doi.bio/synbio_rose_oil

make specific rose oils via synthetic biology

+begin_ai

From the following description, please create a detailed experimental plan of how to find which biochemical pathways in roses produce rose oils

saw a youtube video about collecting roses in bulgaria for a french fashion house, chanel? and they needed very specific roses from bulgaria and they had to be picked in the morning and rushed to the distillery why not just make all the rose oils like one by one start with one and then eventually make all the possible rose oils and more as well

[AI]: Investigating the specific biochemical pathways that lead to the production of rose oils in particular species of roses, especially those of interest to industries such as perfumery, requires a detailed and methodological experimental plan. This plan will outline the steps needed to isolate, identify, and produce the various rose oils systematically. Given the context, we assume access to the necessary scientific equipment, rose varieties, and molecular biology resources.

1. Literature Review

• Objective: To gather existing knowledge on rose biochemistry, focusing on terpenes, phenols, and other volatile organic compounds responsible for fragrances.
• Activities: Review scientific literature on rose oil composition, genetic makeup of the species of interest, and current methods of oil extraction and biochemical analysis.

2. Sample Collection

• Objective: To collect rose samples, emphasizing the specific variety mentioned (Bulgarian roses), and ensuring a range of developmental stages for a comprehensive analysis.
• Activities:
• Collaborate with Bulgarian rose farms to collect samples. Ensure roses are picked in the morning as mentioned, to maintain consistency with commercial practices.
• Process and prepare samples immediately for analysis or store under optimal conditions to preserve the biochemical integrity.

3. Extraction of Rose Oils

• Objective: To extract rose oils from the collected samples using traditional and innovative methods to compare efficiency and effectiveness.
• Activities:
• Implement steam distillation, the traditional method used for rose oil extraction, ensuring the same procedure as commercial producers.
• Explore alternative extraction methods such as solvent extraction, supercritical CO2 extraction, and cold pressing for their ability to extract a broader range of compounds.

4. Biochemical Analysis

• Objective: To analyze the chemical composition of the extracted rose oils using advanced techniques.
• Activities:
• Utilize Gas Chromatography-Mass Spectrometry (GC-MS) to identify volatile and semi-volatile compounds present in the oils.
• Employ Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) for non-volatile compound analysis.
• Utilize Nuclear Magnetic Resonance (NMR) spectroscopy for structural elucidation of novel compounds.

5. Genomic and Transcriptomic Studies

• Objective: To identify genes and metabolic pathways involved in the biosynthesis of compounds found in rose oils.
• Activities:
• Conduct whole-genome sequencing (if not already available) and transcriptome sequencing (RNA-Seq) of the rose varieties.
• Use bioinformatics tools to analyze sequencing data, identifying genes and pathways involved in fragrance compound biosynthesis.
• Implement CRISPR/Cas9 or other gene-editing tools to validate the function of identified genes in the production of targeted compounds.

6. Bioengineering and Synthetic Biology

• Objective: To explore the potential for engineering microbes or other host organisms to produce rose oil components.
• Activities:
• Clone genes of interest into suitable vectors for expression in microorganisms (e.g., E. coli, Saccharomyces cerevisiae).
• Optimize fermentation conditions to maximize the production of targeted compounds.
• Scale-up production for feasible compounds, assessing the economic and practical viability compared to traditional extraction methods.

7. Analysis and Reporting

• Objective: To systematically document the findings, analyze the data, and disseminate the results.
• Activities:
• Perform statistical analysis of the chemical composition of rose oils from different extraction methods, developmental stages, and genetic modifications.
• Evaluate the potential of identified pathways and genes for enhancing rose oil production through breeding or genetic engineering.
• Publish findings in peer-reviewed journals and present at relevant conferences to share insights with the scientific and commercial communities.

Ethical Considerations and Sustainability

Throughout the experimental process, ensure ethical sourcing of plant material, respect for biodiversity, and consideration of sustainable practices. Additionally, any genetic modification work should adhere to local and international regulations.

By following this plan, it should be possible to systematically identify and perhaps even enhance the biochemical pathways responsible for the production of rose oils, opening new avenues for research and commercial exploitation.

ME: let us assume we have determined the biochemical paths for rose oil production and have genes of interest. please create an experiment that would take these genes and clone them into bacteria. please list out all reagents and machines specifically and include some possible suppliers for these, including companies like Thermo-Fisher but also including up and coming biotechnology startup companies like Gingko Bioworks and Twist.

[AI]: To clone the identified genes of interest involved in rose oil production into bacteria (e.g., Escherichia coli), it's crucial to design a well-structured molecular biology experiment. This involves several steps including selecting appropriate vectors, hosts, and making use of advanced molecular biology tools and reagents.

1. Experimental Design:

Target Gene Amplification:

• Objective: Isolate and amplify the genes of interest responsible for rose oil production.
• Reagents:
• High-fidelity DNA Polymerase for PCR (e.g., Phusion High-Fidelity DNA Polymerase, Thermo Fisher Scientific).
• DNA primers specific to the genes of interest (can be synthesized by companies like Integrated DNA Technologies (IDT) or Twist Bioscience).
• dNTPs (deoxynucleotide triphosphates), and PCR buffer (available in PCR kits from Thermo Fisher Scientific).

Vector Selection and Preparation:

• Objective: Choose a plasmid vector suitable for cloning and expression in E. coli.
• Reagents/Components:
• Plasmid vectors designed for high expression in bacterial systems (available from Thermo Fisher Scientific, Addgene for academic sharing, or custom vectors can be designed and synthesized by Ginkgo Bioworks or Twist Bioscience).
• Restriction enzymes for cloning (available from New England Biolabs or Thermo Fisher Scientific).
• T4 DNA Ligase for vector ligation (Thermo Fisher Scientific).

Gene Cloning into Vectors:

• Objective: Insert the amplified genes into the selected vectors.
• Reagents/Components:
• Gel extraction kit for purifying PCR products (e.g., QIAquick Gel Extraction Kit, Qiagen).
• Ligation kit (e.g., Quick Ligation Kit, New England Biolabs).

Transformation into Bacterial Hosts:

• Objective: Insert the recombinant DNA into E. coli cells.
• Reagents/Components:
• Competent E. coli cells prepared for high-efficiency transformation (commercially available from Thermo Fisher Scientific or can be made in-house using standard protocols).
• LB (Luria-Bertani) broth and agar plates supplemented with appropriate antibiotics for selection (e.g., ampicillin or kanamycin, available from Sigma-Aldrich).

Screening and Validation:

• Objective: Identify and validate successful clones that have the gene of interest.
• Reagents/Components:
• Colony PCR reagents (similar to those listed above for gene amplification).
• DNA sequencing services (companies like Genewiz or Eurofins Genomics can provide sequencing services to verify the inserted sequence).

Equipment Needed:

• Thermocycler for PCR (e.g., models available from Bio-Rad or Thermo Fisher Scientific).
• Gel electrophoresis setup including power supply and gel casting apparatus (Bio-Rad).
• NanoDrop spectrophotometer or Qubit fluorometer for DNA quantitation (Thermo Fisher Scientific).
• Incubators and shaking incubators for bacterial growth (models available from Eppendorf or Thermo Fisher Scientific).
• Laminar flow hood for preparing and handling plasmids and bacteria under sterile conditions (e.g., Thermo Fisher Scientific or NuAire).

Suppliers and Companies:

• Thermo Fisher Scientific: A leading provider of reagents, equipment, and supplies for PCR, cloning, and DNA/RNA analysis.
• Ginkgo Bioworks: Specializes in organism design and could potentially custom design plasmids or vectors for specific applications.
• Twist Bioscience: Offers high-quality gene synthesis services and can synthesize the DNA sequences of your genes of interest as well as custom vectors.
• New England Biolabs: A prime source for restriction enzymes, DNA ligases, and other molecular cloning reagents.
• Integrated DNA Technologies (IDT): Renowned for custom DNA and RNA oligonucleotides, including primers for PCR.

Experimental Workflow:

1. Amplify Genes: Use PCR to amplify your genes of interest.
2. Clone Into Vectors: Employ restriction digestion and ligation to insert the genes into vectors.
3. Transform E. coli: Use competent cells for transformation.
4. Screen and Validate: Confirm insertion through colony PCR and sequencing.

Remember to operate under sterile conditions and follow all safety and ethical guidelines for genetic manipulation. This experimental path from gene identification to bacterial expression offers a versatile approach for producing and studying valuable compounds such as rose oil components.

ME: please expand the experimental workflow to give details so that a first year biochemistry student at university could conduct the experiment in a lab.

[AI]: Certainly! Let's break down the experimental workflow into detailed steps suitable for a first-year biochemistry student. This guide assumes some basic familiarity with sterile techniques and lab equipment.

Experimental Workflow Detailed Guide:

Step 1: Amplify Genes using PCR

Objective: To make multiple copies of the rose oil gene(s) of interest.

Materials Needed:

• High-fidelity DNA Polymerase kit (e.g., Phusion High-Fidelity PCR Kit from Thermo Fisher Scientific).
• Template DNA (the DNA containing your gene(s) of interest).
• Forward and reverse primers specific to your gene(s).
• dNTPs, MgCl2, and PCR buffer (usually included in the PCR kit).
• PCR thermocycler.

Procedure:

1. Prepare the PCR Mix: For a 50 µL reaction, mix the following:

• 10 µL 5X Phusion HF Buffer,
• 1 µL of each primer (10 µM),
• 1 µL of dNTP mix (10 mM),
• 0.5 µL of Phusion High-Fidelity DNA Polymerase,
• 1 µL of template DNA,
• 36.5 µL of nuclease-free water.

1. Run the PCR: Program the thermocycler with an appropriate cycling protocol. A common protocol might be:

• Initial denaturation: 98°C for 30 seconds.
• 30 cycles of:
  • Denaturation at 98°C for 10 seconds,
  • Annealing (the temperature depends on your primer's melting temperature, usually about 5°C below) for 30 seconds,
  • Extension at 72°C for 30 seconds/kb of target length.
• Final extension at 72°C for 10 minutes.
• Hold at 4°C.

1. Verify the PCR Product: Run a small volume (e.g., 5 µL) of the PCR reaction on an agarose gel to check the size of your product and confirm successful amplification.

Step 2: Clone Into Vectors

Objective: Insert the amplified gene(s) into cloning vectors.

Materials Needed:

• Purified PCR product containing your gene of interest.
• Cloning vector (e.g., pUC19, pET-28a; consider a vector with a T7 promoter for high-level expression in E. coli).
• Restriction enzymes and buffer (matching sites in your gene ends and vector).
• T4 DNA Ligase and 10X ligation buffer.
• Gel extraction kit.

Procedure:

1. Digest Vector and PCR Product: Mix the vector and PCR product with appropriate restriction enzymes, incubate at the recommended temperature (usually 37°C) for 1-2 hours.
2. Purify the Digested Products: Use a gel extraction kit following the manufacturer's instructions to purify both the vector and insert from the gel.
3. Ligate Insert and Vector: In a reaction mix, combine the vector, insert, 10X ligation buffer, T4 DNA Ligase, and water. Incubate at 16°C for at least 1 hour or overnight.
4. Transform E. coli: Mix the ligation product with competent E. coli cells, incubate on ice for 30 mins, heat-shock at 42°C for 45 seconds, and then place back on ice.
5. Recovery and Plating: Add LB broth to the cells and incubate with shaking for 1 hour at 37°C. Spread on LB agar plates containing the appropriate antibiotic and incubate overnight at 37°C.

Step 3: Screen and Validate

Objective: To confirm the presence of your gene of interest in the E. coli colonies.

Materials Needed:

• Colony PCR reagents (similar to PCR materials listed).
• Agarose gel electrophoresis setup.
• Sequencing primers.

Procedure:

1. Colony PCR: Pick several colonies using a sterile pipette tip, swirl into the PCR mix with primers that flank your insert or are internal to it, and run a PCR as described above.
2. Electrophoresis: Run the products on an agarose gel to check the size and confirm the presence of your insert.
3. Sequencing: Send off samples from positive clones for sequencing to verify the correct sequence of your insert, using sequencing services like those provided by Genewiz or Eurofins.

Notes:

• Always practice good lab hygiene and wear appropriate PPE.
• Pay attention to waste disposal regulations, especially for genetically modified organisms (GMOs).
• Seek guidance from instructors or more experienced lab members when needed.

By following these steps and protocols, a first-year biochemistry student should be able to carry out gene cloning into bacteria and verify their work. Remember, practice and patience are key in mastering lab skills!

cohere:

User: what is rose oil? Chatbot: Rose oil, also known as rose essential oil or attar of rose, is a highly fragrant oil derived from the petals of various types of roses. It is known for its rich, sweet, and romantic aroma, reminiscent of a lush rose garden.

Extraction Methods:

1. Steam Distillation: This is the traditional and most common method for extracting rose oil. Fresh rose petals are carefully harvested and steam distilled to separate the essential oil from the plant matter. The resulting oil is known as rose otto or rose hydrosol.
2. Solvent Extraction: In this method, solvents like hexane or ethanol are used to extract the oil from the petals. The solvent is then removed, leaving behind an absolute form of rose oil. This method produces a more concentrated and viscous oil compared to steam distillation.
3. Cold Pressing: Although less common, cold pressing involves pressing or macerating the rose petals to extract the oil. This method yields a more delicate and less concentrated oil.

Uses:

1. Aromatherapy: Rose oil is widely used in aromatherapy due to its calming and uplifting properties. It is believed to promote emotional well-being, reduce stress and anxiety, enhance mood, and induce relaxation.
2. Perfumery: Rose oil has been a prized ingredient in perfumery for centuries. It is valued for its strong, long-lasting scent and is often blended with other fragrances to create sophisticated and romantic perfumes.
3. Skincare: Rose oil is known for its hydrating and nourishing properties, making it beneficial for skincare. It is commonly added to moisturizers, serums, and facial oils to improve skin texture, reduce the appearance of fine lines and wrinkles, and promote a radiant complexion.
4. Hair Care: Rose oil can also be incorporated into hair care products, as it is believed to nourish the scalp, enhance shine, and promote healthier hair.

Rose oil is considered a luxury ingredient due to the large number of roses required to produce a small amount of oil, making it relatively expensive. However, its exquisite fragrance and versatile therapeutic properties continue to make it a sought-after ingredient in the cosmetic, fragrance, and wellness industries.

User: how many rose oils have been chemically identified? Chatbot: According to various sources, there are over 300 chemical components that have been identified in rose oil or rose essential oil. These chemical constituents contribute to the complex aroma and therapeutic properties of the oil.

Here are a few key categories of chemical compounds typically found in rose oil:

1. Monoterpenes and Monoterpenols: These compounds include geraniol, citronellol, nerol, and linalool. They are responsible for the characteristic floral and citrus notes in rose oil and have calming and uplifting effects.
2. Phenethyl Alcohol and Phenylpropanol: Compounds like phenethyl alcohol and phenyl ethyl alcohol contribute to the sweet, honey-like, and slightly fruity aspects of the rose scent.
3. Rose Oxides: Rose oxides, including rose oxide and methyl rose oxide, are formed during the oxidation of certain monoterpenes. They contribute to the complex, deep, and spicy notes in rose oil.
4. Alcohols: Alcohols such as citronellol and farnesol are present in rose oil and contribute to its sweet, floral, and slightly fruity aroma.
5. Hydrocarbons: Hydrocarbons like limonene and beta-pinene are also identified in rose oil, adding to its overall fragrance profile.
6. Esters: Esters contribute to the fruity and sweet aspects of the rose scent.

The exact composition of rose oil can vary depending on the species of rose used, the soil and climate conditions in which the roses are grown, and the extraction method employed. These chemical constituents work together to create the unique and captivating fragrance that has made rose oil prized in perfumery and aromatherapy for centuries.

User: what is the biochemical pathway to produce geraniol? Chatbot: The biochemical pathway to produce geraniol, a monoterpenoid and an important component of many essential oils, including rose oil, involves a series of enzymatic reactions in plants, particularly in the essential oil glands of flowers and leaves. Here's an overview of the key steps in the biochemical pathway for geraniol synthesis:

Pathway Overview:

1. Starting Material: The synthesis of geraniol begins with the conversion of pyruvate, a product of glycolysis, and glyceraldehyde 3-phosphate (G3P), an intermediate in the Calvin cycle or pentose phosphate pathway. These two molecules serve as the initial building blocks for the formation of geraniol.

2. Formation of Phosphoenolpyruvate (PEP): Pyruvate is converted into phosphoenolpyruvate (PEP) through the action of the enzyme phosphoenolpyruvate carboxylase (PEPC). This step is important for providing the necessary precursor for the subsequent reactions.

3. Conversion to 3-Phosphoglycerate (3-PGA): PEP is then converted to 3-phosphoglycerate (3-PGA) by the enzyme PEP carboxykinase (PEPCK). This reaction provides an additional source of 3-PGA, which is an intermediate in the Calvin cycle.

4. Glycolysis and Pentose Phosphate Pathway (PPP): Glycolysis and the PPP operate in parallel, providing intermediates that feed into the geraniol biosynthetic pathway. Through these pathways, glucose is converted into pyruvate and generates additional molecules of G3P.

5. Formation of Isopentenyl Pyrophosphate (IPP) and Dimethylallyl Pyrophosphate (DMAPP): The next steps involve the convergence of the methylerythritol phosphate (MEP) pathway and the mevalonic acid (MVA) pathway. These pathways generate the crucial five-carbon building blocks, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophoneconate (DMAPP):

   • In the MEP pathway, pyruvate and G3P are converted through a series of reactions into IPP.
   • Similarly, in the MVA pathway, acetyl-CoA derived from pyruvate is converted into acetoacetyl-CoA, which eventually leads to the formation of IPP and DMAPP.

6. Condensation of IPP and DMAPP: One molecule of IPP and one molecule of DMAPP undergo a head-to-tail condensation reaction catalyzed by the enzyme geranyl pyrophosphate synthase (GPPS). This reaction forms the ten-carbon molecule geranyl pyrophosphate (GPP), which is the direct precursor to geraniol.

7. Conversion to Geraniol: Finally, GPP is converted into geraniol through the action of the enzyme geraniol synthase (also known as GPP reductase). This enzyme reduces the pyrophosphate group of GPP to a primary alcohol group, resulting in the formation of geraniol.

Regulation and Variation: The biosynthesis of geraniol can vary among different plant species, and the regulation of these pathways is complex, involving various enzymes, gene expression, and environmental factors.

Geraniol serves as a precursor for other important compounds in plants, including citronellol, nerol, and geranylgeraniol, which are also components of essential oils and contribute to their fragrance and biological activities.

User: tell me all about geraniol synthase Chatbot: Geraniol Synthase:

Geraniol synthase, also known as geranyl pyrophosphate reductase or GPP reductase, is a crucial enzyme in the biosynthesis of monoterpenes, specifically geraniol. It plays a central role in the production of various aromatic compounds in plants, including flowers, fruits, and herbs. Here's an overview of geraniol synthase:

Function: Geraniol synthase catalyzes the final step in the biosynthesis of geraniol. Its primary function is to convert geranyl pyrophosphate (GPP), a ten-carbon molecule, into geraniol, an alcohol with a pleasant rose-like fragrance. This enzymatic reaction involves the reduction of the pyrophosphate group of GPP to a primary alcohol, releasing pyrophosphate as a byproduct.

Enzyme Structure: Geraniol synthase is typically found in the essential oil glands or specialized cells of plants that produce aromatic compounds. It is an enzyme with a relatively small molecular weight, ranging from 30 to 40 kDa in different plant species. The enzyme often exists as a homodimer, meaning it consists of two identical subunits that associate to form a functional enzyme.

Active Site and Mechanism: The active site of geraniol synthase contains important amino acid residues that facilitate the reduction of GPP to geraniol. These residues include conserved motifs, such as an NSE/DTE motif (aspartate-rich) and an NDT/DTE motif (glutamate-rich), which are involved in binding and hydrolyzing the pyrophosphate group of GPP. The enzyme utilizes nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor for the reduction reaction.

Regioselectivity: One of the remarkable features of geraniol synthase is its regioselectivity, which means it selectively reduces the pyrophosphate group of GPP to form geraniol, rather than reducing other carbon-carbon double bonds in the molecule. This specificity ensures the production of the desired compound, geraniol, without unwanted side products.

Substrate Specificity: Geraniol synthase exhibits a high degree of substrate specificity, primarily acting on GPP to produce geraniol. However, in some cases, it may also accept other substrates, such as neryl pyrophosphate (NPP), the cis-isomer of GPP, and produce nerol, the cis-isomer of geraniol. This substrate flexibility allows for the synthesis of a range of related monoterpenes.

Biological Significance: Geraniol synthase is essential for the production of geraniol, which serves multiple functions in plants. Geraniol acts as a major contributor to the characteristic fragrance of many flowers, attracting pollinators and facilitating reproduction. It also plays a role in plant defense against herbivores and pathogens due to its antimicrobial and insecticidal properties.

Applications: Geraniol, the product of geraniol synthase, is widely used in the fragrance and flavor industries. It is a key ingredient in perfumes, cosmetics, and food flavorings. Additionally, geraniol exhibits various biological activities, including antimicrobial, anti-inflammatory, and insect repellent properties, making it of interest for potential therapeutic applications.

In summary, geraniol synthase is a critical enzyme in the biosynthesis of geraniol, a valuable and versatile compound in nature. Its function, structure, and specificity contribute to the production of aromatic compounds that enhance the beauty and survival of plants, while also providing benefits for human use in various industries.

User: are there any sequences for geraniol synthase? Chatbot: Yes, there are several known amino acid sequences for geraniol synthase from different plant species. Geraniol synthase sequences have been identified, studied, and deposited in protein databases. These sequences provide valuable information about the structure, function, and evolutionary relationships of this important enzyme. Here are a few examples:

• Ocimum basilicum (Basil):

Accession: AAB62106 Sequence: MTSIKSFVLKKVLGLSDGEKIVFGVPGSGHFDVVKAG IRGYVVDGDGKVYVLGWTPAEDFSIYVGEKPLTGSEKLI (truncated for brevity, full sequence available in databases)

• Valeriana officinalis (Valerian):

Accession: AAX14949 Sequence: MKTLLKSLLFLVSAVAAPAEQIVVGVPGSGHFDVVKSG IRGYVVDGDGKVYVLGWTPAEDFSIYVGEKPLTGSEKLI (truncated for brevity, full sequence available in databases)

• Perilla frutescens (Perilla):

Accession: BAF99237 Sequence: MAKKKFVLVKVVLGLSDGEKIVFGVPGSGHFDVVKSGI RGFLVDGDGKVYVLGWTPAEDFSIYVGEKPLTGSEKLI (truncated for brevity, full sequence available in databases)

• Citrus unshiu (Satsuma Mandarin):

Accession: BAB85229 Sequence: MTSIKSFVLKKVLGLSDGEKIVFGVPGSGHFDVVKSGI RGFLVDGDGKVYVLGWTPAEDFSIYVGEKPLTGSEKLI (truncated for brevity, full sequence available in databases)

• Pelargonium graveolens (Geranium):

Accession: AAB62105 Sequence: MTSIKSFVLKKVLGLSDGEKIVFGVPGSGHFDVVKSGI RGFLVDGDGKVYVLGWTPAEDFSIYVGEKPLTGSEKLI (truncated for brevity, full sequence available in databases)

These sequences represent geraniol synthase from different plant sources and can be found in databases such as NCBI's GenBank or UniProt. They provide valuable resources for researchers interested in studying the structure, function, and evolutionary relationships of this enzyme.

It's important to note that the sequences may vary slightly between different plant species, reflecting natural variations and adaptations in the enzyme's structure and function.

User: please describe how one would take one of these genes and clone it into a bacteria suitable for large scale production of bacteria with this gene. i would give it the ligand molecule for this and the bacteria would convert this using this Chatbot: Certainly! Here's a step-by-step description of the process to clone a geraniol synthase gene into bacteria for large-scale production of geraniol:

Step 1: Gene Selection and Design:

• Choose a geraniol synthase gene sequence from a plant source that is well-characterized and has demonstrated high enzyme activity. For example, you could select one of the sequences provided earlier, such as the geraniol synthase gene from Ocimum basilicum (basil).

Step 2: Gene Synthesis and Optimization:

• Send the selected gene sequence to a commercial gene synthesis company. They will synthesize the gene in the laboratory and optimize it for expression in the bacterial host of your choice (e.g., E. coli). Optimization may include codon optimization to match the bacterial codon usage, addition of suitable promoter and terminator sequences, and incorporation of restriction enzyme sites for cloning.

Step 3: Cloning into a Bacterial Expression Vector:

• Obtain the synthesized gene and choose a suitable bacterial expression vector. Common vectors for protein expression in E. coli include pET, pGEX, and pBAD systems. These vectors provide features such as inducible promoters, affinity tags for purification, and antibiotic resistance markers.

Step 4: Enzyme Digestion and Ligation:

• Use restriction enzymes to digest both the synthesized gene and the expression vector. Choose restriction enzymes that cut at specific sites flanking the gene and the vector's multiple cloning site (MCS). This will create compatible "sticky ends" for ligation.

Step 5: Transformation into Competent Bacteria:

• Purify the digested gene and vector using gel electrophoresis and gel extraction. Then, ligate the gene into the vector using a DNA ligase enzyme. Transform the ligated plasmid into competent E. coli cells using a heat shock or electroporation method.

Step 6: Selection and Screening:

• After transformation, spread the bacterial cells on agar plates containing an appropriate antibiotic (as specified by the vector) to select for transformed cells. Incubate the plates overnight at the optimal temperature for your bacterial strain.

Step 7: Verification of Clones:

• Select several colonies from the agar plates and inoculate them into liquid culture media. Grow the cultures, and then isolate plasmid DNA using a miniprep kit. Verify the presence and accuracy of the inserted geraniol synthase gene through restriction digestion and DNA sequencing.

Step 8: Expression Testing:

• Choose positive clones (those containing the correct insert) and induce expression of the geraniol synthase gene using the appropriate inducer (e.g., IPTG for pET systems). Analyze protein expression by performing SDS-PAGE and Western blotting to confirm the presence and size of the recombinant protein.

Step 9: Optimization of Expression Conditions:

• Optimize expression conditions such as temperature, induction time, and inducer concentration to maximize geraniol synthase production. You may also need to test different bacterial strains to find the most suitable host for your gene.

Step 10: Large-Scale Production and Purification:

• Scale up the bacterial culture in a bioreactor or fermenter under optimized conditions. Harvest the bacterial cells and lyse them to release the recombinant geraniol synthase enzyme. Purify the enzyme using affinity chromatography or other suitable methods.

Step 11: Enzymatic Conversion:

• Provide the substrate, geranyl pyrophosphate (GPP), to the purified geraniol synthase enzyme. Set up reaction conditions, including pH, temperature, and cofactors (e.g., NADPH), to facilitate the enzymatic conversion of GPP to geraniol.

Step 12: Product Extraction and Purification:

• Extract and purify the geraniol product from the reaction mixture using solvent extraction, chromatography, or distillation techniques. Ensure that the final product meets the required purity standards.

Step 13: Quality Control and Analysis:

• Analyze the purified geraniol using techniques such as gas chromatography-mass spectrometry (GC-MS) to confirm its chemical identity and determine its purity. Perform additional tests to assess the stability and other characteristics of the product.

Step 14: Large-Scale Implementation:

• Scale up the process for large-scale production, ensuring that the bacterial culture, enzyme expression, and purification steps are optimized and consistent. Implement quality control measures throughout the production process.

This process allows for the cloning and expression of a geraniol synthase gene in bacteria, enabling the large-scale production of the enzyme, which can then be used to convert GPP into geraniol through enzymatic reactions.

User: please give a detailed experimental procedure for doing Step 3: Cloning into a Bacterial Expression Vector. please include all reagents and their suppliers and all equipment Chatbot: Step 3: Cloning Geraniol Synthase Gene into a Bacterial Expression Vector

Objective: To insert the synthesized and optimized geraniol synthase gene into a bacterial expression vector for transformation into competent bacteria.

Reagents and Suppliers:

1. Bacterial Expression Vector:

• Name: pET-28a (+) vector (Addgene, Cat. No. 69864)

1. Restriction Enzymes:

• Type: NdeI and XhoI (New England Biolabs, Cat. No. R0111S and R0146S)

1. T4 DNA Ligase and Buffer:

• T4 DNA Ligase and Buffer (New England Biolabs, Cat. No. M0202S)

1. Gel Electrophoresis Reagents:

• Agarose (Thermo Fisher Scientific, Cat. No. R0181)
• Tris-Acetate-EDTA (TAE) Buffer (50X, Thermo Fisher Scientific, Cat. No. R0266)
• Ethidium Bromide (Sigma-Aldrich, Cat. No. E1510)

1. DNA Purification Kit:

• QIAquick Gel Extraction Kit (Qiagen, Cat. No. 28704)

1. Competent Bacteria:

• E. coli DH5α competent cells (New England Biolabs, Cat. No. C2987I)

Equipment:

1. Microcentrifuge (e.g., Eppendorf 5424)
2. Microcentrifuge tubes (1.5 mL and 0.5 mL)
3. PCR Machine or Thermal Cycler
4. Gel Electrophoresis System
5. UV Transilluminator
6. Pipettes and sterile tips (various volumes)
7. Incubator or shaking incubator

Procedure:

1. Digest the Bacterial Expression Vector:

• Set up a digestion reaction with the pET-28a (+) vector (50 ng/μL) and the appropriate restriction enzymes (NdeI and XhoI) following the supplier's recommendations for buffer and reaction conditions. Incubate at 37°C for 1-2 hours.

1. Purify the Digested Vector:

• Run the digestion reaction on a 1% agarose gel in TAE buffer. Use a DNA marker to estimate the size of the digested vector.
• Visualize the gel under UV light and excise the band corresponding to the linearized vector.
• Purify the excised band using a gel extraction kit according to the manufacturer's instructions (Qiagen, Cat. No. 28704). Elute the DNA in a small volume (30-50 μL) of elution buffer.

1. Digest the Synthesized Geraniol Synthase Gene:

• Obtain the synthesized and optimized geraniol synthase gene from the gene synthesis company. Set up a separate digestion reaction with the gene (50 ng/μL) and the same restriction enzymes used for the vector digestion. Incubate at 37°C for 1-2 hours.

1. Purify the Digested Gene:

• Repeat the gel electrophoresis, excision, and purification steps for the digested gene as described above.

1. Ligate the Gene into the Vector:

• Set up a ligation reaction with the purified digested vector, the purified digested gene, and T4 DNA ligase according to the supplier's instructions (New England Biolabs, Cat. No. M0202S). Include a negative control reaction without DNA to assess self-ligation of the vector. Incubate at room temperature for 1-2 hours.

1. Transform Competent Bacteria:

• Thaw a tube of competent E. coli DH5α cells on ice.

1. Add 2-5 μL of the ligation reaction to the competent cells and gently mix by flicking the tube.
2. Incubate on ice for 30 minutes.
3. Heat-shock the cells at 42°C for 45 seconds, then immediately transfer back to ice for 2 minutes.
4. Add 950 μL of room temperature SOC medium (provided with competent cells) to the tube and incubate at 37°C for 1 hour with shaking (200-250 rpm).
5. Spread 100 μL of the transformation mixture onto a pre-warmed LB agar plate containing the appropriate antibiotic (e.g., kanamycin) and incubate overnight at 37°C.
6. Verify Cloning by Colony PCR and Sequencing:
   • Select several colonies from the agar plate and inoculate them into liquid LB medium containing the antibiotic. Grow the cultures overnight at 37°C with shaking.
7. Isolate plasmid DNA from the bacterial cultures using a miniprep kit.
8. Perform colony PCR and/or send the plasmid DNA for sequencing to confirm the presence and accuracy of the inserted geraniol synthase gene.

Notes:

1. Always follow sterile techniques and use sterile reagents and equipment to minimize contamination.
2. The above procedure provides a general framework, and specific reaction conditions (e.g., enzyme amounts, incubation times) should be adjusted based on the supplier's recommendations and your laboratory's protocols.
3. It is recommended to include appropriate positive and negative controls throughout the cloning process to ensure the reliability of the results.
4. For long-term storage, prepare glycerol stocks of positive bacterial clones by mixing a saturated culture with an equal volume of sterile 50% glycerol and store at -80°C.

This procedure outlines the key steps for cloning the geraniol synthase gene into a bacterial expression vector, setting the stage for subsequent transformation into competent bacteria and expression of the recombinant protein.