iGEM 2025 — Team mbg-duth


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[MBG-DUTH](home.html)

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* [Home](home.html)
* Team

  [People](people.html)
  [Attributions](attributions.html)
* [Awards](awards.html)
* Project

  [Description](description.html)
  [Engineering](engineering.html)
  [Implementation](implementation.html)
  [Contribution](contribution.html)
  [Safety](safety.html)
* Wet Lab

  [Experiments](experiments.html)
  [Results](results.html)
  [Lab Book](lab-book.html)
  [New Composite Part](new-composite-part.html)
* Dry Lab

  [Structural Analysis](structural-analysis.html)
  [Model](model.html)
* Human

  [Overview](overview.html)
  [Silver Human Practices](silver-human-practices)
  [Integrated Human Practices](human-practices.html)
  [Education & Communication](education.html)
  [Collaboration](collaboration.html)

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# Engineering Success

On the Engineering Success Page we explain how our team used the design–build–test–learn cycle to refine our approach, optimize parts, and achieve reliable, efficient experimental results.

### Quick Overview

#### 📜 Introduction

Turning a big biological waste problem into a sustainable solution through iterative design and collaboration.

[Take Me There](#introduction)

#### 🧪 Wet Lab

Iterative Engineering and Human Practices shaped Chitinator et al. into a practical and responsible project.

[Take Me There](#wet)

#### 💻 Dry Lab

Models and simulations guided our design, predicting outcomes and reducing trial-and-error in the lab.

[Take Me There](#dry)

#### ☰ Table Of Contents

* [Introduction](#introduction)
* [Wet Lab Aspect](#wet)
* [Dry Lab Aspect](#dry)

## 📜 Introduction

**Outline:**  Turning a big biological waste problem into a sustainable solution through iterative design and collaboration.

![Description](https://static.igem.wiki/teams/5994/images/wet-dry.webp)

Figure 1. The Wet and Dry Lab Aspects of our Project are tightly interconnected.

Every bold project begins with a question: *can we turn nature’s challenges into humanity’s solutions?*

For **Chitinator et al.**, the challenge was immense, mountains of chitin waste, a molecule as resilient as it is abundant, standing as an untapped resource. Our vision was not simply to degrade it, but to reimagine it: to transform what was once discarded into a foundation for sustainable agriculture.

To achieve this, we embraced the **Design–Build–Test–Learn (DBTL) cycle**, not as a rigid framework, but as the heartbeat of our project. Our first designs were ambitious, a single operon, carrying the full arsenal of chitinases. Yet as we built, tested, and learned, that vision evolved into something stronger: a modular system, adaptive and efficient, born from the synergy of iteration.

For us, engineering success was never linear. It was an *infinite dialogue* between the **Dry Lab and the Wet Lab**, two circles of thought and practice that constantly exchanged insights, shaping and reshaping our project. Just like an *infinity loop*, predictions from the Dry Lab flowed into experiments in the Wet Lab, and results from the Wet Lab returned to refine our models. This endless cycle of feedback and improvement became the core of our engineering journey.

Our story is not just about enzymes and plasmids. It is about how *iteration turned into infinity*, and how through this perpetual exchange, waste was turned into value, and vision into impact.

## 🧪 Wet Lab Aspect

**Outline:** Iterative Engineering and Human Practices shaped Chitinator et al. into a practical and responsible project.

![Description](https://static.igem.wiki/teams/5994/images/mandala.webp)

Figure 2. The Mandala

The Wet Lab cycles of our project came together not as isolated steps, but as an interconnected whole, much like a **mandala**. In many traditions, a mandala symbolizes wholeness, balance, and the journey from the center outward, gaining complexity and meaning with each layer.

For us, every cycle of **Design–Build–Test–Learn** was a new concentric ring in our mandala: from the initial vision of soil application, to the operon design, modular constructs, host optimization, and finally the implementation as **BioActivator**. Step by step, our mandala expanded, capturing the iterative nature of engineering in synthetic biology.

This visualization highlights that our **Engineering Success** was not about one perfect experiment, but about the harmony of multiple cycles that, when combined, formed a complete picture.

We present our cycles in two parts:

* **Completed Cycles** – the steps we have already performed and from which we have drawn key lessons.
* **Implementation Cycles** – the steps now in progress or planned, which extend our engineering path into the near future.

Wet Lab Cycles
▼

### Cycle 1 – The Big Operon (Completed)

**Design**
At the very beginning, we envisioned constructing a large operon that would encode three different chitin-degrading enzymes: an *endochitinase*, an *exochitinase*, and an *N-acetylglucosaminidase*. Together, these would provide a “one-shot” solution for complete chitin hydrolysis.

**Crossroad**
As we planned this, it became clear that the operon would be too large to clone and express efficiently. Large constructs are unstable and difficult to maintain in plasmid backbones. In addition, literature suggested that many hosts already have endogenous N-acetylglucosaminidase activity, making one part of our design redundant.

**Redesign**
We shifted to a modular strategy. Instead of an oversized operon, we designed a fusion enzyme that combined one endochitinase and one exochitinase joined by a flexible linker.

**Build**
Fusion constructs were designed in silico and made compatible with **E. coli** and later **Bacillus subtilis**.

**Test**
Bioinformatic evaluations confirmed that the fusion constructs were stable and synthesizable, unlike the oversized operon.

**Learn**
We learned that more is not always better. Modularity and efficiency are critical for feasible engineering. This pivot defined the genetic backbone of Chitinator et al.

### Cycle 2 – Lack of Secretion in pET-28a (Completed)

**Design**
We cloned our fusion enzyme into **pET-28a(+)** for expression in **E. coli BL21**. This vector has a strong T7 promoter and is widely used for protein overexpression.

**Crossroad**
Induction with IPTG led to strong expression, but the protein stayed inside the cells because pET-28a lacks a secretion signal. This blocked us from testing activity in the culture medium.

**Redesign**
We adapted our strategy:

* Use cell lysis followed by disk diffusion and zymogram assays to test activity.
* At the same time, prepare to move into alternative pET vectors that include secretion signals, such as pET-26b, which carry the pelB leader sequence for periplasmic export.

**Build**
Cultures were induced, lysed, and crude extracts were applied to functional assays. Cloning plans into secretion-capable vectors were also drafted.

**Test**
SDS-PAGE confirmed intracellular expression. Functional assays (disk diffusion and zymograms) gave indirect activity evidence.

**Learn**
We understood that secretion is essential for scalable testing. pET-28a was useful for initial expression but limited for functional screening, so we planned to switch to secretion-compatible vectors.

### Cycle 3 – Expression Optimization (Completed)

**Design**
During expression trials in **E. coli BL21**, we encountered inconsistent solubility and yield depending on IPTG levels and sonication efficiency.

**Crossroad**
We had to decide whether to redesign the construct (e.g., adding solubility tags) or optimize culture and induction conditions first.

**Redesign**
We opted to fine-tune conditions:

* Test a gradient of IPTG concentrations.
* Vary induction times.
* Adjust sonication cycles to improve protein release without denaturation.

**Build**
Multiple cultures were induced with different IPTG doses and harvested at different time points. Sonication cycles were applied systematically.

**Test**
SDS-PAGE was performed across conditions to compare protein expression and solubility.

**Learn**
We identified more favorable conditions for protein expression and recovery, showing that optimization can solve issues without immediate genetic redesign.

### Cycle 4 – PCR Optimization (Completed)

**Design**
To clone the insert into secretion-compatible plasmids (**pWB26**, **pHT43**), we needed to amplify it. However, our primers had large melting temperature (Tm) differences.

**Crossroad**
We had to decide whether to redesign primers (losing time and resources) or test amplification through optimization.

**Redesign**
We chose to run a gradient PCR, spanning a range of annealing temperatures, to empirically find conditions where both primers could work.

**Build**
Gradient PCR reactions were set up using the existing primer set.

**Test**
Clear bands of the expected size were observed at all tested temperatures.

**Learn**
Gradient PCR rescued the cloning process without primer redesign. This confirmed that the insert was robustly amplifiable, allowing us to move forward confidently.

### Cycle 5 – Multi-Stage Transition to Bacillus subtilis with pHT43 and Beyond (Planned)

**Design**
While **E. coli** is excellent for rapid prototyping, it does not fully reflect our final application needs. To strengthen our screening pipeline, we plan a stepwise transition:

* Test secretion in E. coli using a vector such as pET-26b(+) which carries a secretion signal or pET-28a(+) (adding a secretion signal), allowing us to better screen activity directly in the culture medium.
* Transfer the construct into **Bacillus subtilis 168** using pHT43, a secretion-competent plasmid with the amyQ signal peptide, for more realistic activity assays in the final chassis.
* In a later phase, adapt the construct into a food-grade Bacillus vector without antibiotic resistance markers, paving the way for agricultural use.

**Crossroad**
The choice was whether to jump directly into Bacillus expression, or to design a layered pipeline: first validate secretion in E. coli, then move into Bacillus subtilis, and finally prepare an antibiotic-free vector for safe agricultural deployment.

**Redesign**
We opted for this multi-step approach, since it combines the speed of E. coli with the realism of Bacillus subtilis, while ensuring long-term biosafety.

**Build**
We will prepare PCR products and cloning strategies for each stage:

* Subcloning into pET-26b(+) or again in the initial vector with a secretion signal for secretion screening in E. coli.
* Cloning into pHT43 for secretion assays in Bacillus subtilis 168.
* Future design of an antibiotic-free Bacillus plasmid for real-world application.

**Test**
Each stage will involve activity assays:

* E. coli: secretion checked via supernatant assays (disk diffusion, zymograms).
* B. subtilis/pHT43: zone-of-clearance on colloidal chitin plates.
* B. subtilis antibiotic-free: performance testing under application-like conditions.

**Learn**
We expect this staged strategy to demonstrate that secretion is achievable in both chassis, while preparing a safe, antibiotic-free vector for agricultural deployment. This stepwise design balances scientific rigor, biosafety, and practical implementation.

### Cycle 6 – Sprayable BioActivator for Garlic

**Design**
Our implementation goal is to develop a sprayable BioActivator tailored for garlic cultivation. Farmers already apply chemical fertilization to meet nutrient needs, but garlic requires additional spray treatments throughout its growth due to fungal vulnerability. Our BioActivator is designed not to replace fertilizers, but to complement them by acting as a biological spray with both growth-promoting and antifungal properties.

**Crossroad**
Farmers rely on repeated chemical sprays to protect garlic crops, but these increase costs, leave residues, and may harm soil health. We needed a product that integrates smoothly into their existing spraying schedules after chemical fertilization, offering a safer and more sustainable option.

**Redesign**
We plan to formulate a cell-free chitin hydrolysate (oligosaccharides and GlcNAc) that:

* Is sprayable with standard equipment (low viscosity, nozzle-safe).
* Stimulates garlic plant defense pathways, reducing fungal infections.
* Enhances soil vitality and crop resilience without replacing fertilizer inputs.
* Fits into the garlic crop cycle as a post-fertilization spray, within the same windows farmers already use for fungicide applications.

**Build**
Pilot bioreactor runs will generate BioActivator, followed by downstream steps (sterile filtration, buffering to pH 6–7). Formulations will be tested for sprayability and tank-mix compatibility with conventional sprays.

**Test**
Planned assays include:

* Analytical: GlcNAc quantification, oligosaccharide DP profiling, viscosity, sterility.
* Application: nozzle compatibility tests, leaf coverage, and mixing trials with chemical sprays.
* Biological: greenhouse garlic trials to monitor growth and fungal resistance (Fusarium proliferatum, Sclerotium cepivorum) under BioActivator treatment.

**Learn**
We expect to validate the BioActivator as a dual-purpose biological spray: boosting garlic growth and providing antifungal protection. By fitting into existing farming practices after chemical fertilization, it will increase farmer acceptance and demonstrate a clear real-world pathway for sustainable agriculture.

Human Practices Interference
▼

### HP Cycle 1 – Biosafety & Farmer Concerns

**Design**
At the beginning, our plan was to engineer a living biofertilizer, releasing GMOs directly into the soil to promote crop growth.

**Crossroad**
Feedback from farmers and biosafety experts revealed strong resistance: “We do not want genetically modified organisms in our soil.” Concerns about ecological disruption and regulation made this approach socially and environmentally risky.

**Redesign**
We shifted toward a bioreactor-based approach, where engineered microbes are contained under controlled lab conditions and only their safe products (bioactivators) are applied to the soil.

**Build**
This decision shaped our Wet Lab workflow, focusing on maximizing enzyme expression and activity in vitro rather than engineering survival traits in soil microbes.

**Test**
Bioactivators could now be safely tested in agricultural scenarios without releasing GMOs, increasing farmer acceptance.

**Learn**
Human Practices taught us that biosafety and social trust are non-negotiable, pushing us toward a safer, more acceptable design.

### HP Cycle 2 – Circular Bioeconomy Framing

**Design**
Initially, we presented our project mainly as a biotechnological tool for chitin degradation.

**Crossroad**
Discussions with sustainability experts highlighted that the project could be undervalued unless placed within a larger narrative of circular bioeconomy, where waste is recycled into value.

**Redesign**
We reframed Chitinator et al. as part of the circular bioeconomy, turning seafood and agricultural chitin waste into nutrient-rich bioactivators that enhance soil health and reduce reliance on synthetic fertilizers.

**Build**
All communication materials, presentations, and even experimental design began emphasizing the “waste-to-value” principle and environmental respect.

**Test**
When shared with stakeholders, this framing resonated strongly, making the project appear not only scientifically innovative but also economically and ecologically relevant.

**Learn**
HP guided us to understand that storytelling and framing are just as important as technical design in making biotechnology meaningful and impactful.

### HP Cycle 3 – Choice of Garlic Cultivation

**Design**
Our system could, in theory, be applied to many crops.

**Crossroad**
Conversations with Greek farmers revealed that garlic cultivation, particularly Greek white garlic, is economically important, culturally symbolic, and highly vulnerable to fungal infections such as *Fusarium proliferatum* and *Sclerotium cepivorum*.

**Redesign**
We selected garlic cultivation as our case study, focusing our bioactivators on this emblematic crop. This made our project locally relevant and globally recognizable.

**Build**
Wet Lab experiments and communication materials highlighted garlic as the first target application, linking bioactivators to plant defense and soil vitality.

**Test**
Feedback from farmers and experts confirmed that this choice made the project more convincing, showing a clear path from lab innovation to agricultural benefit.

**Learn**
HP directed us to root our innovation in a real-world agricultural challenge, ensuring that our design responds to actual farmer needs.

### HP Cycle 4 – Farmers’ Needs for Nutrient Units

**Design**
We initially thought of presenting bioactivators mainly as sustainable alternatives to synthetic fertilizers.

**Crossroad**
Through discussions with agronomists and farmers, we learned that growers calculate their inputs in nitrogen units and expect every fertilizer or activator to clearly meet plant nutritional needs. Without quantifiable values, acceptance would be limited.

**Redesign**
We adapted our concept: instead of positioning bioactivators as a “replacement” for fertilizers, we framed them as complementary inputs that improve soil health and crop resilience while working alongside nitrogen-based fertilization.

**Build**
Communication materials and lab plans began to emphasize measurable impacts, e.g., not only degradation of chitin waste but also its contribution of carbon, nitrogen, and phosphorus back to the soil.

**Test**
Feedback confirmed that this framing made bioactivators more understandable and appealing to farmers, who now saw them as practical rather than abstract tools.

**Learn**
HP reminded us that real-world adoption depends on farmer language and expectations; to gain trust, we must present bioactivators in terms that align with agricultural practice, like nutrient units.

### HP Cycle 5 – Complementary Use as Biological Sprays

**Design**
At first, bioactivators were considered as a possible substitute for chemical fertilizers.

**Crossroad**
Feedback from farmers and agronomists showed that this framing did not match reality: garlic cultivation already depends on chemical fertilization for nutrients, but the real problem is fungal vulnerability, which requires repeated chemical sprays. Farmers were interested in biological alternatives for protection, not in abandoning fertilizers altogether.

**Redesign**
We reframed bioactivators as biological sprays/boosters: applied to garlic crops not to replace fertilizers, but to complement them by strengthening plants against fungal infections and improving soil vitality.

**Build**
Our Wet Lab strategy and communication materials began to highlight garlic as a case study where bioactivators are applied after or alongside chemical fertilization, in order to reduce dependency on fungicides and increase resilience.

**Test**
Farmers responded positively to this repositioning, seeing bioactivators as a practical, biological solution that integrates with their current practices rather than replacing them.

**Learn**
HP reminded us that innovation succeeds when it fits into existing agricultural workflows. By presenting bioactivators as biological sprays, we aligned with farmer needs and increased the real-world applicability of our project.

## 💻 Dry Lab Aspect

**Outline:** Models and simulations guided our design, predicting outcomes and reducing trial-and-error in the lab.

![Description](https://static.igem.wiki/teams/5994/images/labyrinth.webp)

Figure 3. The Labyrinth

Our Dry Lab journey can be best described as navigating a **labyrinth**. At the entrance, we faced the complexity of countless chitinase candidates spread across evolutionary branches. This was our **phylogenetic model**, a vast network of possible paths, where each branch represented a different enzyme family.

Guided by this analysis, we realized that the most promising route lay in selecting an **endochitinase** and an **exochitinase** from the same *glycoside hydrolase* family. Their evolutionary relatedness suggested structural similarities and a greater likelihood of functional compatibility, making them ideal candidates for fusion. In this way, **phylogenetics** did not simply map the maze; it provided the reasoning behind our enzyme choice.

Yet, phylogenetics alone could not lead us out of the labyrinth. To find the true exit, we needed a guiding thread: **structural modeling**. By testing linker flexibility, folding predictions, and stability simulations, we identified which fusion constructs could realistically function.

Thus, our Dry Lab combined two layers: **phylogenetics** gave us the map of possible routes, while **structural modeling** acted as the compass and guiding thread. Together, they allowed us to escape the maze of uncertainty and deliver optimized constructs to the **Wet Lab**.

Model
▼

### Cycle 1 - Choosing the right enzyme

**Design**
We aimed to identify the optimal enzyme set for complete chitin degradation. In front of us stood a vast number of candidate chitinases, distributed across multiple glycoside hydrolase (*GH*) families. The key question was: *which enzymes should we select?*

**Crossroad**
The sheer abundance of options created uncertainty. Random selection risked inefficient or unsuitable enzymes for our project. We needed an objective, bioinformatics-based strategy to guide the decision.

**Redesign**
We turned to publicly available data from the European Nucleotide Archive (*ENA*). This allowed us to gather large collections of chitinase sequences and apply two complementary analyses:

* *Ecological mapping* of the samples (hosts, environments, clinical data).
* *Phylogenetic reconstruction*, to reveal evolutionary relationships among enzymes and identify patterns of convergence or specialization.

This dual approach provided a more reliable framework for judging which enzymes were truly suitable.

**Build**
By integrating ecological and phylogenetic insights, we concluded that the *GH18* family represented the most reliable choice: it exhibits broad ecological distribution, strong diversification, and a history of use in biotechnological applications. We therefore selected specific GH18 enzymes (*BBa\_K4349000* – endochitinase, *CfcI* – exochitinase).

**Test**
This decision was validated by the convergence of both data streams: *GH18 enzymes* emerged as generalist, ecologically stable, and experimentally verified across diverse environments.

**Learn**
From this process, we learned that enzyme selection is not merely a matter of literature mining or chance, but must be grounded in the integration of ecological and phylogenetic data. This step marked a turning point, shaping our strategy for linking dry lab with wet lab.

### Cycle 2 – From Proteins to DNA

**Design**
We initially planned to work with protein sequences of chitinases. The rationale was that protein information reflects function more directly and would provide high-quality phylogenetic trees, free from the noise of synonymous mutations.

**Crossroad**
In practice, significant obstacles arose:

* The number of curated protein sequences was *limited* compared to DNA.
* Annotations were *heterogeneous*, with incomplete metadata and unclear GH family assignments.
* Alignments proved *unstable*, especially across distant GH families, leading to questionable phylogenetic reconstructions.

This impasse forced us to reconsider our initial strategy.

**Redesign**
Instead of restricting ourselves to the few available protein sequences, we turned to DNA sequences from the ENA, which offered:

* *Greater diversity* and a larger number of samples.
* *Better coverage* across ecosystems and hosts.
* Integration with *ecological metadata* (country, isolation source, host).

This allowed us to build comprehensive datasets for each GH family, suitable for more reliable phylogenetic reconstruction.

**Build**
We developed pipelines to parse raw ENA records, extracting DNA sequences along with their associated ecological information. This approach enabled large-scale analyses without the data gaps we would have faced with proteins.

**Test**
Phylogenetic reconstructions based on DNA datasets produced much clearer trees, with well-supported clades and the ability to link evolutionary patterns to ecological contexts.

**Learn**
We learned that the “ideal” dataset is not always the most theoretically clean (*proteins*), but rather the one that is more complete and integrable with metadata. Shifting to DNA provided richer and reliable results, laying the foundation for connecting phylogenetics with ecology in our model.

Structural Validation
▼

### Cycle 1 – The Linker Dilemma

**Design**
Initially, multiple linker strategies were envisioned to connect the *endochitinase* and *exochitinase* domains into a single, functional composite enzyme. These included both flexible (G₆S)₃ linkers and rigid motifs such as EAAAK, KEKE, Proline-rich, and ELP sequences. The goal was to determine which configuration would allow independent folding of both enzymes while preserving synergistic catalytic activity.

**Crossroad**
Early modeling revealed that rigid linkers, though structurally stable, restricted the natural movement required for cooperative activity between the enzymes. The Proline-rich and ELP linkers, in particular, caused excessive rigidity or instability. This forced a reconsideration of linker flexibility versus structural stability.

**Redesign**
The design focus shifted toward flexible linkers that could preserve both enzymatic integrity and motion. The (G₆S)₃ sequence was selected for further analysis, as its glycine and serine composition offered both flexibility and solubility.

**Build**
All linker variants were modeled *in silico* using Swiss-Model and ColabFold (AlphaFold2), followed by visualization in PyMOL and motif validation with ScanProsite. Each structure was evaluated for steric clashes, folding quality, and domain orientation.

**Test**
Validation metrics (Ramachandran plots, MolProbity scores, pLDDT) demonstrated that the (G₆S)₃ construct exhibited minimal steric hindrance and high stereochemical quality. The catalytic motifs remained accessible and unaltered.

**Learn**
This phase confirmed that flexibility is essential for cooperative chitin degradation. The (G₆S)₃ linker was finalized as the optimal design, offering the best balance between domain mobility, solubility, and folding precision.

### Cycle 2 – Structural Validation and Comparative Analysis

**Design**
To ensure the predicted fusion protein was structurally feasible, detailed validation was performed combining homology modeling (Swiss-Model) and deep learning (AlphaFold2) predictions.

**Crossroad**
Initial models highlighted discrepancies in the linker regions of rigid constructs. Drops in IDDT confidence and poor local folding indicated strain. These limitations prompted a more rigorous validation of the flexible linker.

**Redesign**
Models were re-run and refined using hybrid pipelines, integrating multiple templates and quality metrics. Comparative visualization confirmed the flexible linker allowed optimal spatial proximity between domains.

**Build**
High-quality 3D models were constructed, colored by domain, and assessed for active-site accessibility. Clashscores and geometric deviations were recorded.

**Test**
Quantitative validation showed >90% residues in favored Ramachandran regions, MolProbity score ~2, and Clashscore at the 91st percentile, confirming realistic atomic geometry.

**Learn**
Independent folding with intact catalytic sites validated the core rationale of the fusion design. The flexible linker not only preserved activity but also improved predicted stability compared to rigid designs.

### Cycle 3 – From Modeling to Implementation

**Design**
The final structural validation informed the future wet-lab phase by confirming that the construct could be expressed without steric or folding issues, paving the way for secretion optimization in *Bacillus subtilis*.

**Crossroad**
While the computational results were strong, the next bottleneck identified was the absence of a secretion signal for extracellular enzyme release, crucial for future implementation in the bio-activator system.

**Redesign**
A plan was proposed to integrate an appropriate secretion signal peptide into the construct, ensuring extracellular chitin degradation in *B. subtilis*.

**Build**
Future iterations will use the validated (G₆S)₃ fusion backbone, incorporating secretion and promoter elements within expression vectors optimized for *Bacillus*.

**Test**
Functional assays (chitin plate degradation, enzymatic kinetics) will evaluate real-world catalytic efficiency and confirm computational predictions.

**Learn**
The structural validation served as the foundation for the next experimental phase, transforming computational insights into tangible, testable synthetic biology applications.

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##### Contact

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###### Home

* [Home](#top)

###### Team

* [People](people.html)
* [Attributions](attributions.html)

###### Project

* [Description](description.html)
* [Engineering](engineering.html)
* [Implementation](implementation.html)
* [Contribution](contribution.html)
* [Safety](safety.html)

###### Wet Lab

* [Experiments](experiments.html)
* [Results](results.html)
* [Lab Book](lab-book.html)
* [New Composite Part](new-composite-part.html)

###### Human

* [Overview](overview.html)
* [Silver Human Practices](silver-human-practices.html)
* [Integrated Human Practices](human-practices.html)
* [Education & Communication](education.html)
* [Collaboration](collaboration.html)

###### Dry Lab

* [Structural Analysis](structural-analysis.html)
* [Model](model.html)

###### Awards

* [Awards](awards.html)

© 2025 - Content on this site is licensed under a
[Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available
[here](https://gitlab.igem.org/2025/mbg-duth).

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[MBG-DUTH](home.html)

☰

* [Home](home.html)
* Team

  [People](people.html)
  [Attributions](attributions.html)
* [Awards](awards.html)
* Project

  [Description](description.html)
  [Engineering](engineering.html)
  [Implementation](implementation.html)
  [Contribution](contribution.html)
  [Safety](safety.html)
* Wet Lab

  [Experiments](experiments.html)
  [Results](results.html)
  [Lab Book](lab-book.html)
  [New Composite Part](new-composite-part.html)
* Dry Lab

  [Structural Analysis](structural-analysis.html)
  [Model](model.html)
* Human

  [Overview](overview.html)
  [Silver Human Practices](silver-human-practices)
  [Integrated Human Practices](human-practices.html)
  [Education & Communication](education.html)
  [Collaboration](collaboration.html)

🌞
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# Experiments

On the Experiments Page, we summarize all the laboratory procedures, protocols and methods used throughout our project.

### Quick Overview

#### 🔎 What This Page Contains

[Take Me There](#introduction)

#### LB Broth Preparation Protocol

[Take Me There](#lbbroth)

#### LB Agar Preparation Protocol

[Take Me There](#lbagar)

#### Competent Bacteria For Glycerol Stock

[Take Me There](#stock)

#### Transformation

[Take Me There](#trans)

#### PCR

[Take Me There](#pcr)

#### PCR Clean-Up

[Take Me There](#clean)

#### Agarose Gel

[Take Me There](#agarose)

#### Plasmid Isolation And Nanodrop

[Take Me There](#nano)

#### Bacterial Lysis And Lysis Buffer Preparation

[Take Me There](#lysis)

#### SDS-PAGE Protocol

[Take Me There](#sds)

#### Colloidal Chitin Preparation Protocol

[Take Me There](#prot)

#### Colloidal Chitin Agar Preparation Protocol

[Take Me There](#agp)

#### Chitinase Activity Assay

[Take Me There](#assay)

#### ☰ Table Of Contents

* [What This Page Contains](#introduction)
* [LB Broth Preparation Protocol](#lbbroth)
* [LB Agar Preparation Protocol](#lbagar)
* [Competent Bacteria For Glycerol Stock](#stock)
* [Transformation](#trans)
* [PCR](#pcr)
* [PCR Clean-Up](#clean)
* [Agarose Gel](#agarose)
* [Plasmid Isolation And Nanodrop](#nano)
* [Bacterial Lysis And Lysis Buffer Preparation](#lysis)
* [SDS-PAGE Protocol](#sds)
* [Colloidal Chitin Preparation Protocol](#prot)
* [Colloidal Chitin Agar Preparation Protocol](#agp)
* [Chitinase Activity Assay](#assay)

## 🔎 What This Page Contains

**Outline:** Here is explained what this Page contains and how you should navigate it.

The lab procedures for our project include a series of standard molecular biology and microbiology
experiments designed to support our research objectives. These protocols cover media preparation, bacterial
transformation, plasmid isolation, PCR amplification, gel electrophoresis, protein analysis, and chitinase activity
assays. Together, these methods enabled the growth and maintenance of bacterial cultures, the introduction and
verification of genetic constructs, and the expression and characterization of target proteins necessary for
the birth of Chitinator et al.. Each step of the lab process has been organized into a separate card in the
overview panel, allowing you to easily browse and select the specific procedure you wish to read and learn more
about.

Details about the experimental process can be also found on our [Lab Book Page](lab-book.html)

## LB Broth Preparation Protocol

* Weigh the required amount of LB Broth powder (according to manufacturer’s instructions)
* Dissolve the powder in distilled water up to the desired volume
* Sterilize by autoclaving at 120 °C for 15 minutes
* Allow to cool
* Store in bottles at 4 °C until use

## LB Agar Preparation Protocol

* Weigh the required amount of LB Agar powder (according to manufacturer’s instructions)
* Dissolve the powder in distilled water up to the desired volume
* Sterilize by autoclaving at 120 °C for 15 minutes
* Allow to cool
* Store in bottles at 4 °C until use

## Competent Bacteria For Glycerol Stock

* LB agar plates supplemented with antibiotic were inoculated with 50 μL of bacterial stock and incubated at 37 °C
* Inoculation of a single colony in 5 mL LB broth. Incubation at 37 °C
* Addition of 1 mL culture to 100 mL LB broth (1:100 dilution). Incubation until OD₆₀₀ 0.4–0.6
* On ice for 15 min for growth arrest
* Division of culture into two 50 mL Falcon tubes. Centrifugation at 3000 × g for 15 min at 4 °C
* Removal of supernatant. Resuspension of pellets in 50 mL ice-cold 50 mM CaCl₂ for each pellet
* Gentle inversion of Falcon tubes. Placement on ice every 10 inversions to prevent temperature drop
* Incubation on ice for 20 min
* Centrifugation at 3000 × g for 5 min at 4 °C. Resuspension of pellets in 4 mL 50 mM CaCl₂ with 15% glycerol in the same way
* Transfer of 200 μL from the Falcon tubes into fresh Eppendorf tubes, under flame
* Rapid transfer of each Eppendorf tube into a bath of absolute ethanol pre-chilled at –80 °C
* Verification of complete freezing of all tubes; disposal of incompletely frozen samples. Storage at –80 °C
* Alternative procedure: immersion of Eppendorf tubes in liquid nitrogen within a dry vessel in the absence of absolute ethanol

## Transformation

* Mixing of 100 μL competent cells with 100 ng plasmid DNA
* Incubation on ice for 30 min
* Heat shock at 42 °C for 1.5 min
* Incubation on ice for 5 min
* Addition of 400 μL LB pre-warmed at 37 °C. Incubation at 37 °C with shaking for 1 h
* Centrifugation at 3000 × g for 10 min. Removal of 200 μL supernatant to increase cell concentration
* Plating on LB agar plates supplemented with appropriate antibiotic. Incubation for 16–18 h at 37 °C

## PCR

PCR Reaction Setup (Total volume: 95 μL)

Polymerase used: NEB Taq 2× standard buffer master mix

* Forward primer (10 μM): 0.5 μL was added → 3.5 μL in scaled reactions.
* Reverse primer (10 μM): 0.5 μL was added → 3.5 μL in scaled reactions.
* Polymerase (Master Mix): 19.5 μL was included → 80 μL in scaled reactions.
* Plasmid DNA: For each 25 μL reaction, 2.5 μL plasmid DNA was added.
* Nuclease-free water: 9 μL was added → 63 μL in scaled reactions.

(Volumes were adjusted proportionally for multiple reactions.)

According to NEB, the recommended plasmid DNA amount was 1 pg – 10 ng. The plasmid stock concentration was 2000 ng/μL (2 μg/μL). So for each 25 μL reaction, 2.5 μL plasmid DNA was added.

Primer Dilution (from IDT, 100 μM stock)

* Forward primer: 1 μL of primer diluted in 9 μL nuclease-free H₂O → 10 μM working stock.
* Reverse primer: 1 μL of primer diluted in 9 μL nuclease-free H₂O → 10 μM working stock.

Due to the high variability of the primers, a temperature gradient was performed. Gradient annealing temperatures applied: 61°C, 62°C, 63°C, 64°C, 65°C, 66°C.

Thermocycling Conditions (30 cycles)

* Initial denaturation: Samples were heated at 94°C for 30 sec.
* Denaturation: 94°C for 15–30 sec.
* Annealing: 45–68°C for 15–60 sec.
* Extension: 68°C for 1 min/kb.
* Final extension: 68°C for 5 min.
* Hold: 4°C.

## PCR Clean-Up

* Mixture of 100 μL PCR product and 200 μL Buffer NTI
* Mixture transferred onto NucleoSpin spin column
* Centrifuged at 11,000 × g for 30 sec
* Flow-through discarded
* 700 μL of Buffer NT3 (supplemented with 100% ethanol) added to the column
* Centrifuged at 11,000 × g for 30 sec
* The previous step was repeated once more
* Flow-through discarded
* Centrifugation of column without any added liquid at 11,000 × g for 1 min
* Incubation at 70°C for 5 minutes on the thermoblock
* Flow-through discarded
* Transfer of the column into a 1.5 mL Eppendorf
* 25 μL of elution buffer NE were added
* Incubation at room temperature for 1 min
* Centrifugation at 11,000 × g for 1 min
* Step repeated two more times
* Fresh preheated buffer added at 70 °C and incubated for 5 min
* Centrifugation at 11,000 × g for 1 min
* Eluted DNA stored at −20 °C

## Agarose Gel

* Weighing of 0.5 g agarose
* Dissolution in 50 mL TAE buffer
* Heating in microwave until complete dissolution
* Addition of 2.5 μL EtBr when the solution has cooled down
* Pouring into casting tray and placement of comb
* Solidification at room temperature
* Mixing of 5 μL sample from each Eppendorf with 1 μL loading dye
* Loading onto gel
* Electrophoresis at 90 V for 30–40 min

## Plasmid Isolation And Nanodrop

* Cells collected by centrifugation at 11,000 × g, 10 μL LB-BL21
* Supernatant discarded
* Addition of resuspension buffer A1 and complete resuspension of cell pellet
* Addition of lysis buffer A2
* Gentle inversion 6–8 times, not vortexing
* Incubation at room temperature for 5 min
* Addition of neutralization buffer A, gentle inversion 6–8 times, avoiding vortexing
* Centrifugation at 11,000 × g for 10 min to clarify lysate
* Transfer of cleared supernatant onto NucleoSpin spin column placed in a collection tube
* Centrifugation at 11,000 × g for 1 min
* Flow-through discarded, spin column placed into a new collection tube
* Addition of 500 μL wash buffer AW1 to the spin column
* Centrifugation at 11,000 × g for 1 min
* Flow-through discarded
* Addition of 600 μL wash buffer AW2 to the spin column
* Centrifugation at 11,000 × g for 1 min
* Centrifugation repeated at 11,000 × g for 2 min to remove residual ethanol
* Spin column placed into a clean 1.5 mL Eppendorf tube
* Addition of 50 μL preheated elution buffer AE to the center of the membrane
* Incubation at 70 °C in a thermal block for 2 min
* Centrifugation at 11,000 × g for 1 min to elute plasmid DNA
* Nanodrop measurement

## Bacterial Lysis And Lysis Buffer Preparation

Preparation of lysis solutions:

* NaCl: 10 mM, 330 μL
* Tris HCl: 1 mM, 66 μL
* EDTA: 100 mM, 2000 μL
* Triton X-100: 0.5 v/v, 500 μL
* H2O: 79 mL

Division of 100 μL bacterial culture into:

* Two 50 mL Falcon tubes containing 40 mL culture each
* Two 15 mL Falcon tubes containing 10 mL culture each

Calculation of required lysis buffer:

* For 2 mL culture: 6 mL lysis buffer needed
* For 10 mL culture: 150 μL lysis buffer needed

Bacterial Lysis:

* Centrifugation of Falcon tubes without lysis buffer
* Discarding of supernatant and addition of LB medium to the pellet
* Gentle vortexing to resuspend the pellet
* Incubation at room temperature for 30 min
* Placement on ice for 10 min
* Sonication 3 times for 30 sec at 70% amplitude; 2 min rest on ice between each cycle
* Centrifugation at 15,000 × g for 30 min at 4 °C
* Separation of supernatant from the pellet

## SDS-PAGE Protocol

* Preparation of protein samples: proteins mixed with SDS sample buffer (containing SDS, β-mercaptoethanol, glycerol, bromophenol blue, and Tris-HCl)
* Heating of samples at 95 °C for 5 min to denature proteins
* Assembly of polyacrylamide gel in electrophoresis apparatus
* Loading of samples and protein ladder into wells
* Addition of running buffer to gel tank
* Electrophoresis at constant voltage (e.g., 120 V) until dye front reaches bottom of gel
* Removal of gel from apparatus
* Staining of gel with Coomassie Brilliant Blue or other protein stain
* Destaining to visualize protein bands

## Colloidal Chitin Preparation Protocol

* Weigh 20 g chitin powder into a glass beaker
* Add 350 mL cold concentrated HCl slowly while stirring
* Incubate at 4 °C for 24 h to allow partial depolymerization
* Filter the suspension through glass wool into 2 L pre-cooled ethanol (–20 °C) with vigorous stirring
* Centrifuge at 10,000 × g, 20 min, 4 °C
* Wash the resulting pellet repeatedly with dH₂O until the pH becomes neutral
* Lyophilize (freeze-dry) the washed colloidal chitin, or store it as a suspension at –20 °C

## Colloidal Chitin Agar Preparation Protocol

* Resuspend colloidal chitin in dH₂O to 1% (w/v)
* Homogenize by passing 5× through a hand homogenizer or by sonication until a milky suspension forms
* Prepare LB agar (1.6% agar) and autoclave
* Mix equal volumes of molten LB agar (cooled to ~50 °C) with colloidal chitin suspension to obtain final 0.5% colloidal chitin agar
* Pour plates (~20 mL per Petri dish) and allow them to solidify
* Store plates at 4 °C until use

## Chitinase Activity Assay

* Well diffusion assay: Punch wells into agar, load with culture supernatant or cell lysate
* Wait for at least 48 hours

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###### Wet Lab

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* [Awards](awards.html)

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# Results

On the Results Page we present the outcomes of our experiments, highlighting the performance, reliability, and insights gained from our iterative experimental process.

### Quick Overview

#### 📜 Introduction

A brief recap of the project's goals, methodology, and the context for our experimental results.

[Take Me There](#introduction)

#### 📊 Results

Detailed experimental outcomes, including key metrics, data visualization, and performance evaluation.

[Take Me There](#results)

#### 💬 Discussion

Interpretation of results, comparison with expectations, and implications for future work.

[Take Me There](#discussion)

#### ☰ Table Of Contents

* [Introduction](#introduction)
* [Results](#results)
* [Discussion](#discussion)

## 📜 Introduction

**Outline:** A brief recap of the project's goals, methodology, and the context for our experimental results.

Our project set out to create a reliable chitinase expression system, using an iterative design–build–test approach to guide each step. We combined molecular cloning, protein expression, and activity testing to see what worked and what could be improved. The results not only confirmed that our system was functioning as intended but also gave us valuable insights to refine and strengthen the project moving forward.

Below, we present the detailed results of our experiments, followed by a discussion of their significance and the insights they provide for improving the system.

## 📊 Results

**Outline:** Detailed experimental outcomes, including key metrics, data visualization, and performance evaluation.

**A comprehensive set of experiments was carried out to evaluate the progress of the experimental workflow, from transformation to activity testing.(figure 3)**

For the sake of logical flow and clarity, the results are presented in an order that differs slightly from the actual sequence of laboratory procedures. Specifically, the data are discussed beginning with antibiotic selection plates, SDS-PAGE expression analysis, and chitinase activity assays, followed by the results of agarose gel electrophoresis and NanoDrop spectrophotometry. This arrangement allows for a more coherent presentation of findings, progressing from protein-level validation to genetic-level confirmation.

Transformation on selective plates confirmed that the plasmid was **successfully taken up and maintained** in bacterial cells. Plasmid purification followed by NanoDrop analysis verified that the extracted DNA had both **high concentration** and **appropriate purity**, ensuring its suitability for downstream applications. PCR amplification under gradient conditions consistently produced a distinct **~2.25 kb band**, which after clean-up was clearly validated by agarose gel electrophoresis, confirming the **accuracy and reproducibility** of the amplification. At the protein level, IPTG induction followed by SDS-PAGE revealed distinct bands at the **expected molecular weight**, demonstrating **successful expression** of the composite construct. Finally, preliminary activity assays with colloidal chitin agar did not yield obvious halos of clearance, but they provided critical feedback by highlighting *assay sensitivity limitations* and guiding the integration of a **secretion signal** into the design.

**Together, these results demonstrate **stable transformation**, **reliable DNA preparation and amplification**, **robust protein expression**, and important insights for improving extracellular activity in future stages of the project.**

![Description](https://static.igem.wiki/teams/5994/images/fig1.webp)

Figure 1: Results Overview Summary of the main experimental outcomes, including transformation verification (antibiotic selection plates), protein expression (SDS-PAGE), preliminary functional testing (chitinase activity plates), DNA amplification (agarose gel PCR verification), and plasmid quality assessment (NanoDrop analysis).

### 1. Antibiotic selection plates

To confirm successful transformation, cultures were plated on LBAgar supplemented with the appropriate antibiotic. Growth of colonies on selective plates indicated the presence of transformed bacteria carrying the plasmid.

![Description](https://static.igem.wiki/teams/5994/images/fig2.webp)

Figure 2: BL21 (DE3) untransformed cells plated on LB agar supplemented chloramphenicol. No with colonies kanamycin and were observed, confirming the effectiveness of the antibiotic selection.

![Description](https://static.igem.wiki/teams/5994/images/fig3.webp)

Figure 3: BL21 (DE3) competent cells transformed with the construct and plated on LB agar supplemented with kanamycin and chloramphenicol. Distinct colonies were observed, indicating successful transformation and plasmid uptake.

### 2. SDS-PAGE

To verify expression of chitinase proteins, samples were analyzed by SDS-PAGE. Protein bands of the expected molecular weight were detected, confirming induction of target protein expression.

![Description](https://static.igem.wiki/teams/5994/images/figure4.webp)

Figure 4: SDS-PAGE analysis of protein expression SDS-PAGE was performed to evaluate induction of the composite protein. On the gel, samples correspond to before IPTG induction and after IPTG induction at different timepoints, showing the appearance of a band at the expected molecular weight region. Protein marker is indicated in the ladder lane (highlighted by circle), and the band corresponding to the calculated molecular weight of the target protein is observed in the predicted region.

### 3. Chitinase Activity Plates

To assess enzymatic activity, culture supernatants were applied to colloidal chitin agar. Distinct halos of clearance around wells indicated chitinase activity.

![Description](https://static.igem.wiki/teams/5994/images/fig5.webp)

Figure 5: Colloidal chitin agar plate loaded with lysate before IPTG induction. No clearance zones were expected or observed.

![Description](https://static.igem.wiki/teams/5994/images/fig6.webp)

Figure 6: Colloidal chitin agar plate loaded with lysate 2 h after IPTG induction. No distinct halos of clearance were visible.

![Description](https://static.igem.wiki/teams/5994/images/fig7.webp)

Figure 7: Colloidal chitin agar plate loaded with lysate 24 h after IPTG induction. No significant clearance was observed, consistent with low detectable activity under these conditions.

### 4. Agarose Gel (PCR Verification)

PCR products were resolved on a 1% agarose gel to evaluate amplification success. A distinct band at ~2.25 kb was observed, consistent with the expected insert size.

![Description](https://static.igem.wiki/teams/5994/images/fig8.webp)

Figure 8: Agarose gel electrophoresis of PCR products, showing a distinct band at ~2.25 kb, consistent with the expected insert size and confirming successful amplification.

### 5. NanoDrop Analysis – Plasmid DNA

Purified plasmid DNA was assessed by NanoDrop spectrophotometry. The concentration and purity (A260/280, A260/230) indicated successful extraction and suitability for PCR.

![Description](https://static.igem.wiki/teams/5994/images/fig9.webp)

Figure 9: NanoDrop spectrophotometric analysis of purified plasmid DNA. The concentration and purity ratios (A260/280, A260/230) indicated successful extraction and suitability for downstream applications such as PCR.

## 💬 Discussion

**Outline:** Interpretation of results, comparison with expectations, and implications for future work.

### Antibiotic Selection Plates

* **Experimental Plate (Figure 3):** This plate contained BL21 E. coli that had been transformed with the plasmid of interest and plated on LB Agar supplemented with Kanamycin and Chloramphenicol. Growth of distinct colonies was observed, confirming that only the transformed cells survived under selective pressure. The morphology of the colonies (round, off-white, and well-defined) is also consistent with E. coli growth. The absence of background lawn growth further supports that non-transformed cells were eliminated by antibiotic selection.
* **Blank Control Plate (Figure 2):** This plate contained LB agar with antibiotics but no bacteria inoculated. As expected, no colonies were observed, confirming that the medium was sterile and that growth on the experimental plate resulted specifically from transformed bacteria rather than contamination.

### SDS-PAGE

In the gel (figure 4), three conditions were tested: **before IPTG induction, 2 h after IPTG, and 24 h after IPTG**. Clear differences in protein expression were observed across these timepoints. At **2 h post-induction, a strong band appeared** at the expected molecular weight region (indicated by the circles next to the protein ladder). This band became even more pronounced after **24 h**, demonstrating a marked **increase in expression levels**.

Importantly, the bands marked by the circles correspond to the **molecular weight region where the fusion protein (composite part) is expected**. Some proteins did not migrate strictly according to their predicted molecular weight, which is a known occurrence in SDSPAGE due to **protein structure and charge**, but the consistent appearance of the induced band across replicates strongly supports the **correct expression of the construct**.

Overall, these results demonstrate that **the composite part is successfully expressed upon IPTG induction**. The **protein is clearly detectable**, accumulates over time, and its expression **does not appear to be toxic for the host cells**, as normal growth and induction were maintained.

This constitutes a major result for our project, confirming that **our designed genetic construct functions as intended**.

### Chitinase Activity Plates

Three colloidal chitin agar plates were prepared, into which cell lysates from three different induction conditions were loaded:

* **Before IPTG induction** (Figure 5)
* **2 hours after induction** (Figure 6)
* **24 hours after induction** (Figure 7)

As expected, **no clearance zones** were visible in the sample before induction, since *no chitinases* had been expressed at that stage. However, **no clear halos** were observed in the induced samples either, at 2 hours or at 24 hours post-induction. Even after staining and examining the plates under *UV light*, **no distinct results** were obtained. This lack of visible activity may be explained by the **instability of crude lysates**, which are easily degraded and therefore lose enzymatic activity, as well as by the **quality and purity of the colloidal chitin substrate**, which may have reduced enzyme accessibility. Furthermore, the well diffusion assay itself has relatively **low sensitivity**, which may limit the detection of low levels of activity.

Although **no obvious activity** was detected in these assays, the outcome is still significant, as it highlighted the need to improve **extracellular availability** of the enzymes. For this reason, in the **Implementation**, we have incorporated a **secretion signal**, aiming to facilitate export of the chitinases into the medium and enhance both their **activity** and **detectability** in future assays.

### Agarose Gel (PCR Verification)

**PCR amplification** of the target insert was carried out using a **temperature gradient** to optimize annealing conditions. The products were analyzed on a **1% agarose gel** (Figure 8), and across all tested gradient temperatures, the outcome was identical: a single, clear band appeared at **~2.25 kb**, corresponding precisely to the expected size of the insert. The uniform presence of this distinct band in every reaction demonstrates that the PCR was **highly specific**, with no evidence of nonspecific products or background smearing. The clarity and consistency of the bands across all conditions indicate that the primers were **well designed**, the template plasmid was of **good quality**, and the PCR clean-up was successful, removing any potential inhibitors. Importantly, the reproducibility of the result across the gradient shows that the amplification is **robust** and not dependent on a narrow temperature window, which increases confidence in the reliability of the method.

This result constitutes a **key milestone** for the project. It confirms that the insert can be consistently amplified in **high quality and yield**, providing the foundation for downstream cloning and expression experiments. The strong and uniform band pattern not only validates the experimental design but also demonstrates that the molecular tools developed for the project are **functional and effective**. In other words, the PCR verification establishes that our **target gene** is ready for subsequent integration into the composite construct and supports the overall feasibility of our implementation strategy.

### NanoDrop Analysis – Plasmid DNA

Purified plasmid DNA was analyzed using a **NanoDrop spectrophotometer** (Figure 9) to determine **concentration** and **purity**. The spectrum displayed a clear absorbance peak at **260 nm**, characteristic of nucleic acids, with a smooth baseline indicating the absence of major contaminants. The measured DNA concentration was **~109 ng/μL**, sufficient for downstream molecular biology applications. Purity ratios were within the acceptable range for high-quality DNA: the **A260/280 ratio** was 1.95, close to the ideal value of ~1.8–2.0 for pure DNA, indicating minimal protein contamination. The **A260/230 ratio** was 2.08, also within the expected range (2.0–2.2), suggesting that residual salts or organic compounds were not present in significant amounts.

Overall, the **NanoDrop results** confirm that the plasmid extraction was successful and yielded DNA of both **high concentration** and **purity**. The plasmid preparation was therefore deemed suitable for **PCR amplification** and subsequent **cloning procedures**.

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###### Home

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###### Project

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* [Safety](safety.html)

###### Wet Lab

* [Experiments](experiments.html)
* [Results](results.html)
* [Lab Book](lab-book.html)
* [New Composite Part](new-composite-part.html)

###### Human

* [Overview](overview.html)
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* [Education & Communication](education.html)
* [Collaboration](collaboration.html)

###### Dry Lab

* [Structural Analysis](structural-analysis.html)
* [Model](model.html)

###### Awards

* [Awards](awards.html)

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