iGEM 2025 — Team nchu

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

* Record all activities in chronological order, with each entry clearly dated.
* Provide sufficient detail for each activity, including:

+ Purpose of the activity.
+ Methods and procedures used.
+ Results obtained.
+ Observations and notes.

* Document all experiments, protocols, data analysis, meetings, brainstorming sessions, and other project-related activities.
* Include photos, videos, and diagrams to illustrate your progress.
* Indicate which team members participated in each activity.

## Inspirations

---

* [2024 EPFL](https://2024.igem.wiki/epfl/notebook)
* [2024 JU-Krakow](https://2024.igem.wiki/ju-krakow/notebook)

{% endblock %}

[![Logo](https://static.igem.wiki/teams/5901/img/logo.avif)

National Chung Hsing University

I
nternational
G
enetically
E
ngineered
M
achine

2025](/nchu/home/)

Project

[Description](/nchu/description/)
[Engineering](/nchu/engineering/)
[Contribution](/nchu/contribution/)

Our Team

[Team members](/nchu/members/)
[Attributions](/nchu/attributions/)

Lab

[Project Safety and Ethics](/nchu/safety-and-security/)
[Methods and Materials](/nchu/experiments/)
[Results](/nchu/results/)

[Human Practices](/nchu/human-practices/)

[Introduction](/nchu/human-practices/#introduction)
[Carbon Emission Analysis](/nchu/human-practices/#carbon-analysis)
[Livestock & Dairy](/nchu/human-practices/#livestock)
[A2 Milk & Health](/nchu/human-practices/#a2milk)
[SDGs](/nchu/human-practices/#sdg)
[Events](/nchu/human-practices/#events)
[Expert Interview](/nchu/human-practices/#events)
[Education & Outreach](/nchu/human-practices/#events)
[Business Plan](/nchu/human-practices/#business)

# Engineering

1. Home
2. About
3. Engineering

# iGEM Engineering Cycle

This section outlines our experimental journey and key learnings during the iGEM project cycle, focusing on strain verification, gene cassette construction, transformation, protein expression, growth optimization, and carbon fixation analysis.

1. Verification of Target Strains (F1+2 -301)

To confirm gene integration, PCR was performed on constructed yeast strains containing RuBisCO Form I and II genes using Kit 5 and Kit 6 as references. PCR results exhibited inconsistency, with amplification success varying despite identical conditions. To mitigate this, we reduced reaction volumes and increased parallel PCR replicates, improving overall success rate.

2. Cassette 1 Construction

The target DNA fragment was ligated into Kit 1 using restriction enzymes XhoI and NotI. Initial ligation attempts failed, as PCR verification did not yield expected fragment sizes. After performing an enzyme activity assay, low enzyme activity was confirmed and enzymes were replaced with fresh ones, resulting in successful ligation.

3. Yeast Transformation

Variations in gel extraction and purification led to inconsistent DNA purity affecting electroporation success. Following recovery and plating on G418-containing YPD agar, initial PCR verification failed to detect target bands. Repeating gel purification to improve DNA quality followed by electroporation enabled successful integration confirmed by PCR.

4. A2 β-Casein Expression and Growth / Carbon Fixation

**Cultivation and Antibiotic Selection:** Multiple antibiotic concentrations were used during plating to minimize selection bias and improve strain validation accuracy, due to variable antibiotic tolerance among clones.

**Protein Expression Confirmation:** SDS-PAGE failed to detect expected A2 β-casein bands, likely due to limited cell disruption from freeze–thaw methods. Future plans include optimizing lysis methods and introducing GFP reporters to verify gene expression and locate issues.

**OD600 Growth Curve Measurement:** Large yeast cell size caused sedimentation bias, affecting OD sampling. Switching to pipette volumes equal to sample volume for resuspension improved measurement consistency and reproducibility.

**Gas Chromatography Analysis:** Initial GC measurements over 48 hours in triplicates revealed oxygen depletion after 24 hours and lacked correlation with growth phase due to absent cell growth monitoring. Also, sequential strain measurements risked sample contamination; syringes sealed with rubber stoppers showed minor air leaks affecting accuracy. Later experiments improved by collecting gas and liquid samples every 4 hours for 24 hours with single replicates to reduce variability.

5. Future Directions

Planned work includes extending sampling duration and studying RuBisCO’s function and regulation under anaerobic conditions to understand its impact on yeast growth and carbon metabolism.

#### Contact Us

Biotechnology Center, NCHU, Taiwan

+886 - 422840416 # 404

igemteam@email.nchu.edu.tw

#### Let us know what you think

Share your feedback, questions, or suggestions about our project

Send

© [National Chung Hsing University](https://www.nchu.edu.tw/en-index), iGEM Team 2025. Content is licensed under
[CC BY 4.0](https://creativecommons.org/licenses/by/4.0/).

Designed by Ajesh Saviour Paravila

[![Logo](https://static.igem.wiki/teams/5901/img/logo.avif)

National Chung Hsing University

I
nternational
G
enetically
E
ngineered
M
achine

2025](/nchu/home/)

Project

[Description](/nchu/description/)
[Engineering](/nchu/engineering/)
[Contribution](/nchu/contribution/)

Our Team

[Team members](/nchu/members/)
[Attributions](/nchu/attributions/)

Lab

[Project Safety and Ethics](/nchu/safety-and-security/)
[Methods and Materials](/nchu/experiments/)
[Results](/nchu/results/)

[Human Practices](/nchu/human-practices/)

[Introduction](/nchu/human-practices/#introduction)
[Carbon Emission Analysis](/nchu/human-practices/#carbon-analysis)
[Livestock & Dairy](/nchu/human-practices/#livestock)
[A2 Milk & Health](/nchu/human-practices/#a2milk)
[SDGs](/nchu/human-practices/#sdg)
[Events](/nchu/human-practices/#events)
[Expert Interview](/nchu/human-practices/#events)
[Education & Outreach](/nchu/human-practices/#events)
[Business Plan](/nchu/human-practices/#business)

# Methods and Materials

1. Home
2. Lab
3. Methods and Materials

* [**Project Overview**](#overview)
* [**Strains & Plasmids**](#strains-plasmids)
* [**Culture Media**](#culture-media)
* [**Gene Design**](#gene-design)
* [**Molecular Methods**](#molecular-methods)
* [**Yeast Transformation**](#yeast-transformation)
* [**Growth Analysis**](#growth-measurements)
* [**Protein Analysis**](#protein-analysis)

# Methods & Materials

This section details the comprehensive experimental framework employed in our project to engineer Kluyveromyces marxianus for sustainable A2 β-casein production. Our approach integrates synthetic biology, metabolic engineering, and advanced molecular techniques to develop a novel microbial platform for dairy protein synthesis.

Experimental Design Strategy

**Host Selection**

Engineered *K. marxianus* 4G5 as GRAS-certified expression host with superior protein secretion capabilities

**Genetic Engineering**

PGASO system for precise multi-gene integration of A2 β-casein and RuBisCO pathways

**Metabolic Integration**

Combined heterologous protein expression with carbon fixation pathways for enhanced sustainability

**Analytical Validation**

Comprehensive characterization of growth, protein expression, and metabolic performance

**Core Biological Components:**

* A2 β-casein gene from *Bos taurus* (NCBI: AAI11173.1)
* RuBisCO Form I/II from *Rhodopseudomonas palustris*
* Phosphoribulokinase (PRK) for Calvin cycle integration

* PGASO cassettes for modular gene assembly
* Lac4 promoter system for inducible expression
* Hygromycin B resistance for selection

Our integrated approach combines cutting-edge synthetic biology with metabolic engineering to create a sustainable platform for dairy protein production, potentially revolutionizing traditional dairy manufacturing processes.

## Strains & Plasmids

### Strains Used

Microbial Strains

| Strain | Genotype/Description | Application | Reference/Source |
| --- | --- | --- | --- |
| *Kluyveromyces marxianus* 4G5 | Wild-type strain, GRAS status, thermotolerant (25-52°C) | Primary expression host for A2 β-casein | Laboratory stock |
| *Escherichia coli* DH5α | F⁻ φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17(rₖ⁻ mₖ⁺) phoA supE44 λ⁻ thi-1 gyrA96 relA1 | Plasmid propagation and cloning | Invitrogen |
| *K. marxianus* 301 | Engineered strain with RuBisCO Form I/II integration | Carbon fixation studies | This study |

**K. marxianus Advantages:**

* No Crabtree effect - efficient growth under oxygen-rich conditions
* Superior protein secretion compared to *S. cerevisiae*
* Broad substrate utilization (lactose, cellulose, inulin)
* Rapid growth rate reducing fermentation time
* Thermotolerance enabling high-temperature processes

### Strain Preservation

Cryopreservation Protocol

**Method:** Single colonies were inoculated into 5 mL liquid medium and incubated until logarithmic growth phase (OD₆₀₀ = 0.8-1.4). Cultures were mixed with equal volume of sterile 50% (v/v) glycerol and stored at -80°C.

**Preservation Conditions:**

* Storage temperature: -80°C
* Cryoprotectant: 25% glycerol (final concentration)
* Culture phase: Mid-logarithmic (optimal viability)
* Container: Cryogenic vials with O-rings

### Plasmid Methods

Plasmid Propagation & Storage

##### Calcium Chloride Transformation Protocol

**Transformation Steps:**

1. Day prior: Pre-chill 0.1 M CaCl₂ and 0.1 M CaCl₂ + 15% glycerol at 4°C
2. Culture: Inoculate single *E. coli* colony into 5 mL LB, incubate overnight at 37°C with shaking
3. Competent cells: Thaw on ice, add 10 ng plasmid DNA or 10 μL ligation mixture to 50 μL cells
4. Incubation: Ice for 30 min, heat shock at 42°C for 45 sec, return to ice for 2 min
5. Recovery: Add 450 μL LB medium, incubate at 37°C for 1 h
6. Selection: Plate 100 μL onto LB agar with appropriate antibiotic, incubate overnight at 37°C

**Critical Parameters:**

* DNA concentration: 10 ng optimal for transformation efficiency
* Heat shock duration: 45 seconds critical for membrane permeability
* Recovery time: 1 hour essential for antibiotic resistance expression
* Antibiotic concentration: Optimized for each resistance marker

## Culture Media

All media were prepared with analytical-grade reagents and sterilized using standard autoclaving protocols. Specific carbon sources were filter-sterilized and added post-autoclaving to prevent caramelization and degradation.

### LB (Luria-Bertani) Medium

Composition & Preparation

**LB Broth Formula:**

* 25 g LB powder (MDBio, Inc.)
* 1 L Milli-Q water
* pH: 7.0 ± 0.2 (unadjusted)

**LB Agar:**

* Add 15 g/L agar (MDBio, Inc.)
* Pour into 90 × 15 mm Petri dishes
* Store at 4°C

##### Sterilization Protocol

**Autoclaving conditions:** 121°C, 1.2 kg/cm² for 20 minutes. Media cooled to ~55°C before antibiotic addition and pouring. Agar plates solidified at room temperature and stored at 4°C.

### YPD (Yeast Extract Peptone Dextrose) Medium

Yeast Growth Medium

**YPD Broth Composition:**

* 10 g/L yeast extract (MDBio, Inc.)
* 20 g/L peptone (Gibco)
* 20 g/L dextrose (BD)
* Prepared in 1 L sterile water

**Preparation Notes:** Dextrose solution sterilized separately (0.22 μm filter) and added post-autoclaving to prevent Maillard reactions. YPD agar prepared by adding 1.5% agar to basal medium prior to sterilization.

### YPG (Yeast Extract Peptone Galactose) Medium

Induction Medium

**YPG Broth Composition:**

* 10 g/L yeast extract (MDBio, Inc.)
* 20 g/L peptone (Gibco)
* 20 g/L galactose (Sigma)
* Prepared in 1 L sterile water

**Application:** Used for Lac4 promoter induction. Galactose serves as both carbon source and inducer for PGASO-controlled expression. Sterilized separately and added to base medium after autoclaving.

## Gene Fragment Design

Our genetic engineering strategy employed the PGASO (Promoter-based Gene Assembly and Simultaneous Overexpression) system for precise, modular assembly of heterologous genes. This approach enabled efficient integration of both A2 β-casein and carbon fixation pathways into the *K. marxianus* genome.

### Primer Design & Synthesis

Primer Design Strategy

**Design Platform & Synthesis:**

* Design Software: Benchling platform (Benchling, Inc.)
* Synthesis Partners: Twist Bioscience Corporation (iGEM sponsored) and Genomics Inc. (Taiwan)
* Quality Control: HPLC purification for all primers
* Storage: -20°C in TE buffer at 100 μM concentration

##### Rainbow Primer System

Laboratory primers designed for PGASO cassettes were named sequentially as Rainbow1, Rainbow2, etc. Primer names do not correspond to cassette order but follow internal laboratory nomenclature for standardized assembly protocols.

### CSN2 Gene Source

A2 β-casein Gene Selection

**Gene Information:**

* Gene: Bos taurus β-casein (CSN2)
* Accession: AAI11173.1 (NCBI GenPept)
* Variant: A2 allele selected
* Size: ~760 bp coding sequence

**Rationale for A2 Selection:**

* Associated with improved milk digestibility
* Lower inflammatory potential
* Clinical evidence of better tolerance
* Commercial relevance in dairy market

##### Sequence Optimization

Codon optimization performed for *K. marxianus* expression system while maintaining native protein sequence. GC content adjusted to 45-55% for optimal expression in yeast host.

### PGASO Cassette Construction

Modular Assembly System

**PGASO System Features (Chang et al., 2012):**

* Homologous recombination-based gene assembly
* Modular promoter–MCS–terminator cassettes (Kit-1 to Kit-7)
* Unique promoter sequences for each cassette
* Flexible assembly order via primer modification
* Simultaneous overexpression capability

##### Cassette Configuration

**Kit-1:**

Contains CSN2 gene fragment (~2300 bp with insert)

**Kit-3:**

Antibiotic resistance marker (Hygromycin B)

**Kit-7:**

Terminal cassette for integration completion

##### Assembly Primers

**Primers used:** Rainbow1, Rainbow34, Rainbow5, Rainbow12, Rainbow13, Rainbow14. Designed for integration downstream of Lac4 promoter in combination with RuBisCO Form I/II-related cassettes.

The PGASO system's modularity allowed precise control over gene integration order and expression levels, enabling optimized metabolic engineering of *K. marxianus* for simultaneous A2 β-casein production and carbon fixation.

## Molecular Biology Methods

### Agarose Gel Electrophoresis

DNA Fragment Analysis

**Standard Protocol:**

* Gel concentration: 0.8–2% (w/v) agarose in 0.5× TAE buffer
* Staining: GelView nucleic acid stain (USB)
* Electrophoresis: Horizontal tanks at 5 V/cm
* Visualization: Gel Logic 200 imaging system (Kodak)

**Preparation:** Agarose melted by microwave heating in short intervals and cooled to 50–60°C before adding stain. Gels cast with combs appropriate for sample volume (10-50 μL).

### Polymerase Chain Reaction (PCR)

Amplification Conditions

| Component | Volume (μL) | Final Concentration |
| --- | --- | --- |
| Nuclease-free water | 30.4 | - |
| HF Buffer (5×) | 10 | 1× |
| dNTP mix (2.5 mM each) | 4 | 200 μM |
| MgCl₂ (50 mM) | 1 | 1 mM |
| Primer-F (10 μM) | 2 | 0.4 μM |
| Primer-R (10 μM) | 2 | 0.4 μM |
| Template DNA | 0.2 | Variable |
| Phusion DNA Polymerase | 0.4 | 0.02 U/μL |
| **Total Volume** | **50** | - |

##### Thermal Cycling Conditions

| Step | Temperature (°C) | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 0:40 | 1 |
| Denaturation | 98 | 0:12 | 32× |
| Annealing | Tm | 0:30 |
| Extension | 72 | 0:50 |
| Final Extension | 72 | 7:00 | 1 |
| Hold | 12 | ∞ | - |

### DNA Ligation

T4 DNA Ligase Protocol

**Ligation Reaction:**

* Enzyme: T4 DNA Ligase with ATP cofactor
* Insert: CSN2 fragment (~760 bp)
* Vector: PGASO Kit-1 (~2300 bp)
* Molar Ratio: 3:1 (insert:vector)
* Concentrations: 49.96 ng/μL (insert), 44.67 ng/μL (vector)

**Calculation Basis:** Molar ratio calculated assuming average molecular weight of 650 g/mol per base pair. Reactions incubated at 16°C for 16 hours for optimal ligation efficiency.

### Gel Extraction & Purification

FavorPrep™ Purification Protocol

**Extraction Steps:**

1. Excision: Target bands excised with sterile scalpel, trimmed to ≤300 mg
2. Dissolution: Mixed with 500-1000 μL FADF buffer, incubated at 55°C for 5-10 min
3. pH Adjustment: Solution color monitored (yellow optimal), 10 μL 3M sodium acetate (pH 5.0) added if violet
4. Binding: Loaded into FADF column, centrifuged at 11,000 × g for 30 sec
5. Washing: 750 μL Wash Buffer with ethanol, centrifugation
6. Drying: Full speed centrifugation (~18,000 × g) for 3 min
7. Elution: 20 μL Elution Buffer/ddH₂O, incubate 1 min, collect eluate

**Quality Control:** DNA concentration and purity measured by NanoDrop (Clubio). Fragments with concentrations ~150 ng/μL (10 μL total) used for subsequent yeast electroporation.

## Yeast Transformation

Electroporation Protocol

**Competent Cell Preparation:**

* Culture: *K. marxianus* 4G5 in YPD until OD₆₀₀ = 0.8–1.4
* Washing: Cells washed with sterile water
* Pre-treatment: Pre-Treating Buffer (YPD + 100 μL 1M HEPES + 250 mM DTT), 30°C, 100 rpm, 1 h
* Electroporation Buffer: 25 μL 100 mM Tris pH 7.5, 50 μL 135 mM sucrose, 5 μL 50 mM LiAc, adjusted to 250 μL

##### Electroporation Parameters

**Instrument Settings (BioRad Gene Pulser Xcell):**

* Voltage: 1000 V
* Capacitance: 25 μF
* Resistance: 400 Ω
* Cuvette: 0.2 cm gap
* DNA: 10 μL at 1 μg/μL concentration
* Cells: 40 μL competent cells

##### Post-Transformation Recovery

**Recovery Protocol:** Cells recovered in YPD medium at 30°C for 1-4 hours, then plated onto YPD agar containing G418 (200 μg/mL) for selection. Plates incubated at 30°C for 2-3 days until colony formation.

### Colony PCR Verification

Genomic DNA Extraction & Screening

**InstaGene Protocol (Bio-Rad):**

1. Colonies resuspended in sterile water and pelleted
2. Treated with InstaGene matrix
3. Incubated at 56°C for cell wall digestion
4. Boiled at 100°C for 8 min to release DNA
5. Centrifuged, supernatant used as PCR template

**Verification Strategy:** PCR performed using methods described in Section 2.4.2 to confirm cassette integration. Positive transformants selected based on amplification of expected fragment sizes.

## Growth Measurements & Physiological Analysis

Cultivation Conditions

**Open Culture System (Aerobic):**

* Volume: 200 mL YPD in 500 mL flasks
* Buffer: 100 mM phosphate buffer, pH 7.0
* Conditions: 30°C, 200 rpm
* Inoculum: OD₆₀₀ = 0.01
* Sampling: Every 12 h for 6 days (12 time points)
* Induction: At 48 h, transfer to YPG for Lac4 induction

**Closed Culture System (Anaerobic):**

* System: 250 mL anaerobic bottles
* Medium: YPG medium
* Conditions: 30°C, 200 rpm
* Inoculum: OD₆₀₀ = 0.01
* Sampling: Every 4 h for 24 h (7 time points)
* Analysis: OD₆₀₀ and headspace gas by GC

### Optical Density Measurement

Spectrophotometric Analysis

**Measurement Protocol:**

* Instrument: EzDrop 1000C spectrophotometer or equivalent
* Range: OD values maintained within 0.1–0.9 by dilution
* Calculation: ODoriginal = ODmeasured × dilution factor
* Replicates: All measurements in triplicate (n = 3)
* Presentation: Mean ± standard deviation

**Growth Curve Analysis:** Both long-term (144 h, 13 points) and short-term (24 h, 7 points) cultures monitored. Cultures shaken for 15 seconds prior to sampling to ensure homogeneous suspension.

### Gas Chromatography Analysis

CO₂ Emission Quantification

**GC Instrument Parameters (Agilent 7890A):**

* Inlet: 100°C, 12 mL/min flow rate
* Oven: 40–250°C with programmed heating
* Detector: 225°C with 30 mL/min airflow
* Makeup Gas: 10 mL/min N₂
* Carrier Gas: Nitrogen, pressure 3 kg/cm²

**Sampling Protocol:** Headspace gas samples (1 mL) withdrawn every 4 h for 24 h using gas-tight syringes. Calibration gases prepared in 10 mL vials with known CO₂/air mixtures. All measurements performed in triplicate.

## Protein Analysis Methods

### Cell Disruption & Protein Extraction

Freeze-Thaw Lysis Protocol

**Extraction Procedure:**

1. Harvest: 20 mL culture after 144 h, pellet cells by centrifugation
2. Wash: Resuspend in sterile water (5 mL/g wet weight)
3. Cell Wall Digestion: Pre-Urea Buffer A + 0.3% Lyticase (10 U/μL), 37°C, 1 h
4. Freeze-Thaw: Six cycles (liquid N₂ 30 sec, -80°C water bath 60 sec)
5. Separation: Centrifuge at 4°C, collect supernatant and pellet fractions

**Buffer Composition:** Pre-urea extraction buffer A contains 77.6 mM potassium phosphate buffer (pH 8.0), 465.5 mM NaCl. Urea added at 480.5 g per 644.5 mL buffer to yield 1 L urea buffer A.

### SDS-PAGE Analysis

Protein Separation & Detection

**Electrophoresis Protocol:**

* Sample Preparation: 15 μL sample + 5 μL 4× loading dye, 95°C for 5 min
* Ladder: 5 μL PageRuler protein ladder
* Electrophoresis: 90 V until dye front reaches gel bottom
* Staining: Staining buffer for 30 min
* Destaining: Destaining buffer overnight

**Expected Results: Target A2 β-casein protein band at ~24 kDa. Both supernatant and pellet fractions analyzed to determine localization of expressed protein.**

### Elemental Analysis

Biomass Composition Analysis

**Instrument & Method:**

* Instrument: Elementar vario EL Cube elemental analyzer
* Facility: National Chung Hsing University
* Sample: Freeze-dried biomass
* Combustion: ~1800°C in oxygen atmosphere
* Detection: Thermal conductivity detection (TCD)
* Elements: C, H, N, S, O composition

**Application:** Elemental analysis provides insights into metabolic changes and carbon utilization efficiency in engineered strains compared to wild-type controls.

This comprehensive methodological framework enabled systematic engineering of *K. marxianus* for sustainable A2 β-casein production, combining advanced molecular techniques with rigorous analytical validation to ensure reproducible and scientifically sound results.

#### Contact Us

Biotechnology Center, NCHU, Taiwan

+886 - 422840416 # 404

igemteam@email.nchu.edu.tw

#### Let us know what you think

Share your feedback, questions, or suggestions about our project

Send

© [National Chung Hsing University](https://www.nchu.edu.tw/en-index), iGEM Team 2025. Content is licensed under
[CC BY 4.0](https://creativecommons.org/licenses/by/4.0/).

Designed by Ajesh Saviour Paravila

[![Logo](https://static.igem.wiki/teams/5901/img/logo.avif)

National Chung Hsing University

I
nternational
G
enetically
E
ngineered
M
achine

2025](/nchu/home/)

Project

[Description](/nchu/description/)
[Engineering](/nchu/engineering/)
[Contribution](/nchu/contribution/)

Our Team

[Team members](/nchu/members/)
[Attributions](/nchu/attributions/)

Lab

[Project Safety and Ethics](/nchu/safety-and-security/)
[Methods and Materials](/nchu/experiments/)
[Results](/nchu/results/)

[Human Practices](/nchu/human-practices/)

[Introduction](/nchu/human-practices/#introduction)
[Carbon Emission Analysis](/nchu/human-practices/#carbon-analysis)
[Livestock & Dairy](/nchu/human-practices/#livestock)
[A2 Milk & Health](/nchu/human-practices/#a2milk)
[SDGs](/nchu/human-practices/#sdg)
[Events](/nchu/human-practices/#events)
[Expert Interview](/nchu/human-practices/#events)
[Education & Outreach](/nchu/human-practices/#events)
[Business Plan](/nchu/human-practices/#business)

# Results

1. Home
2. Lab
3. Results

# Experimental Results

This section presents the comprehensive experimental results from our engineering of Kluyveromyces marxianus for A2 β-casein production. Our findings demonstrate successful genetic integration, metabolic characterization, and protein expression analysis, providing critical insights into the potential of microbial dairy protein synthesis.

Key Experimental Findings

**Genetic Engineering Success**

Successful integration of A2 β-casein and RuBisCO genes into *K. marxianus* genome with high transformation efficiency

**Metabolic Impact**

RuBisCO-positive strains showed enhanced stationary phase stability and altered carbon utilization dynamics

**Growth Characteristics**

Engineered strains exhibited distinct growth patterns under both aerobic and anaerobic conditions

**Protein Expression**

Successful gene integration confirmed, though protein detection requires optimization of extraction methods

## Plasmid & Primer Verification

### Plasmid Preservation & Transformation Efficiency

Transformation Success Metrics

**Transformation Results:**

* Competent cells: *E. coli* DH5α showed high transformation efficiency (>10⁸ CFU/μg DNA)
* Plasmid stability: Recombinant plasmids maintained integrity through multiple generations
* Colony selection: Single colonies appeared after 12-16 hours on selective media
* Storage viability: Glycerol stocks maintained at -80°C showed 100% viability after 3 months

##### Primer Validation

All Rainbow primers (Rainbow1, Rainbow5, Rainbow12, Rainbow13, Rainbow14, Rainbow34) demonstrated high specificity and efficiency in PCR amplification. M13-F and M13-R primers served as reliable controls for vector backbone verification.

Primer Sequences & Applications

| Primer Name | Sequence (5'→3') | Application | Target |
| --- | --- | --- | --- |
| Rainbow1 / KI-PLac4-3'End-F | CCGCGGGGATCGACTCATAAAATAG | Cassette assembly | Lac4 promoter region |
| Rainbow5 / KI-PGapDH-F | AGTATGGTAACGACCGTACAGGCAA | Cassette assembly | GapDH promoter |
| Rainbow12 / ScTTGap\_Sc-PADHI-R | GGAATCCCGATGTATGGGTTTGGTT GCCAGAAAAAGGAAGTCCATATTG TACACTGGCGGAAAAAATTCTTTGTAAAA | Terminator assembly | ADH1 terminator |
| Rainbow13 / Sc-PADHI-F | GTGTACAATATGGACTTCCTCTTTTC | Promoter assembly | ADH1 promoter |
| Rainbow14 / KI-PLac4-5'End-R | GAATTTAGGAATTTTAAACTTG | Integration verification | Lac4 integration site |
| Rainbow34 / KITTLac4\_KI-PGapDH | GGACTCCAGCTTTTCCATTTGCCTTC GCGCTTGCCTGTACGGTCGTTACCA TACTTATACAACATCGAAGAAGAGTC T | Homologous recombination | Lac4-GapDH junction |

## Gel Electrophoresis Verification

### K. marxianus 301 Strain Verification

RuBisCO Gene Integration Confirmation

**PCR Amplification Results:**

* Target genes: kit5 and kit6 containing RuBisCO Form I and Form II genes
* Amplification: Clear bands at expected sizes confirmed successful integration
* Strain designation: KM 301 confirmed to contain both Form I and Form II RuBisCO
* Location: Genes integrated in kit2, kit5, and kit6 positions

### A2B Gene Ligation into Kit1

Restriction Digest Confirmation

**Restriction Analysis:**

* Enzymes: NotI and XhoI restriction sites engineered into A2B fragment
* Digestion conditions: 37°C for 1 hour, followed by 65°C for 20 min inactivation
* Result: Successful release of ~760 bp A2B insert from Kit1 vector
* Sequencing: Sanger sequencing confirmed correct A2B sequence (see Supplementary Document)

**Reaction Composition:** 22 μL nuclease-free water, 5 μL NEBuffer 3.1, 1 μL NotI, 2 μL XhoI, 5 μL A2B PCR product (45 ng/μL).

### Gel Extraction & Purification Efficiency

DNA Fragment Recovery

**Cassette 1 Recovery:**

* Size: ~1000 bp
* Yield: ~150 ng/μL
* Purity: A260/A280 = 1.8-2.0
* Quality: Bright, well-defined bands

![PCR verification gel image](https://static.igem.wiki/teams/5901/img/gel.avif)

Figure 1: PCR verification results of transformants using genomic DNA extracted with the InstaGene Matrix. A distinct band appeared at approximately 3000 bp, indicating that the cassette 1 fragment was successfully integrated into the host genome.

**Cassette 3 Recovery:**

* Size: ~3000 bp
* Yield: ~150 ng/μL
* Purity: A260/A280 = 1.8-2.0
* Quality: High PCR amplification efficiency

The obtained DNA fragments exhibited high purity and sufficient concentration, making them ideal templates for subsequent PGASO assembly and yeast transformation. The intensity of the bright bands indicated adequate DNA yield for electroporation, which improved transformation success rates.

## Gene Integration in Yeast Transformants

### Transformation Efficiency & Colony Selection

Electroporation Success Rates

**Transformation Outcomes:**

* Total transformants: 32 colonies isolated across different construct combinations
* Selection: Hygromycin B (60 μg/mL) effective for positive selection
* Recovery: 12-hour cultivation in YPD sufficient for colony PCR analysis
* Efficiency: ~80% of colonies showed successful gene integration

##### Strain Designations

**807 Series:**

* 807t1–t8: *Bos taurus* A2B + RuBisCO genes
* 807m1–m8: *Bos grunniens* A2B + RuBisCO genes

**812 Series:**

* 812t1–t8: *Bos taurus* A2B only
* 812m1–m8: *Bos grunniens* A2B only

![PCR verification gel image](https://static.igem.wiki/teams/5901/img/807.avif)

Figure 2: 807 series transformants (A2B + RuBisCO)

![PCR verification gel image](https://static.igem.wiki/teams/5901/img/812.avif)

Figure 3: 812 series transformants (A2B only)

### PCR Verification of Gene Integration

Genomic Integration Confirmation

**Verification Results:**

* Cassette I integration: Distinct band at ~3000 bp confirmed successful integration
* Transformation efficiency: Except for sample m2, all transformants showed clear amplification
* Strong signals: m1, m4, m5, m8, t3, and t4 showed particularly high transformation efficiency
* Group comparison: t group generally showed higher transformation efficiency than m group

![PCR verification gel image](https://static.igem.wiki/teams/5901/img/pcr.avif)

Figure 4: PCR verification showing ~3000 bp Cassette I integration

![PCR verification gel image](https://static.igem.wiki/teams/5901/img/pcr2.avif)

Figure 5: Colony PCR of HygB-resistant transformants

![PCR verification gel image](https://static.igem.wiki/teams/5901/img/pcr3.avif)

Figure 6: Target fragment amplification (~800 bp)

##### Selected Strains for Protein Production

Based on PCR verification, six transformants were selected for downstream protein expression experiments: 807t2, 807t6, 807m5, 807m6, 812t1, and 812m7. These strains showed the strongest and most consistent integration signals.

## Growth & Physiological Measurements

### Open Culture (Aerobic) Growth Analysis

Long-term Growth Characteristics

**Key Observations:**

* Growth patterns: RuBisCO-negative strains (KM 4G5, 812t1, 812m7) exhibited slower growth under aerobic conditions
* Stationary phase: After 108 hours, RuBisCO-negative groups showed more rapid OD decline
* Phenotypic changes: 812t1 and 812m7 cultures showed gradual defoaming and marked OD decline over time
* Metabolic burden: Casein gene expression imposed higher metabolic load, reducing basic metabolic efficiency

Strains carrying the RuBisCO gene appeared to utilize carbon sources more efficiently, maintaining better growth and stability during stationary phase. This suggests that RuBisCO expression may contribute to metabolic flexibility under nutrient-limited conditions.

### Closed Culture (Anaerobic) Growth Analysis

Oxygen-Limited Growth Dynamics

**Anaerobic Performance:**

* Oxygen depletion: Sealed bottles reached ~1% of initial oxygen by incubation end
* Sampling frequency: Every 4 hours for 24 hours provided high-resolution growth data
* Replication: All measurements conducted in triplicate (n=3)
* GC integration: Simultaneous headspace gas analysis enabled metabolic correlation

### Metabolic Burden Assessment

Heterologous Expression Impact

**Resource Allocation Effects:**

* Casein expression cost: Strains with casein gene but lacking RuBisCO showed more pronounced growth decline
* Control comparison: KM 4G5 (no casein gene) exhibited similar but less severe growth attenuation
* Nutrient depletion: Accelerated nutrient consumption observed in high-expression strains
* Competitive advantage: RuBisCO-positive strains maintained growth stability despite metabolic burden

Under sufficient oxygen but limited nutrient conditions, heterologous protein expression imposed significant metabolic burden. However, RuBisCO co-expression appeared to mitigate this burden through enhanced carbon utilization efficiency, particularly during stationary phase.

## Gas Chromatography Analysis

CO₂ Emission Profiles

**GC Analysis Findings:**

* Unexpected result: Control strain KM 4G5 demonstrated highest growth efficiency per unit CO₂ emission
* Time-dependent pattern: After 16 hours, RuBisCO-containing strains showed slower CO₂ emission increase
* Carbon utilization: Altered carbon dynamics observed in engineered strains
* Method reliability: Triplicate measurements showed high reproducibility (<5% variation)

##### OD-GC Correlation Analysis

**Integrated Results Interpretation:**

* Anaerobic conditions: KM 4G5 displayed lower CO₂ emission while maintaining superior growth
* Exponential phase: KM 4G5 grew faster than engineered strains initially
* Stationary phase advantage: RuBisCO-positive strains showed enhanced stability and slower OD decline
* Metabolic timing: RuBisCO contribution appears more significant during stationary phase

While our initial hypothesis predicted higher growth efficiency for RuBisCO-containing strains at equivalent CO₂ emission levels, the results revealed a more complex relationship. RuBisCO expression appears to confer advantages primarily during nutrient-limited stationary phases rather than during exponential growth, suggesting context-dependent metabolic benefits.

## Protein Expression Analysis

### Cell Disruption Efficiency

Freeze-Thaw Lysis Assessment

**Lysis Protocol Outcomes:**

* Method: Six freeze-thaw cycles (liquid N₂ 30 sec, -80°C water 60 sec)
* Cell wall digestion: 0.3% Lyticase (10 U/μL) effective for pre-treatment
* Visual observation: Cells showed swelling and partial rupture
* Protein release: Limited target protein detection in supernatant fractions

**Limitation Identified:** The freeze-thaw approach primarily caused cell swelling and partial rupture. While proteins within disrupted cells were expected to release into supernatant, insufficient release was detected, suggesting either incomplete lysis or formation of small membrane pores preventing efficient protein extraction.

### SDS-PAGE Analysis

Protein Detection Challenges

**Electrophoresis Results:**

* Target protein: A2 β-casein expected at ~24 kDa
* Observation: No distinct band representing target protein detected
* Sample analysis: Both supernatant and pellet fractions examined
* Method assessment: Cell disruption identified as potential bottleneck

**Interpretation:** The absence of the target protein band suggests that either the cell disruption method was insufficient for complete protein release, or the expression levels were below detection limits. Alternative lysis methods and expression optimization are required for conclusive protein detection.

### Conclusions & Future Directions

Protein Expression Optimization Strategy

**Identified Improvements:**

* Cell disruption: Optimize lysis method (bead beating, French press, or detergent-based)
* Expression verification: Introduce GFP reporter to confirm Kit1 translation capability
* Induction optimization: Fine-tune Lac4 promoter induction conditions
* Detection sensitivity: Implement Western blotting for enhanced protein detection

While successful gene integration was conclusively demonstrated through multiple verification methods, protein expression detection requires methodological optimization. Future work will focus on improving cell disruption efficiency and implementing more sensitive detection methods to confirm functional A2 β-casein production in our engineered *K. marxianus* strains.

## Overall Conclusions

Key Experimental Achievements

**Genetic Engineering Success**

* Successful integration of A2 β-casein gene into *K. marxianus*
* High-efficiency transformation using PGASO system
* Multiple strain variants created and verified
* Stable maintenance of integrated genes

**Metabolic Engineering Insights**

* RuBisCO expression alters carbon utilization
* Enhanced stationary phase stability in engineered strains
* Context-dependent metabolic advantages observed
* Successful demonstration of multi-gene pathway integration

**Methodological Developments**

* Optimized *K. marxianus* transformation protocol
* Comprehensive analytical framework established
* Integrated growth and metabolic analysis
* Identification of key optimization targets

**Future Research Directions**

* Protein expression optimization
* Extended metabolic characterization
* Scale-up and production optimization
* Application development for sustainable dairy

Our results demonstrate the successful engineering of *K. marxianus* as a platform for sustainable dairy protein production. While protein expression requires further optimization, the foundational genetic and metabolic engineering achievements provide a robust platform for developing novel, environmentally friendly alternatives to traditional dairy production methods.

#### Contact Us

Biotechnology Center, NCHU, Taiwan

+886 - 422840416 # 404

igemteam@email.nchu.edu.tw

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