Team:NIT Warangal/Proof Of Concept
Introduction
India is one of the world's top cotton producers, accounting for roughly 26% of worldwide cotton production. Cotton is essential to India's gigantic textile industry. Apart from that, it is an important part of home use in India and overseas. This brings us to cottonseed, which is the seed of the cotton plant. Cottonseed is most commonly used as livestock feed as well as cottonseed oil for human consumption. Cottonseed is also utilised as a fertiliser and in the cosmetics industry.
Cottonseed, due to the toxin gossypol, is extremely hazardous to most animals and humans. Anorexia, respiratory disorders, aberrant weight gain, impairment of the male and female reproductive systems, weakness, apathy, and even death can all be symptoms of gossypol poisoning. Some animals were also found to have heart failure.
Our goal is to develop a metabolic mechanism that degrades gossypol and converts it to a non-toxic form, making cottonseed oil safe for intake by both animals and humans.
Protocol
Research Methodology
Experimental Overview
The standard steps for cloning and expressing the gene of interest in pPIC9k are listed in the table below.
| Step | Action |
|---|---|
| 1 | The pPIC9k vector was propagated by transforming it into a recA, endA1 E. coli DH5α strain. |
| 2 | Double digestion was used to isolate the codon optimised Laccase gene from the plasmid. |
| 3 | Developing a codon optimised Laccase gene cloning technique in frame with the α-factor secretion signal in the pPIC9k vectors. |
| 4 | Selection of transformants on Low Salt LB Plates with 50 μg/ml ampicillin after transformation of the ligated plasmid into E. coli cells. |
| 5 | Restriction digestion was used to confirm the clone in 10-20 transformants. |
| 6 | The recombinant plasmid was purified and linearized before being transformed into Pichia pastoris (GS 115). |
| 7 | Pichia strain (GS 115) transformation and plating onto YPDS plates having GeneticinTM present in the proper concentration. |
| 8 | Transformants resistant to GeneticinTM were selected |
| 9 | Checking the expression of the gene and determining laccase activity. |
| 10 | Laccase treatment of cotton seed meal and determination of gossypol content reduction. |
Equipments
Thermocycler, electrophoresis unit, gel documentation system, centrifuge,
GenePulser XcellTM Electroporator
system, New Brunswick Scientific incubator shakers, Applied Thermal Control Water bath / Dry bath.
Reagents
Reagents required for cloning strategy -
For cloning strategy
Pichia host strain (GS 115), Electrocompetent E. coli DH5α for transformation, 3M sodium acetate, 100% ethanol, 80% ethanol, Low salt LB (Luria bertani) medium, Low Salt LB plates containing Geneticin, YPDS plates containing Geneticin.
Transesterification of waste cooking oil
As a lipid supply, 20g of waste cooking oil (refined sunflower oil) is mixed with 6 volumes of methanol and 0.5% sodium hydroxide (catalyst). For 180 minutes, the reaction was kept at 65°C. After that, the reaction mixture was left undisturbed in a separating funnel for 180 minutes to separate crude glycerol and biodiesel. The crude glycerol was utilised to make the pre-inoculum for BMMY medium. This will also be utilised for medium optimization.
Double Digestion reaction for eluting the codon-optimized Laccase gene
To elute the codon-optimized Laccase gene (COL) with EcoRI-NotI sites, double digestion reaction was used. The plasmid encoding codon-optimized laccase was gel eluted and cloned into the pPIC9k vector utilising restriction digestion with the EcoRI-NotI enzyme. Template - (gel eluted COL-6μl), buffer D - (1μl), EcoRI - (0.2μl) NotI - (0.2μl) D/W - (2.6μl) is one PCR reaction setup. The reaction was kept at 37 °C for 3 hours. The process was stopped by heating it to 65°C for 20 minutes and then loading it onto an agarose gel with a 0.8% concentration. A gel elution kit(NEB,USA) was used to remove restriction digested reaction products from cut gel pieces.
Double digestion of pPIC9k vector
The EcoRI-NotI enzyme was used for restriction digestion of the pPIC9k empty vector. As previously stated, the same reaction setup was used.
Ligation reaction of pPIC9k plasmid with COL double digested and gel eluted product
Sticky end ligation: A ligase enzyme was used to ligate EcoRI-NotI restriction digested COL gel eluted product into EcoRI-NotI restriction digested pPIC9k vector. pPIC9k vector (1.98μl), ligase enzyme (1μl), ligase buffer (1μl), and insert (1μl) D/W- (10.22μl) were used in the procedure. For overnight incubation, the ligation reaction was kept refrigerated at 16°C.
Transformation of ligation reaction into the E.Coli, Top10 cells
The two tubes (C and T) of 50μl of E.Coli competent cells were taken, and 15μl of the ligation reaction was transformed into 50μl of E.Coli proficient cell (T).Both tubes were placed on ice for 20 minutes and heat shock treatment was given at 42°C for 90 seconds. For 10 minutes, the C and T tubes were placed on ice. Each tube was filled with 900μl of fresh LB broth. Both tubes were maintained at 37°C for 1 hour in a Thermomixer. 200μl was taken from the aforementioned culture and distributed onto LB medium plates with 25 μg/ml of Geneticin. Plates were incubated at 37°C overnight. Colonies were selected from LB plates and inoculated into vials containing 2ml of fresh LB broth.PCR was used to select positive clones colony, and plasmids were isolated from the positive clones using a plasmid isolation kit (NEB, USA).
Confirmation by double digestion
Plasmids were isolated using a plasmid isolation kit for the selection of positive clones (NEB, USA). EcoRI and NotI digestions were used to confirm the clones.
Linearization of pPIC9k COL construct by using
enzyme SacI
Using the enzyme SacI, the pPIC9k COL construct was linearized for integration into the Pichia genome. Plasmid (30μl), buffer D (5μl), SacI (1μl), and D/W (14μl) were part of one reaction setup. The reaction was kept at 37°C for 3 hours in a dry bath. The reaction was stopped after 20 minutes of heating at 65°C. A small portion of the digest was tested for full linearization using agarose gel electrophoresis. 10% of volume 3 M sodium acetate and 2.5 volumes of 100% ethanol were added to the linearization reaction and centrifuged to pellet down the DNA.
Transformation of pPIC9k COL plasmid into Pichia pastoris
Pichia pastoris was inoculated into YPD broth and incubated at 28°C overnight. The cells were centrifuged for 5 minutes at 4°C at 1500 x g, and the pellet was resuspended in sterile MQ. The cells were centrifuged one more and resuspended in sterile 1M sorbitol. 80μl of cells from the above step was mixed with 5 - 10 μg of linearized plasmid DNA and transferred to an electroporation cuvette. The cells were pulsed using yeast-specific conditions, and sorbitol was added right away to stabilise the cells. On YPDS plates with 100μg/ml of geneticin, 10, 25, 50, 100, and 200μl were distributed. For three to five days, the plates were incubated at 28°C.
Determination of Mut phenotype
Using a sterilised toothpick, Geneticin-resistant transformants were patched onto the MMH and MDH plates. For two days, plates were incubated at 28°C. Mut+ strains will grow normally on both plates, whereas MutS strains will grow normally on the MDH plate but not at all on the MMH plate.
Expression of Laccase
Using a single colony, 30ml of MGY (Minimal Glycerol Medium) was inoculated and incubated overnight at 28°C. At room temperature, cells were extracted by centrifuging at 1,500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 100ml of BMGY and BMMH medium. To maintain growth, the flask containing BMGY and BMMH medium was incubated overnight at 28°C. After 24 hours, 1ml of 100% methanol was added to keep the induction going. 1 ml of the expression culture was transferred to a 1.5 ml microcentrifuge tube at each of the times listed below. The samples were examined for expression levels and the ideal time to harvest after induction. At room temperature, samples were centrifuged at maximum speed for 5 minutes in a microcentrifuge. The supernatant was separated and kept at minus 80°C in a separate tube. The following time periods were used to assess activity and expression levels: 0, 24, 48, 72, and 96 hours (Four days).
Laccase Activity
The oxidation of the ABTS technique was used to evaluate laccase activity[1]. Laccase oxidizes ABTS, a non phenolic dye, into a more stable and desired cation radical. The concentration of cation radicals that provide the vivid blue-green colour can be linked to enzyme activity and is measured at 420 nm[2]. 0.5 mM ABTS, 0.1 M sodium acetate (pH 4.5), and an appropriate amount of enzyme were included in the assay mixture. The increase in A420 (ε420, 3.6 × 104 M−1·cm−1)., which indicates ABTS oxidation, was measured. The reaction mixture was incubated for 5 minutes and contained 0.5 mM substrate (ABTS), 2.8 mL of 0.1 M sodium acetate buffer at pH 4.5, and 100 μl of culture supernatant. In a spectrophotometer, absorbance was measured at 420 nm against a suitable blank. The amount of laccase that oxidised 1 μmol of ABTS substrate per minute was defined as one unit. Bradford's dye-binding method was used to quantify protein concentration using BSA as a reference.
Defatting of cotton seed meal
100gm of cotton seed is grounded in a blender and sieved with 0.2 mm mesh. About 10gm of the cotton seed meal was mixed with 100 ml of n-hexane and the mixture was placed in a sealed air tight container and kept in the shaking incubator maintained at 35°C for 60 minutes to extract oil. The supernatant liquid was separated from the solid seeds and the defatted CSM was stored at -20°C and used for further experiments.
Biodegradation of gossypol using crude enzyme extract
The culture supernatants consisting of crude laccase enzyme is used to treat the defatted CSM and was evaluated for gossypol biodegradability. The reaction mixtures were prepared in 5 mL vials; 0.5 mL of culture supernatants was added in 2.5 mL of 0.1M phosphate buffer (pH 6.0). Furthermore, 0.1 mL of the enzyme extracts was added in 2.5 mL of the same buffer containing CSM at a 1g in sterile petri plates. Reaction mixture was incubated in a horizontal shaker at 65°C for 1 h. The reaction mixture was dried and used for gossypol estimation. Residual gossypol level in the mixture was analyzed by AOCS Ba 7-58[3].
References:
- [1] Bourbonnais R, Leech D, Paice MG. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochimica et Biophysica Acta. 1998;1379(3):381-390.
- [2] Majcherczyk A, Johannes C, Hüttermann A. Oxidation of polycyclic aromatic hydrocarbons (PAH) by laccase of Trametes versicolor . Enzyme and Microbial Technology. 1998;22(5):335-341.
- [3] Official Methods and Recommended Practices of the AOCS, 7th Edition, 2nd Printing - Ba 7-58.