iGEM 2025 — Team bologna

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Scientific research thrives on transparency and reproducibility. Our laboratory notebook serves as a comprehensive chronicle of our experimental journey, documenting every protocol, observation, success, and setback encountered throughout the project. This detailed record not only ensures the traceability of our results but also provides valuable insights for future teams building upon our work. Here you will find our day-to-day laboratory activity that contributed to shaping HERO and HELMET project, advancing our understanding of *Rhodococcus opacus* as a chassis for bioremediation.

## HERO Notebook

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## HELMET Notebook

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# ENGINEERING SUCCESS

Parts

Software

Hardware

Education

Science Communication

### 🧬 Parts

### Genetic Component Engineering

Our parts engineering focused on developing and optimizing genetic constructs for expression in *Rhodococcus*. Through multiple iterations, we addressed challenges in construct amplification, assembly methods, and promoter characterization.

#### Iteration 1: Q5 Amplification + Homology with Fusogenic Region

◀

DESIGN

##### 🔍 DESIGN

The construct was designed for amplification using Q5 high-fidelity polymerase, with homology arms flanked by a region for fusion PCR. PCR primers for ROCI 1 homology region were designed for accuracy, minimal error rate, and compatibility with downstream cloning. ROCI 1 region was discovered in paper [1]. Primers for their amplification also added 20bp homology regions with the extremities of linear construct. A deeper insight on LoxPort design can be found on part [BBa\_2566C7GE](https://registry.igem.org/parts/bba-2566c7ge) documentation.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle1-design.webp)

Original LoxPort OF amplification strategy with Q5 polymerase.

##### 🔨 BUILD

PCR amplification was performed using Q5 polymerase, generating the desired homology regions. PCR was also performed to amplify the linear construct.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle1-build.webp)

Agarose gel demonstrating successful PCR amplification of homology regions.

##### 🧪 TEST

PCR confirmed successful amplification of the desired fragments only for homology regions, showing bands of the expected size. No PCR results were obtained for the linear construct, neither with high-GC enhancer buffer addition.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle1-test.webp)

Scheme of successful parts in this iteration.

##### 📊 LEARN

The observed inefficiency suggested that even with Q5’s fidelity, amplification failed, probably due to a mixture of construct length and high GC content. We therefore decided to amplify smaller fragments.

▶

#### Iteration 2: Division into Multiple Parts + Fusogenic Region

◀

DESIGN

##### 📐 DESIGN

To manage construct size and complexity, we designed primers to divide the construct into multiple smaller fragments. The principle was that smaller fragments would be easier to amplify and assemble.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle2-design.webp)

Strategy scheme of second attempt at LoxPort OF amplification with Q5 polymerase.

##### 🔨 BUILD

Fragments were generated by PCR and prepared for fusion PCR.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle2-build.webp)

Agarose gel of PCR obtained LoxPort OF fargments.

##### 🧪 TEST

Although individual fragments amplified correctly, assembling them into a single construct showed inefficient and incomplete results.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle2-test.webp)

Scheme of successful parts in this iteration.

##### 📊 LEARN

The results showed that dividing the construct introduced additional junctions that increased assembly difficulty. This highlighted the tradeoff between fragment manageability and fusion PCR efficiency. Q5 polymerase did not seem to be able to amplify the whole construct. We therefore decided to amplify the construct in a plasmid.

▶

#### Iteration 3: Assembly in pGEM

◀

DESIGN

##### 📐 DESIGN

To amplify the native linear construct, we decided to clone it into pGEM vector. We needed to perform A-tailing and ligation to obtain a plasmid to transform into *E.coli.*

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle3-design.webp)

Strategy scheme of LoxPort OF cloning into pGEM for amplification.

##### 🔨 BUILD

The linear synthesis result was A-tailed following NEB’s klenow protocol. It was then ligated in pGEM vector, with T tailing. Then, transformation in *Escherichia coli* DH5-α was performed with ligation results.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle3-build.webp)

Agarose gel of colony PCR for construct verification.

##### 🧪 TEST

DNA extraction from colonies yielded no plasmid DNA.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle3-test.webp)

Scheme of successful parts in this iteration.

##### 📊 LEARN

No successful transformation was obtained. Also, not much linear construct was left: a working amplification was needed to proceed with the experiments, so we tried PCR with a different polymerase.

▶

#### Iteration 4: Extaq Amplification

◀

DESIGN

##### 📐 DESIGN

An alternative strategy used Extaq polymerase for amplification, anticipating robust amplification even for challenging sequences, followed by fusion PCR with previously obtained plasmids.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle1-design.webp)

Strategy scheme of first attempt at PCR amplification with ExTaq polymerase.

##### 🔨 BUILD

PCR with Extaq successfully generated the desired fragments, on which fusion PCR with homology regions was performed.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle4-build.webp)

Agarose gel of PCR results from Ex-taq polymerase. Among much non-specific signal, some good bands have been isolated by gel extraction.

##### 🧪 TEST

While PCR products were clear and matched expected sizes, attempts to assemble construct and homology regions fragments consistently failed.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle4-test.webp)

Scheme of successful parts in this iteration.

##### 📊 LEARN

The results revealed that successful amplification did not guarantee successful fusion PCR. Sequence context, particularly high GC content and secondary structure, likely interfered with amplification, together with construct length. This suggested the need for sequence optimization and to have the construct directly inserted in a plasmid.

▶

#### Iteration 5: Twist Bioscience Synthesis - Reduced GC + Medium-Copy Plasmid

◀

DESIGN

##### 📐 DESIGN

To overcome previous challenges, the construct was redesigned with reduced GC content, already fused homology regions and was synthesized directly in a plasmid by Twist Bioscience. LoxPort construct ([BBa\_2566C7GE](https://registry.igem.org/parts/bba-2566c7ge)) was the result of this optimization. It was placed in a medium-copy backbone to limit toxicity, while allowing sufficient yield.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle5-design.webp)

Strategy scheme of plasmid amplifiction of synthetic LoxPort construct.

##### 🔨 BUILD

The optimized construct was synthesized and delivered in plasmid form, bypassing the challenges of PCR-based amplification and in vitro assembly.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle5-build.webp)

Agarose gel of restriction enzyme digestion for plasmid verification.

##### 🧪 TEST

The synthesized plasmid worked as expected, remaining stable in the chassis and yielding reliable DNA for downstream applications.

##### 📊 LEARN

This success confirmed that high GC content and sequence complexity were key barriers in previous cycles. The iteration demonstrated that codon optimization and backbone choice are critical parameters, feeding directly into future design strategies for complex constructs.

▶

#### Iteration 6: Construct Integration in pKSAC45

◀

DESIGN

##### 📐 DESIGN

LoxPort construct ([BBa\_2566C7GE](https://registry.igem.org/parts/bba-2566c7ge)) was designed to be integrated into the pKSAC45 backbone, with the goal of performing double recombination inside *Rhodococcus*. The choice of pKSAC45 [2] was based on its compatibility with the target organism and its ability to carry the desired genetic elements for stable integration.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle6-design.webp)

Strategy scheme of synthetic LoxPort cloning in pKSac45, for *Rhodococcus* double recombination.

##### 🔨 BUILD

Utilizing SphI and EcoRI enzymes, the construct was successfully cloned into pKSAC45. This modified plasmid was then introduced into *E. coli* for amplification and plasmid preparation.

After plasmid DNA extraction failure of pKSac+LoxPort, PCR was attempted to see whether recombination happened.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle6-build.webp)

Agarose gel electrophoresis of PCR amplification on different LoxPort fragments for integration verification.

##### 🧪 TEST

Transformation in *E. coli* yielded colonies resistant to kanamycin, indicating that the plasmid was likely present and functional. Unfortunately, the following miniprep attempts consistently failed to recover sufficient DNA. Only one attempt showed plasmid extraction, but sequencing proved that it was a self-ligated form of pKSac45.

PCR reactions have been attempted to test whether recombination occurred and if it was site-specific or not.

![](https://static.igem.wiki/teams/5716/engineering/parts/parts-cycle6-test.webp)

Scheme of successful parts in this iteration.

##### 📊 LEARN

The discrepancy between antibiotic resistance and failed plasmid extraction suggested that the plasmid might have been integrated in *Escherichia coli* DH5-α genome. This revealed limitations in using pKSAC45 as a backbone for amplification in this chassis. A redesigned approach may involve using a medium copy plasmid backbone better suited for *E. coli* propagation. Also, linear plasmid tansformation approach may be a possibility, as described by De Lorenzo et al. (2018) [1]

▶

### Successful Promoter Constructs (p2, pB2, pB3)

#### Constitutive Promoter Characterization

◀

DESIGN

##### 📐 DESIGN

A set of constitutive promoters was selected to evaluate their relative strengths in driving expression of sfGFP in *Rhodococcus*. Reporter system was based on pTip-QT1 plasmid. The reporter gene sfGFP [BBa\_25BE2EBO](https://registry.igem.org/parts/bba-25be2ebo) was based on part [BBa\_J428326](https://registry.igem.org/parts/bba-j428326), and domesticated by removal of SapI and BsrGI restriction sites for cloning inside the MCS.

Promoter set was developed starting from the paper from Round et al. 2019 [3]. We chose to study p2 and p10 promoters, since they showed a stronger constitutive activity than pNit in *Rhodococcus jostii* RHA1. We applied the same optimization strategy used in the paper for p10 promoter (shortening of tested sequence) on both p10 and p2 promoters, creating M2 and M6 from p10 (as performed in the paper), as well as pB2 and pB3 as novel promoters form p2.

Furthermore, we analyzed pLac, whose constitutive activity in *Rhodococcus* was identified in 2015 by Hetzler et al [4].

Each promoter was designed to be amplified by PCR. Primers added lg-10 RBS from pTip [2] right downstream of the promoter, and added BsrGI and NcoI restriction sistes, for substituting the resulting promoter+RBS fragment to native pTip promoter and RBS.

Positive control was performed with pNit-QT1 plasmid, carrying the widely used in *Rhodococcus* pNit constitutive promoter, on which MCS sfGFP was cloned, following the same approach as pNit-QT1 cloning with NcoI and NotI restriction enzymes.

![Promoter optimization map](https://static.igem.wiki/teams/5716/design/p2-promoter-optimization.webp)

Figure 6. SnapGene map of the three promoters, highlighting the different primers used.

##### 🔨 BUILD

sfGFP was amplified, domesticated and flanked by NcoI and NotI restriction sites by PCR with sfGFP\_f ([BBa\_25TME224](https://registry.igem.org/parts/bba-25tme224)) and sfGFP\_r ([BBa\_25RAR337](https://registry.igem.org/parts/bba-25rar337)) primers . It was cloned in MCS of pTip-QT1 and pNit-QT1 plasmids, using NcoI and NotI enzymes.

Promoters were amplified by PCR with specific primers (which added a standard RBS and flanked the sequence with BsrGI and NcoI restriction sites) and cloned in pTip+sfGFP reporter plasmid, replacing native promoter and RBS.

Reporter plasmids were first transformed into *E. coli* by heat-shock, selected on ampicillin plates, extracted and verified by plasmid purification and digestion. Validated constructs were then transformed by electroporation into *Rhodococcus opacus* PD630 and plated on tetracycline-containing plates.

![Promoter gel electrophoresis](https://static.igem.wiki/teams/5716/results/restriction-promoters.webp)

Gel electrophoresis performed on enzymatic digestion of reporter plasmids used for *Rhodococcus opacus* PD630 transformation. pNit + sfGFP was digested with NcoI and NotI restriction enzymes, pTip-based reporter plasmids were cut with NcoI and BsrGI restriction enzymes. All bands are as expected.

##### 🧪 TEST

PCR amplification gave poor results for p10 and its derivative M2 and M6, so they were discarded.

Colonies obtained from *Rhodococcus opacus* PD630 transformations have been re-streaked and re-cultured to ensure stability. For each promoter construct, four colonies were randomly selected, grown in liquid medium to OD₆₀₀ = 0.5, and 3 for each promoter were tested in technical triplicates for fluorescence. Promoters p2, pB2, pB3 an pLac produced measurable expression. A detailed protocol can be found at [experiments page](https://2025.igem.wiki/bologna/experiments#fluorescence).

![Promoter strength graph](https://static.igem.wiki/teams/5716/results/promoters-to-pnit.avif)

Bar graph showing constitutive promoter strength in *Rhodococcus opacus* PD630.

##### 📊 LEARN

The results demonstrated that while several constitutive promoters drove detectable sfGFP expression, differences showed due to technical limitations. Specifically, p10 amplification failed because primers from the literature were not compatible with our PCR conditions. pB3 promoter showed weaker activity than expected, probably because the optimization strategy used removed important regions, like -10. These failures suggest the need for primer redesign and improved annealing temperatures. The cycle highlighted both successful constitutive promoter characterization and optimization, as well as critical redesign steps for future iterations.

Anyways, promoters in the library showed consistent fluorescence among replicates and different strength between each other, opening up to a possibility to study more promoters using techniques established here.

▶

#### Parts Engineering: Key Takeaways

* **Sequence optimization is critical:** High GC content and secondary structures were major barriers overcome through professional synthesis with codon optimization
* **Backbone selection matters:** Different backbones show varying compatibility with chassis organisms and cloning methods
* **Fragment strategy affects efficiency:** Smaller fragments improve amplification but can decrease assembly efficiency
* **Promoter characterization success:** Successfully validated multiple constitutive promoters (p2, pB2) for *Rhodococcus* expression
* **Primer validation is essential:** Literature primers require experimental validation for new PCR conditions

### 💻 Software:

#### Iteration 1 - Excluding currency metabolites

◀

DESIGN

##### 🔍 DESIGN

Once our KEGG network was available, we started looking for some paths. Sadly, they were full of unrealistic shortcuts through
currency metabolites. Inspired by the work of Kim et al [5], we created a list of currency metabolites to exclude from the graph.

##### 🔨 BUILD

We created a list of 58 metabolites to remove from the graph, available on our GitLab repository in the folder *data/*

![Example path](https://static.igem.wiki/teams/5716/engineering/kg-connectivity.avif)

Node connectivity distribution. Additional to literature, this provided useful insights on which metabolites to exclude.

##### 🧪 TEST

The median of the average path lengths increased from around 3 to around 8. Similar results were obtained also by Kim et al.

![Path lenght after filtering](https://static.igem.wiki/teams/5716/engineering/kgpath-lengths-filtered.avif)

KEGG pathway lengths after filtering

##### 📊 LEARN

Introducing a small bias by excluding some arbitrary currency metabolites allows to create a realistic metabolic representation.

▶

#### Iteration 2 - Biological Meaningful Paths

◀

DESIGN

##### 🔍 DESIGN

Even though removing currency metabolites greatly improved the topology of the network, pathways proposed were still not meaningful.
Other subtle shortcuts were still present. We then decided to implement a penalty system to assign weights to edges.

##### 🔨 BUILD

Penalty is composed as a sum of the following:

* Annotation level: KEGG white edge (+0), KEGG white edge (+2), RetroPath2.0 prediction (+4)
* Chemical dissimilarity using Tanimoto distance (+3 for chemical dissimilarity >=99%)
* Molecular weight change penalty: huge gains or losses in molecular weight are penalized (0-3)
* RetroPath2.0 Score, only for predicted reactions (0-4)

The lowest the penalty, the highest the biological relevance.

![Example path](https://static.igem.wiki/teams/5716/engineering/mw-pen-plot.avif)

Values of the MW change penalty as function of MW Loss or Gain. To favor degradation, the same % change is more penalized when it is gained.

##### 🧪 TEST

Several empirical tests for known paths confirmed the value of this system.

![Example path](https://static.igem.wiki/teams/5716/engineering/example-path.avif)

Example path from Terephthalic acid to Succinyl-CoA generated by our tool

##### 📊 LEARN

Introding a small a certain degree of bias is necessary to obtain biologically relevant paths.
Further improvements might be represented by a "path penalty", considering more than one reaction at a time,
or by integrating the concept of "module", like KEGG Modules.

▶

### 🔧 Hardware: Device Development

#### Iteration 1 – Optimization for lipase solubilization

◀

DESIGN

##### 🔍 DESIGN

The initial aim was to achieve complete solubilization of lipase while maintaining its catalytic activity. Preliminary tests indicated that lipase exhibited partial insolubility and aggregation under standard buffer conditions; consequently, a systematic investigation of both chemical and physical factors influencing solubility was planned.

##### 🔨 BUILD

Different buffer systems were evaluated, including sodium phosphate and Tris-HCl at different concentrations and pH values: none of these conditions provided full solubilization.
 Subsequent trials incorporated several additives, such as glycerol, Tween 20, Triton X-100, DMSO, and isopropanol, alongside various physical treatments (shaking, orbital waves, vortexing). Despite these modifications, lipase remained only partially soluble, and aggregation persisted.
 Further optimization involved testing bovine serum albumin (BSA) at different concentrations as a potential stabilizing agent.

##### 🧪 TEST

A buffer formulation consisting of 1.5% BSA in 1 M Tris-HCl (pH 8) enabled complete solubilization of lipase without detectable aggregation: the enzyme remained stable under incubation and ready for subsequent enzymatic assays.

##### 📊 LEARN

The findings demonstrated that the presence of BSA plays a crucial role in preventing aggregation and enhancing enzyme stability, likely by shielding hydrophobic regions and improving dispersion. The optimized buffer composition was established as the standard condition for all further experiments, providing a reproducible and reliable foundation for analyses.

▶

#### Iteration 2 – Lipase activity assay

◀

DESIGN

##### 🔍 DESIGN

After achieving full solubilization, the next objective was to quantify and optimize lipase activity using *p*-nitrophenyl palmitate (pNPP) as the substrate: the goal was to identify the enzyme concentration providing a stable, linear and sensitive colorimetric response.

##### 🔨 BUILD

Activity assays were conducted using a Varioskan multimode plate reader set at 410 nm, employing a concentration range of 10–350 U/mL of lipase: each reaction was performed under identical conditions, with appropriate controls lacking enzyme or substrate.

##### 🧪 TEST

At concentrations below 100 U/mL, the enzymatic activity was minimal, whereas concentrations above 200 U/mL resulted in rapid saturation of the absorbance signal.
 An optimal performance was observed at 125 U/mL, yielding a strong and linear response within the measurable range.

![](https://static.igem.wiki/teams/5716/engineering/hardware/chimica1.webp)
![](https://static.igem.wiki/teams/5716/engineering/hardware/chimica2.webp)

Lipase activity essay results.

##### 📊 LEARN

This optimization confirmed 125 U/mL as the ideal working concentration for lipase under the established assay conditions: excessive enzyme loading was shown to compromise linearity through signal saturation.

These insights provide key parameters for integrating lipase into the complete enzymatic cascade and for guiding the refinement of enzyme ratios in subsequent calibration and device assembly stages.

▶

#### Iteration 3 – Calibration curve with palmitic acid

◀

DESIGN

##### 🔍 DESIGN

The design of this cycle focuses on establishing a robust calibration curve for the biosensor using palmitic acid as a model compound. This includes defining the range of TAGs concentrations to be tested, selecting appropriate enzymatic assay conditions, and setting the parameters for data collection to ensure high precision and reproducibility.

##### 🔨 BUILD

This phase would involve preparing the calibration samples with different concentrations of palmitic acid, setting up the enzymatic reactions under the defined conditions, and configuring the detection system for quantitative signal acquisition.

##### 🧪 TEST

To be continued!

##### 📊 LEARN

To be continued!

▶

#### Iteration 4 – Paper-based biosensor optimization

◀

DESIGN

##### 🔍 DESIGN

The aim of this cycle is to optimize the performance of the paper-based biosensor. The design phase includes outlining different enzyme immobilization strategies (e.g., covalent binding, physical adsorption) and identifying possible improvements in signal detection, such as alternative colorimetric or optical readouts.

##### 🔨 BUILD

This phase would consist of fabricating paper-based prototypes incorporating the selected immobilization approaches and preparing the setups for comparative evaluation of the different sensing formats.

##### 🧪 TEST

To be continued!

##### 📊 LEARN

To be continued!

▶

### 📚 Education: Outreach & Learning Programs

#### Iteration 1 - University lessons

◀

DESIGN

##### 🔍 DESIGN

We designed interactive lessons For university students enrolled in the elective courseViral and Microbial Biotechnology (link al corso) during their Bachelor’s degree in Biotechnology aimed at keeping students engaged, encouraging participation, testing their knowledge, and reinforcing concepts through explanations.

##### 🔨 BUILD

The lessons included interactive elements such as quizzes (prepared with Quizziz) and **hands-on plasmid** construction exercises while explaining our project.

[Project presentation at University](https://static.igem.wiki/teams/5716/engineering/education/ita-version-project-presentation-university.pdf)
[QUIZ for Univesity lecture](https://static.igem.wiki/teams/5716/engineering/education/ita-version-project-presentation-university.pdf)

##### 🧪 TEST

This strategy effectively captured students’ attention. However, sometimes quiz solutions did not work as intended, as **interruptions** during explanations caused a loss of focus.

##### 📊 LEARN

The software used for the quizzes proved cumbersome. In the future, we would structure this part more carefully beforehand or include **physical** materials that allow students tosee and interact with the concepts directly, enhancing their understanding and engagement.

▶

#### Iteration 2 - High school students

◀

DESIGN

##### 🔍 DESIGN

After facing several challenges while teaching university students, we realized we needed a new strategy to explain the field of synthetic biology in a **simpler** and more **engaging** way. Since high school students are less specialized than university students, we had to find an approach that could make complex concepts accessible without losing their attention. The best solution we found — since quizzes wouldn’t be as effective — was to create a board game!

##### 🔨 BUILD

Before preparing the lessons, we focused on designing the board game itself. Our goal was to clearly represent lab activities and the steps required to bring synthetic biology to life. We started by playtesting the game within our team to refine its dynamics, and then involved PhD students from our lab to ensure that the scientific content was accurate and relevant. Later, the lessons were structured to introduce students to the theoretical concepts underlying the game’s dynamics, so they could better understand the scientific principles behind each action.

[Synthetic biology lecture for High School](https://static.igem.wiki/teams/5716/engineering/education/eng-version-synthetic-biology-lecture-hs.pdf)

##### 🧪 TEST

We tested the game with fifth-year high school students during synthetic biology classes. After a short introduction to the fundamentals — revisiting core concepts such as the central dogma and DNA — we explained the game’s rules. Each session lasted about two hours: the first 30 minutes were dedicated to explanations, followed by about one and a half hours of gameplay.

##### 📊 LEARN

The game includes a large amount of information — after all, representing lab life in a board game is no easy task!It is designed to be played for at least an hour, mirroring the reality that even a single laboratory procedure can take hours to complete. During the first lessons, we learned how to organize playtesting sessions more efficiently by dividing each class into three groups of three students. This setup allowed us to run complete game sessions smoothly and ensure that all students could fully engage with the material. This interactive approach proved far more effective than the one used with university students.

▶

#### Iteration 3 - Feedback form

◀

DESIGN

##### 🔍 DESIGN

After noticing some issues in previous university lessons, we decided to introduce the feedback form for high school students to collect suggestions, improve future sessions and make them more engaging. We therefore designed a **feedback form**specifically aimed at evaluating how well our lessons were received. The questionnaire was implemented only for the high school lessons.

##### 🔨 BUILD

form was created using **Google Forms**and was designed to assess students’ interest and understanding of the topic before and after the lesson. It was divided into four sections:

* Section A – Interest and Knowledge: gauging prior knowledge and initial interest.
* Section B – Methods and Activities: evaluating how the board game helped understanding.
* Section C – Overall Experience: whether the lesson was enjoyable and what could be improved.
* Section D – Future Improvements: suggestions for further enhancement.

[Students survey results](https://static.igem.wiki/teams/5716/engineering/education/ita-version-survey-students.pdf)

##### 🧪 TEST

The questionnaire was administered immediately after the high school lesson, with participation being completely voluntary.

##### 📊 LEARN

Students provided very useful feedback. Out of around 40 participants, we received **21 completed responses**, which allowed us to confirm the excellent results we had observed during the sessions. A summary of the results is included in the annexed data.

[Students satisfaction](https://static.igem.wiki/teams/5716/engineering/education/students-satisfaction.pdf)

▶

### Lab workshops

#### Iteration 1 - Teacher training course

◀

DESIGN

##### 🔍 DESIGN

We designed the first set of experimental activities for laboratory training courses aimed at high school teachers. Since the available equipment was more **limited** than in our usual work environment, we needed to plan a **quick, simple, and reliable** project that could both explain the principles of synthetic biology and teach teachers new skills to bring back to their classrooms.

##### 🔨 BUILD

The lessons were divided into three parts: bioinformatics, synthetic biology, and experimental activity. For the bioinformatics module, we created a **ChimeraX-based session** to visualize and compare the amino acid sequences of GFP and its derivatives, showing how specific mutations can shift fluorescence emission spectra.
 In the synthetic biology section, we introduced key concepts and assembly techniques, using a quiz to engage teachers before the practical part.
 The experimental activity focused on applying Gibson Assembly to construct a plasmid containing GFP and mCherry, as well as on **designing primers** for specific PCR reactions. Each part of the experiment was pre-prepared by the team, so teachers could focus on performing the core procedures in the lab.

[High School teachers presentation](https://static.igem.wiki/teams/5716/engineering/education/ita-version-professors-presentation.pdf)

##### 🧪 TEST

The full 10-hour session was divided into 2.5 hours of bioinformatics, 2.5 hours of synthetic biology, and 5 hours of experimental work. The experimental section presented some challenges — the Gibson Assembly had not been pre-tested, resulting in transformed cells expressing only GFP rather than the complete assembled construct. The primers we designed for amplifying STRs from human genomic DNA worked, but the gel resolution was low.

![](https://static.igem.wiki/teams/5716/engineering/education/sfgfp-prof1.webp)
![](https://static.igem.wiki/teams/5716/engineering/education/sfgfp-prof2.webp)
![](https://static.igem.wiki/teams/5716/engineering/education/piastre-prof.webp)

Plates from lab workshops for High School teachers.

##### 📊 LEARN

We learned that the theoretical aspects of synthetic biology can be effectively **adapted** for teachers and, by extension, their students. However, the experimental part requires **optimization**. Gibson Assembly should be fine-tuned to ensure reliable results even when performed by less experienced users, and gel electrophoresis conditions must be adjusted depending on the desired resolution to improve visualization of PCR products.

▶

#### Iteration 2 - Complementary Oprional Project (COP)

◀

DESIGN

##### 🔍 DESIGN

We designed a30-hour POC (Complementary Operational Programme)for fourth-year high school students, aimed at introducing motivated and aware participants to synthetic biology and the main steps required to apply it in the lab. Building on the structure of the previous teacher training course, we prepared a program combining lessons in bioinformatics**,**synthetic biology**,** andhands-on experimental work.

##### 🔨 BUILD

The teacher training course served as a pilot for this program. The lessons will follow a similar structure, featuring simplified introductory bioinformatics sessions compared to the teacher version, and a fully lab-based experimental plan for the synthetic biology module. Given the 30-hour duration, students will also have the opportunity to experience the preparation and setup phase of the experiments, which had been omitted from the teacher course due to time constraints. To ensure the experimental module works smoothly, we will test Gibson Assembly protocols in advance, optimizing all steps for classroom conditions. If the assembly does not succeed as expected, we have a backup plan: we ordered a plasmid from **IDT** that allows expression of mCherry under the control of a different antibiotic than the GFP already included in the educational kit, so students will still be able to observe the desired results.

##### 🧪 TEST

To be continued in January!

##### 📊 LEARN

To be continued in January!

▶

### Surveys

#### Iteration 1 - Assessment of public knowledge on bioremediation and biotechnology

◀

DESIGN

##### 🔍 DESIGN

We created a survey to assess the public's knowledge and interest in our project, bioremediation, and our chassis—our preferred future application field.

##### 🔨 BUILD

The survey included specific questions about bioremediation knowledge and public perception. We deliberately varied terminology to identify potential biases, such as reactions to terms like "GMO" and "bacteria."

##### 🧪 TEST

The survey was distributed through social media channels to evaluate public perception. We analyzed the responses and created data visualizations to identify key trends.

[Survey results plots](https://static.igem.wiki/teams/5716/engineering/education/survey-result.pdf)

##### 📊 LEARN

The survey revealed significant gaps in public knowledge about bioremediation. We identified strong negative biases, particularly regarding bacteria and GMOs, which are often perceived as dangerous or undesirable due to societal misconceptions and lack of accurate information.

▶

#### Iteration 2 - Improving public perception of bioremediation and GMOs through social media

◀

DESIGN

##### 🔍 DESIGN

Based on the survey findings, we identified a need to address misinformation and challenge common biases. We decided to create an Instagram reel addressing the most controversial questions and concerns raised by respondents.

##### 🔨 BUILD

We analyzed the survey results to identify critical misconceptions. We then produced a direct-response video where team members took turns addressing these key questions and concerns in an accessible, conversational format.

##### 🧪 TEST

We evaluated the effectiveness of our educational approach by tracking audience engagement metrics, including views, likes, shares, comments, and overall interaction patterns. To see what we did go to [to our Instagram page](https://www.instagram.com/p/DOOSV4UjD9N/)!

##### 📊 LEARN

The video received 594 views and 25 likes. While engagement was moderate, we recognized that our distribution strategy could have been more effective. Since the original survey was distributed through private channels and the response video was posted on a different platform (our Instagram profile), many survey respondents may not have seen our educational content. Despite this limitation, the direct-response format proved effective for addressing misconceptions and challenging biases rooted in insufficient public knowledge about biotechnology and bioremediation.

▶

### 🗣 Science Communication

#### Iteration 1 – First period

◀

DESIGN

##### 🔍 DESIGN

We designed our science communication strategy to share engaging content on social media. The model was based on successful scientific dissemination accounts, creating mostly carousel like posts.

##### 🔨 BUILD

The communication content was created and integrated into our Instagram profile and posted on a biweekly basis. It served as a testing ground, where the outreach strategy could be tested and observed in action.

##### 🧪 TEST

The effectiveness of our communication strategy was assessed through audience interactions, including likes, shares, comments, and engagement trends.

##### 📊 LEARN

We observed low engagement and a slow growth in follower, questioning whether the type and amount of content that we were creating was most fitting for our audience. These learnings were used to redesign and refine our communication strategy for improved effectiveness in future posts.

▶

#### Iteration 2 – Second period

◀

DESIGN

##### 🔍 DESIGN

We contacted several science influencers asking for advice and guidance in the production of new types of content that could be more engaging for our audience. Stefano Bertacchi showed interest in the project and became our official advisor for communication.

##### 🔨 BUILD

With the help of Stefano, we created new types of content that integrated also memes and new trends, expanding our platforms to tik tok and LinkedIn. We started posting more frequently and posts were also re-shared on Stefano’s socials to expose them to a bigger audience.

##### 🧪 TEST

The effectiveness of our communication strategy was assessed through audience interactions, including likes, shares, comments, and engagement trends and growth in followers.

##### 📊 LEARN

We observed that by posting more content our page reached a broader audience, and therefore more interactions, likes and comments. This was a positive result, but we were noticing an imbalance between the actual scientific content and the more light-hearted content, so we had to create something new.

▶

#### Iteration 3 – Third period

◀

DESIGN

##### 🔍 DESIGN

We observed what other teams were posting, and we saw that we weren’t using Instagram stories at their full potential. We decided to create a weekly column called ‘Thursday Science Pills’ where we talked about the most interesting articles of the week.

##### 🔨 BUILD

We created a set of four stories containing the title of the article and a small description of the contents.

##### 🧪 TEST

The effectiveness of our new content was assessed through audience interactions and likes.

##### 📊 LEARN

We saw our page grow both in followings, interactions and likes and establishing a balance between scientific and funny contents.

▶

## References

▼

* 1. DeLorenzo DM, Rottinghaus AG, Henson WR, Moon TS. Molecular Toolkit for Gene Expression Control and Genome Modification in*Rhodococcus opacus* PD630. *ACS Synthetic Biology*. 2018;7(2):727-738. doi: <https://doi.org/10.1021/acssynbio.7b00416>
* 2. Nakashima N, Tamura T. Isolation and Characterization of a Rolling-Circle-Type Plasmid from *Rhodococcus erythropolis* and Application of the Plasmid to Multiple-Recombinant-Protein Expression. *Applied and Environmental Microbiology*. 2004;70(9):5557-5568. doi: <https://doi.org/10.1128/aem.70.9.5557-5568.2004>
* 3. Round JW, Roccor R, Eltis LD. A biocatalyst for sustainable wax ester production: re-wiring lipid accumulation in*Rhodococcus*to yield high-value oleochemicals. *Green Chemistry*. 2019;21(23):6468-6482. doi: <https://doi.org/10.1039/c9gc03228b>
* 4. Hetzler S, Bröker D, Steinbüchel A. Saccharification of Cellulose by Recombinant *Rhodococcus opacus* PD630 Strains. *Applied and Environmental Microbiology*. 2013;79(17):5159-5166. doi: <https://doi.org/10.1128/aem.01214-13>

{% endblock %}

{% extends "layout.html" %}
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{% block title %}EXPERIMENTS{% endblock %}
{% block hero\_image %} ![HERO Experiments](https://static.igem.wiki/teams/5716/header/experiments.webp) {% endblock %}
{% block menu\_title %} Experiments {% endblock %}
{% block menu\_content %}

[Research Overview](#overview)

[Protocols Quick Access](#protocolsbuttons)

[Producing pKSAC45 + the construct](#exp1)

[Promoter Characterization Study](#exp2)

[PROTOCOLS](#protocols)

{% endblock %}
{% block page\_content %}

## Research Overview

We followed two parallel research lines. The first aimed to integrate our designed construct *LoxPort* into the genome of *Rhodococcus opacus* PD630. The second focused on characterizing a set of constitutive promoters in *R. opacus* to evaluate their strength and suitability for gene expression.
Below, you can find the common protocols we used for both research lines.
 Whether you have never held a pipette in your life or you already know these techniques inside out, we encourage you to take a look!

**Why read our protocols?** Our protocols not only describe how to perform each procedure, but also explain how they work, the scientific rationale behind them, and include useful tips and tricks for troubleshooting when things go wrong.

## General Protocol Quick Access

---

[**High-fidelity PCR with Q5 polymerase**](#pcr-q5)
[**Agarose gel electrophoresis**](#gel-electrophoresis)
[**NucleoSpin Gel and PCR Clean-Up**](#gel-cleanup)
[**Ligation**](#ligation)
[**Bacterial transformation**](#transformation)
[**Miniprep plasmid extraction**](#miniprep)
[**PCR with ExTaq polymerase**](#pcr-taq)
[**Agarose plate preparation**](#plate-prep)
[**Primer design**](#primer-design)
[**Restriction Enzymes Digestion**](#resenz)

## Producing pKSAC45 + the construct

---

The designed construct, synthesized in a medium-copy plasmid, was introduced into *E. coli* cells using the standard heat-shock transformation protocol. Transformed cells were plated on selective agar plates containing ampicillin and IPTG. After colony growth, a single colony was picked and cultured.

Plasmid DNA was purified from this culture using a MidiPrep kit from Macherey-Nagel. An additional wash step was performed to reduce salt content and improve the final DNA concentration.

Next, the construct was ligated into the suicide plasmid pKSAC45 using the SphI and EcoRI restriction enzymes. The construct, carrying the required homology arms, enables double-crossover homologous recombination once delivered into *R. opacus*. The pKSAC45 backbone serves as the non-replicative vector for integration.

Cells were plated on kanamycin-selective agar plates, and plasmids were subsequently isolated using a MiniPrep kit from Macherey-Nagel.

[**A-tailing**](#a-tailing)

## Promoter Characterization Study

---

A set of constitutive promoters was selected to evaluate their relative expression strengths: p2, pB2, pB3, and pLac (see more in the [Design page](https://2025.igem.wiki/bologna/design)).

Promoter pLac was amplified by PCR from a vector containing an IPTG-inducible selection system. Promoters p2, pB2, and pB3 were amplified by PCR from the *Rhodococcus jostii* RHA1 genome; in the case of pB2 and pB3, partial fragments of the native promoter were used. All promoters were cloned with restriction sites for NcoI and BsrGI.

The sfGFP coding sequence was also amplified by PCR, introducing mutations to remove undesired restriction sites (BsrGI and SapI) and adding NcoI and NotI recognition sites. To construct the reporter system, the pTip plasmid was digested, and its original promoter was replaced with one of the selected promoters driving sfGFP expression through restriction cloning.

The recombinant plasmids were first introduced into *E. coli* via the standard heat-shock transformation protocol. Transformed cells were plated on ampicillin-containing agar plates, and plasmid DNA was subsequently purified using a MiniPrep kit from Macherey-Nagel.

Validated constructs were then electroporated into *Rhodococcus* and plated on tetracycline-selective agar plates. After colony growth, approximately 100 colonies were picked, re-streaked, and re-cultured to ensure clonal stability. From each promoter construct, four colonies were randomly selected and grown in liquid medium until reaching an OD₆₀₀ of 0.5.

Finally, the fluorescence of these cultures was measured, providing a comparative assessment of promoter strength across the different constitutive elements.

[**sfGFP fluorescence measurement**](#fluorescence)
[**Electroporation transformation**](#electroporation)

## Determination of enzyme kinetic parameters through spectrophotometric analysis

---

The first step in the construction of a biosensor is the study of the behavior of the enzyme we are working with, which in our case is Porcine Pancreatic Lipase. To design a functional and optimized sensor, it is essential to determine the enzyme’s activity with respect to a standard substrate, pNPP. This is achieved through a lipase assay test as seen in the protocol below.

Once we have an idea of the minimum enzyme concentration required to obtain a significatively different signal from the control, we can consider using a substrate more similar to our target analyte. In this case, the chosen substrate is palmitic acid, which is degraded into fatty acids by lipase. However, since we are the one chosing the concentration of the substrate, we can use this knowledge to assess the accuracy of the second assay.

This is the goal of the next protocol, currently under development, where, unlike in the first experiment, we will prepare solutions with varying substrate concentrations instead of varying enzyme concentrations. This will allow us to calculate the Michaelis-Menten constant and other useful kinetic parameters.

Once this phase is completed, we can move on to the next step in the biosensor workflow: optimizing the method in liquid phase

[**Lipase activity assay**](#lipase-activity-assay)

## PROTOCOLS

Each protocol section is expandable and contains three main sections: Description, Quick Protocol, and Complete Protocol PDF.

##### High-fidelity PCR with Q5 polymerase

Used to amplify DNA fragments with high accuracy, minimizing mutations during amplification.

###### **Quick protocol:**

* Prepare reaction mixes in PCR tubes as follows **on ice:**

![](https://static.igem.wiki/teams/5716/lab/experiments/q5-1.webp)

* Template DNA quantity is described here:

![](https://static.igem.wiki/teams/5716/lab/experiments/q5-2.webp)

* Set the thermocycler with the following protocol:

![](https://static.igem.wiki/teams/5716/lab/experiments/q5-3.webp)

##### Agarose gel electrophoresis

Separates DNA fragments by size to verify PCR products or digestion results.

###### **Quick protocol:**

* **Gel casting**

+ Melt 0.35g in 50 ml TAE buffer, in a 250 ml Erlenmeyer flask.
+ Cool down till touchable hot.
+ Add 2.5 μl EtBr to reach 0.5μg/ml final concentration.
+ Cast the gel in a sealed tray, and place the desired well comb.
+ Cool down for 20-30 minutes at RT.
+ [optional]: cool down at 4°C for 10 minutes.

* **Sample preparation**

+ Add 6x loading dye to correctly concentrated DNA (for PCR verification, use 2 μl of product and 8 μl of bi-distilled water).
+ Load the whole sample mix in a well.
+ Load MW marker in an external well.

* **Run Conditions**

+ For PCR or digestion verification: constant 100 mV, roughly 35 min.
+ For gel extraction: constant 80 mV, roughly 50 min

##### NucleoSpin Gel and PCR Clean-Up

Purifies DNA fragments from PCR reactions or agarose gels for downstream applications.

During our experiments, we utilized **Macherey-Nagel’s kit**. The protocol on the company’s website can be found [here](https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf).

##### Ligation

Joins DNA fragments together, typically inserting an amplified construct into a plasmid backbone.

###### **Quick protocol:**

* Assemble the reaction **on ice** following the table

![](https://static.igem.wiki/teams/5716/lab/experiments/ligation.webp)

* Incubate for 16h overnight or 10 minutes at room temperature, we assessed that also ligation incubated at 4°C over-weekend performed well.
* OPTIONAL: inactivate enzymes at 65°C for 10 minutes [1].
* Store at -20°C for further use.

##### Bacterial transformation

Introduces recombinant plasmids into *E. coli* or other bacteria for propagation and expression.

###### **Quick protocol:**

* **First incubation**

+ Defrost an aliquot of competent cells
+ Add 400pg to 100ng of plasmid
+ Incubate on ice for 30’

* **Heat Shock**

+ Transfer for 2’ at 42°C
+ Place back on ice for 2’

* **Cell Recovery**

In a sterile environment:+ Add 1 ml sterile LB
+ Incubate 45’ at 37°C with shaking

* **Plating**

In a sterile environment:+ [OPTIONAL] Plate 100μl onto selective plates
+ Pellet the remaining suspension
+ Remove all supernatant but ~100μl
+ Resuspend the pellet
+ Spread resuspended pellet onto a selective plate

##### Miniprep plasmid extraction

Isolates plasmids amplified in *E. coli* cells

During our experiments, we utilized **Macherey-Nagel’s kit**. The protocol on the company’s website can be found [here](https://www.mn-net.com/media/pdf/45/51/02/Instruction-NucleoSpin-Plasmid.pdf).

##### PCR with ExTaq polymerase

Performs routine DNA amplification where high fidelity is not essential, e.g., for screening colonies.

###### **Quick protocol:**

* Prepare reaction mixes in PCR tubes as follows **on ice:**

![](https://static.igem.wiki/teams/5716/lab/experiments/extaq-1.webp)

* Template DNA quantity is described here:

![](https://static.igem.wiki/teams/5716/lab/experiments/extaq-2.webp)

* Set the thermocycler with the following protocol:

![](https://static.igem.wiki/teams/5716/lab/experiments/extaq-3.webp)

##### Agarose plate preparation

Provides a selective growth medium for bacterial cultures, often containing antibiotics.

###### **Quick protocol:**

* Melt sterile LB agar inside the sterile bottle
* Cool down to touchable hot
* In a sterile environment, add antibiotic to reach the desired concentration

![](https://static.igem.wiki/teams/5716/lab/experiments/plate.webp)

* In a sterile environment, rapidly pour roughly 15-20 ml of LB agar in each petri dish
* Make plates cool down in a sterile environment with the lid open, to avoid condensation formation
* Once solid, store **upside down** at 4°C

##### Primer design

Learn how to create short DNA sequences that define the start and end points of PCR amplification

##### A-tailing

Adds a single adenine overhang to PCR-amplified DNA fragments, preparing them for cloning into T-overhang vectors.

During our experiments, we utilized NEB’s Klenow fragment (3’->5’ exo-) protocol. The protocol on the company’s website can be found [here](https://www.neb.com/en/protocols/2013/11/06/a-tailing-with-klenow-fragment-3-5-exo).

##### sfGFP fluorescence measurement

Quantifies reporter gene expression by measuring the fluorescence of superfolder GFP in cells or cultures.

###### **Quick protocol:**

* **Day 1**

+ Under a sterile hood, plate the colonies to be studied onto selective plates (if needed)
+ Grow 2 to 4 days at 30°C with orbital shaking at 150rpm

* **Day 2**

+ Under a sterile hood, pick biomass to fill half of a 10 μl microbiology loop
+ Transfer the biomass in 600 μl LB and vortex thoroughly
+ Sediment for 2 mins
+ Inoculate 300 μl of the supernatant into the pre-inoculum of 10 ml LB (with selection) in a sterile Erlenmeyer flask.
+ Grow for roughly 18 hours at 30°C with orbital shaking at 150rpm

* **Day 3**

+ Measure OD600 with a spectrophotometer
+ Inoculate a new sterile Erlenmeyer flask with 10 ml LB (with selection) with bacteria from the pre-inoculum to reach OD600 = 0.05.
+ Grow at 30°C with orbital shaking at 150rpm till OD600 = 0.5
+ Control OD600 with the spectrophotometer
+ Once OD600 = 0.5 is reached, measure fluorescence and 600nm absorbance on 200 μl of culture using a plate reader

##### Electroporation transformation

Introduces plasmid DNA into bacterial cells by applying an electrical pulse that temporarily permeabilizes the cell membrane.

###### **Quick protocol:**

* **Pre-inoculum**
  In a sterile environment:
  - Plate *Rhodococcus opacus* PD630 on an LB agar plate and wait 2 days to have biomass
  - Inoculate in a flask with 20mL of LB + Gly(0.85%) + Sucrose (1%), O/N at 30°C
* **Inoculum**
  In a sterile environment:
  - Inoculate in the evening the right amount of cells in order to have a starting OD600 equal to 0.02 in a flask with 50mL of LB + Gly(0.85%) + Sucrose(1%)
  - The next morning wash 3 times the cells with H2O, resuspend in 2.5mL and split into aliquots of 200 μl
* **Electroporation**
  Electroporate the cells with set parameters (25 μF, 200 ohm, 2.5 kV)
  In a sterile environment:
  - Add 600 μl of fresh LB to cells and incubate O/N for recovering
* **Plating**
  In a sterile environment:
  - Centrifuge the colture and leave 300 μl of supernatant, resuspend
  - Plate around 150μl onto 2 selective plates
  - Incubate for 2-3 days at 30°C

##### Restriction Enzymes Digestion

Cuts DNA on a specific target sequence, used mainly for cloning and construct verification.
 If you don’t know how much DNA you should cut, please refer to the complete PDF file.

###### **Quick protocol:**

* Assemble the reaction **on ice** following the table:

![](https://static.igem.wiki/teams/5716/lab/experiments/restriction.webp)

* Incubate for times varying from 2 hours to overnight at 37°C (see optimization section).
* OPTIONAL: inactivate enzymes (enzyme specific process, see documentation from the producer if you intend to do so) [1].
* Store at -20°C for further use.

##### Lipase activity assay

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{% extends "layout.html" %}
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{% block title %}RESULTS{% endblock %}
{% block hero\_image %} ![Results](https://static.igem.wiki/teams/5716/header/results.webp) {% endblock %}
{% block menu\_title %} Results {% endblock %}
{% block menu\_content %}

[LoxPort Insertion](#loxport)

[Promoter Characterization](#promoters)

[Biosensor Characterization](#biosensor)

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Synthetic Biology
Hardware Production

## Insertion of LoxPort into *Rhodococcus opacus* PD630

---

Our goal was to build and test a system based on our construct, called LoxPort. This cre recombinase expressing construct was designed to perform site-specific recombination on lox sequences, to facilitate gene insertion. The construct was designed to be integrated in *Rhodococcus opacus* PD630 genome via double homologous recombination. Several changes were made to our original plan to improve amplification, assembly, and plasmid stability.

Using **Q5 high-fidelity polymerase**, we successfully amplified the DNA fragments containing homology regions. The PCR bands matched the expected sizes, confirming correct amplification. However, PCR experiments for linear construct that we first ordered for synthesis never yielded a good amplification.

To make the process easier, the construct was then divided into smaller fragments. Each fragment was amplified correctly, but fusion PCR on them never yielded a full construct. This has been mainly attributed to the complexity of the region to be amplified.

Linear construct amplification was also attempted by cloning into the **pGEM vector**, but no correct recombinant plasmids were obtained. Although verification PCR reactions worked well, but isolation of plasmid DNA gave no results. A similar result was obtained using **ExTaq polymerase**: amplification succeeded, but assembly in both pGEM and Topo vector failed.

To solve these issues, the construct was redesigned with **reduced GC content** and synthesized directly by **Twist Bioscience** in a **medium-copy plasmid**. This optimized version was stable, easy to amplify, and gave reliable DNA yields. This confirmed that the main problems before might have been caused by high GC content and by the choice of plasmid backbone.

Next, we tried integrating the LoxPort construct into the **pKSac45 plasmid**, necessary for double recombination in *Rhodococcus*. Cloning with SphI and EcoRI enzymes produced kanamycin-resistant *E. coli* colonies, but plasmid extraction (miniprep) gave almost no DNA. Colony PCR detected correct junctions in several colonies, especially colony 2, but sequencing showed that this clone only contained a self-ligated pKSac45 plasmid with a small deletion.

![DNA extraction gel](https://static.igem.wiki/teams/5716/results/gel-electrophoresis.webp)

Figure 1 - Agarose gel electrophoresis of DNA extracts. The first two wells show abundance of plasmidic DNA, with very intense bands below 3000bp. Band shape is typical of coiled circular plasmids. The other wells all show a non-specific smear, probably derived from background nucleic acid extraction from E. coli.

![Snapgene alignment](https://static.igem.wiki/teams/5716/results/snapgene-aligned-sequences.webp)

Figure 2 - Snapgene screenshot showing colony 2 extract sequence (below) aligned on pKSac45 sequence. Results indicate a 50bp deletion over MCS region. Restriction enzymes used for cloning were SphI and EcoRI.

The combination of antibiotic resistance, lack of plasmid recovery, and inconsistent PCR results suggested that LoxPort in high-copy plasmids may cause unwanted **Cre recombinase expression** in *E. coli* DH5-α. This expression could trigger recombination and plasmid instability or even integration into the bacterial chromosome. In contrast, the construct remained stable when carried on a medium-copy plasmid, supporting the idea that copy number strongly affects plasmid stability and Cre activity.

Overall, these results suggest that **GC content, secondary structures, and plasmid copy number** are key factors for successful assembly and maintenance of Cre-lox–based constructs in *E.coli*. Future experiments should test LoxPort in medium-copy versions of pKSac45 and may include genome sequencing of resistant colonies to confirm whether plasmid integration has occurred.

## Constitutive Promoter Characterization

---

To extend the available toolkit, several **constitutive promoters** were tested to compare their ability to drive **sfGFP expression** in *Rhodococcus opacus* PD630. The goal was to identify promoters with different expression strengths that could be implemented in the future molecular toolkit of *Rhodococcus opacus* PD630.

Promoters **p2, pB2, pB3, pLac, and pNit** were selected. pLac was obtained from a vector containing an IPTG-inducible system, while p2, pB2, and pB3 were amplified from the *Rhodococcus jostii* RHA1 genome. In contrast, PNit is part of the standard toolkit of *Rhodococcus opacus* PD630 and represents the most well-known constitutive promoter for this bacterium. All promoters were cloned into the pTip plasmid, replacing its native promoter and positioned upstream of the sfGFP gene. The sfGFP sequence was domesticated by removing unwanted restriction sites and adding compatible cloning ends.

![Promoter gel electrophoresis](https://static.igem.wiki/teams/5716/results/restriction-promoters.webp)

Figure 3 - gel electrophoresis performed on enzymatic digestion of reporter plasmids used for *Rhodococcus opacus* PD630 transformation. pNit + sfGFP was digested with NcoI and NotI restriction enzymes, pTip-based reporter plasmids were cut with NcoI and BsrGI restriction enzymes. All bands are as expected.

The reporter plasmids were first assembled and verified in *E. coli* through heat-shock transformation and plasmid purification. After sequence validation, reporter plasmids were electroporated into *Rhodococcus*, plated on tetracycline selective media, and stable colonies were selected. For each promoter, four colonies were grown in liquid culture to an OD₆₀₀ of 0.5, and fluorescence intensity was measured to compare promoter strength.

Promoters **p2, pB2, and pB3** successfully drove sfGFP expression in *Rhodococcus*. Among them, **pB2 showed the highest activity**, p2 produced intermediate fluorescence, and pB3 had the weakest expression. We aimed to study also p10 promoter, but it could not be tested due to primer design issues that prevented successful amplification of it and its optimizations. **pLac promoter** also showed measurable activity comparable to pNit, confirming its function in *Rhodococcus*.

We decided to include the **pNit promoter** in our study as a positive control. Originally described by Nakashima and Tamura (2004), pNit is a well-established constitutive promoter in *Rhodococcus*. Our results confirmed that pNit consistently drives strong sfGFP expression, in agreement with the published data. Therefore, in the table below, the fluorescence values of the other promoters were normalized to pNit activity to allow a reliable comparison of their relative strengths

![Promoter strength graph](https://static.igem.wiki/teams/5716/results/promoters-to-pnit.avif)

Figure 4 – Bar graph showing constitutive promoter strength in *Rhodococccus oapcus* PD630.

##### Promoter Strength Data

| Promoter | Strength (norm. pNit) |
| --- | --- |
| pB2 | **1.20** |
| pNit | 1.00 |
| pLac | 0.93 |
| p2 | 0.60 |
| pB3 | 0.24 |

Overall, this first characterization cycle confirmed that all the tested promoters function constitutively in *Rhodococcus opacus* PD630 at varying intensities, with **pB2 emerging as the strongest** among the new candidates. The experiment also highlighted areas for improvement, like redesigning primers for p10 and optimization of pB3 (which may have lost key regulatory sequences during cloning). These results provide a solid foundation for building a standardized library of *Rhodococcus* promoters based on normalized sfGFP expression data. The table on the right reports the relative fluorescence values normalized to the activity of the pNit promoter

## References

▼

* Nakashima N, Tamura T. Isolation and Characterization of a Rolling-Circle-Type Plasmid from *Rhodococcus erythropolis* and Application of the Plasmid to Multiple-Recombinant-Protein Expression. *Applied and Environmental Microbiology*. 2004;70(9):5557-5568. doi: https://doi.org/10.1128/aem.70.9.5557-5568.2004
* Round JW, Roccor R, Eltis LD. A biocatalyst for sustainable wax ester production: re-wiring lipid accumulation in *Rhodococcus* to yield high-value oleochemicals. *Green Chemistry*. 2019;21(23):6468-6482. doi: https://doi.org/10.1039/c9gc03228b

## Biosensor Characterization

---

For the development and optimization of a colorimetric biosensor for monitoring lipase activity, the initial step focused on identifying the **optimal buffer** for enzyme solubilization and activity. Several buffers were tested, and their effects on enzyme stability and assay performance were evaluated; some buffers caused precipitation or inconsistent activity, while others maintained enzyme stability over time. The combination of **1.5% BSA with 1 M Tris-HCl** was selected for subsequent assays as it provided complete enzyme solubilization and consistent activity measurements.

Lipase activity was assessed using the **p-nitrophenyl palmitate (pNPP) assay**, with enzyme concentrations ranging from 50 to 200 U/mL: absorbance was measured at 410 nm using a Varioskan spectrophotometer. The results showed a dose-response relationship, with maximal activity observed at **125 U/mL**, which was established as the optimal working concentration; higher concentrations (>150 U/mL) displayed minor deviations from linearity, likely due to substrate saturation or aggregation. Replicate measurements confirmed the reliability of the results, with standard deviations within acceptable limits.

![Lipase activity Type II](https://static.igem.wiki/teams/5716/results/lipase1.webp)

Lipase activity assay (Type II, ≥125 units/mg)

![Lipase activity 125 U/mL](https://static.igem.wiki/teams/5716/results/lipase2.webp)

Lipase activity 125 U/mL - Control and optimized conditions

During optimization, initial enzyme preparations occasionally produced inconsistent activity readings: these inconsistencies were attributed to incomplete dissolution or buffer pH variations. Implementation of a **pre-incubation step** and **fine-tuning** of the buffer composition resolved these issues, resulting in reproducible measurements across independent experiments.

Under the optimized conditions, the biosensor generated stable and measurable colorimetric signals corresponding to lipase activity. The linear response observed in the pNPP assay indicated that the system could be reliably employed for enzyme monitoring. These results demonstrate that the biosensor design is robust and that methodological optimizations significantly improved performance and reproducibility.

{% endblock %}