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

2. Sample Collection

3. Extraction of Rose Oils

4. Biochemical Analysis

5. Genomic and Transcriptomic Studies

6. Bioengineering and Synthetic Biology

7. Analysis and Reporting

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:

Vector Selection and Preparation:

Gene Cloning into Vectors:

Transformation into Bacterial Hosts:

Screening and Validation:

Equipment Needed:

Suppliers and Companies:

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:

Procedure:

  1. Prepare the PCR Mix: For a 50 µL reaction, mix the following:
  1. Run the PCR: Program the thermocycler with an appropriate cycling protocol. A common protocol might be:
  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:

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:

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:

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):

  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:

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

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

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

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

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:

Step 2: Gene Synthesis and Optimization:

Step 3: Cloning into a Bacterial Expression Vector:

Step 4: Enzyme Digestion and Ligation:

Step 5: Transformation into Competent Bacteria:

Step 6: Selection and Screening:

Step 7: Verification of Clones:

Step 8: Expression Testing:

Step 9: Optimization of Expression Conditions:

Step 10: Large-Scale Production and Purification:

Step 11: Enzymatic Conversion:

Step 12: Product Extraction and Purification:

Step 13: Quality Control and Analysis:

Step 14: Large-Scale Implementation:

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:
  1. Restriction Enzymes:
  1. T4 DNA Ligase and Buffer:
  1. Gel Electrophoresis Reagents:
  1. DNA Purification Kit:
  1. Competent Bacteria:

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:
  1. Purify the Digested Vector:
  1. Digest the Synthesized Geraniol Synthase Gene:
  1. Purify the Digested Gene:
  1. Ligate the Gene into the Vector:
  1. Transform Competent Bacteria:
  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:
  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.




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