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:

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

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

Colonies resuspended in sterile water and pelleted
Treated with InstaGene matrix
Incubated at 56°C for cell wall digestion
Boiled at 100°C for 8 min to release DNA
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: OD_original = OD_measured × 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:

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

Methods and Materials