acid and ddH2O to final volume. The working solution is diluted from 50x to 1x.
- GEL LOADING DYE, BLUE 6X
  This loading solution consists of one tracking dye for agarose gel electrophoresis and contains SDS, which often results in sharper bands.
- NEB 1KB DNA LADDER MOLECULAR WEIGHT CONTROL
- AQUEOUS SOLUTION OF ETHIDIUM BROMIDE (10MG/ML)

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 Reagents

- Reagents for isolation of plasmid DNA from bacteria: “NucleoSpin® plasmid” kit from Macherey-Nage

### References

1. Snapp EL. Fluorescent proteins: a cell biologist's user guide. Trends Cell Biol. 2009 Nov;19(11):649-55. doi: 10.1016/j.tcb.2009.08.002. Epub 2009 Oct 8. PMID: 19819147; PMCID: PMC2784028.

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}