Biolab

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[edit] Equipment: Introduction

-Incoming Transmission

The minimum set of what you need are: 1) Tools to aspirate and dispense liquids (1 uL to 10 mL); usually you can buy pipettors to accomplish this. 2) Containers to hold liquids (1 uL to 1 L); these usually include various types of flasks, beakers, graduated cylinders, plastic tubes, and petri dishes 3) Machines to heat/cool liquids; these include thermal cyclers (for heating/cooling DNA in a PCR reaction) and shaker incubators (for heating/cooling cells) 4) Machines to sterilize your tubes/chemicals; these include autoclave machines (and sometimes people use pressure cookers) 5) Centrifuges 6) Gel-running equipment, including a power supply, gel electrophoresis box + combs, and gel imager (usually UV light, but there are also blue light imagers) 7) Spectrophotometer to analyze samples

That's pretty much all you need to get a simple bio lab started (infrastructure-wise); you'll need to obviously buy disposable items/chemicals as well.

Most of my equipment that I have right now is on the higher-end of things and is largely automation-friendly, so it's a lot more expensive and nicer than a typical manual bio-lab setup you'd see in academia.

See this DIYBio page for some other tips/items: http://openwetware.org/wiki/DIYbio/FAQ/Equipment

-End of Transmission

Equipment for use in a biolab varies by the intended lab function. A microbiology lab will require at minimum an incubator, a sterile working area, and a pressure cooker for sterilisation. Ideally it would also have a centrifuge and appropriate glassware such as petri dishes and test tubes. A molecular biology (DNA/Protein) lab will require more equipment to handle, visualise and store DNA/Protein. A plant tissue culture lab would resemble a microbiology lab but would have artificial lighting installed in growth chambers.

Here are some examples of easily acquired/made items of equipment for a biotech lab, divided loosely by function. As many labs will require a baseline microbiology setup to function (for example, a DNA manipulation lab will require microbes to carry and safely store DNA), assume that a microbiology lab is the "minimum" lab.

[edit] OSE context

An agroecological approach to producing biobased materials will require a specialized set of protocols and equipment, that will combine agricultural, biochemical, and localized economic knowledge. Methods to store, propagate, optimize, and manipulate biocatalysts for human utility will most likely benefit OSE in the pursuit of post-scarcity, with an ecology intertwined with agriculture, biorefinement of chemicals, and human health.

[edit] Microbiology Lab

A lab that enables the safe growth, storage and handling of microbes, whether bacteria, yeast or other fungi, or single-celled algae, is a microbiology lab. Requirements include sterility, incubation, safe handling/storage, and safe disposal where relevant.

Functions of a microbiology lab include medical diagnostics by traditional culture of blood or skin samples, propagation of agriculturally important strains and species, scale-up of useful strains for fermentation or composting, nutritional fermentation of yeasts and bacteria for consumption, or as a foundation for molecular biology methods like DNA manipulation.

[edit] Incubation

An incubator can be produced using a simple thermostat and a heater, and a well-insulated compartment or container. A simple example is a polystyrene shipping box with a radiative infrared heating mat and a pet thermostat, which can easily and accurately maintain a 30C incubator.

[edit] Sterilisation

A pressure cooker can be used to sterilise heat-stable liquids, solids and equipment by maintaining full temperature and pressure for 20-25 minutes. To confirm sterilisation, chemical indicator tape that changes colour is normally used, although cultures of heat-stable spores could also be used as indicators; after sterilisation, the indicator culture is incubated and observed for growth, which would indicate a failed sterilisation. The usual spore culture used for this is Bacillus stearothermophilis though B.subtilis (below) spores would probably suffice.

For heat-stable equipment, wrapping in metal foil and baking at 200C for 1:20 hours is sufficient. Longer time periods at lower temperatures can be used also if 200C is beyond the reach of available equipment.

Where filters are available, filter sterilisation is an attractive means of sterilising heat-labile liquids such as antibiotic samples. Filters may consist of "candle" filters used for water sterilisation (although the strict requirements of a lab may call for double-filtration), or syringe-powered filter cartridges. It is possible (though never tested) that in-house-produced cellulose filters from kombucha could be used if properly treated and if suitable pressure is applied.

Finally, for heat-labile ingredients, tyndallisation can be used; over three successive days, steam is used to pasteurise the sample. Vegetative (growing) bacterial/yeast/fungal cells are killed during the steaming process, and as new cells germinate over the following two days they are also killed. This process is somewhat gentler than pressure cooking, but more labour intensive and prone to failure.

[edit] Centrifugation

A centrifuge is used to separate cells from a liquid culture, and for transferring cells from one culture sample to another, possibly with "rinsing" steps. The procedure is simple; cells are spun at a high speed so that they are pelleted against the bottom of the sample vial/tube, and the liquid they were suspended in can then be removed with a pipette. The pelleted cells can then be resuspended in a new liquid using agitation with a pipette or inverting/vortexing/flicking/spinning the tube.

DremelFuge is a 3D printed centrifuge rotor that can be fitted to a Dremel multitool or a drill, and is Open Source Hardware.

Blenderfuge is a centrifuge produced by drilling out a rotor for use on a domestic blender appliance or similar.

[edit] Sterile Working Area

A HEPA filter, perhaps repurposed from an automotive or vacuum cleaner or as part of a room air purifier, can be used to direct a sterile airflow onto a surface, providing a sterile working area. Within this area, sterilised samples will likely remain sterile with proper lab methods on the part of the operator.

A bunsen burner or camping burner with a strong blue flame can produce an area of effective sterility, both by cycling air that has been through the flame and by providing a local updraft that prevents downward contamination upon samples and petri dishes.

[edit] Cultures

Essential to a microbiology lab are microbes to be grown within. These could be native or wild species cultivated for study or development, medical samples (handle with care), or laboratory cultures provided by another lab. Laboratory strains deserve special note:

E.coli is the prototypical bacterium, and is the "model organism" of modern bioscience. Contrary to its bad reputation, most strains of E.coli are relatively harmless and can probably be found living quietly inside most mammals. E.coli lab strains are mostly derived from a lab strain called E.coli K12, and are generally too incompetent to survive in the wild (or the gut, for that matter).

Lab strains of E.coli are used in most labs to carry DNA constructs called Vectors, which usually refers to circular DNA molecules called Plasmids. It is as part of these plasmids that most transgenic systems are delivered into E.coli to be read from and processed, or as intermediate constructs on the way to being developed fully in another species. Because E.coli can be forced to stably maintain plasmids within the cell at high copy-numbers of plasmids per cell using antibiotics and encoded antibiotic resistance genes, it has been the main method of choice for storing DNA between uses.

However, the requirement for antibiotics in this use-case renders the use of such antibiotic-resistant plasmids unsuitable for community use; antibiotics are firstly too important to be squandered in this manner, and secondly are too expensive or difficult to produce locally for this purpose. Also, E.coli generally requires refrigeration at very low temperatures to remain stable, typically -80C in an institutional or commercial biolab. To meet this requirement in a community lab would require far too great an expense using a scale of engineering that is far from resilient.

B.subtilis is another model bacterium used in biotechnology and bioscience, though to a much lesser extent than E.coli. B.subtilis offers significant advantages for community use in terms of ease of culture, handling and storage, and there are no known hazardous strains of B.subtilis (although it has some bad relatives that are easily mistaken for it: Anthrax and B.cereus numbering among them).

Because B.subtilis forms stable spores upon starvation, it does not require refrigeration. Delivery of plasmid DNA to B.subtilis is, in principal, easier than with E.coli because B.subtilis has a natural tendency to adopt and use compatible DNA present in the environment (i.e. it can genetically manipulate itself when conditions are suitable). However, the prevailing method of industrial manipulation also employs antibiotic selection. Alternatives could be developed that do not require antibiotic resistance.

B.subtilis has not been as popular as a carrier for DNA because of perceived DNA stability issues; however, it is possible that these stability issues could be sidestepped by mindful design of DNA to omit sites that the B.subtilis topoisomerase recognises.

The primary lab strains of B.subtilis are derived from B.subtilis 168 which, like E.coli K12, are highly domesticated and are generally considered inviable outside the laboratory environment.

[edit] Molecular Biology

This section needs work.

[edit] Central Dogma

The Central dogma is the fundamental process through which life continues itself, whereby information stored in stable DNA is transcribed to flexible RNA that is translated into functional proteins. The molecules of life are linear polymers that are assembled along an almost universal (figuratively speaking) set of rules, so that a linear DNA sequence derives an exact protein polypeptide. With the knowledge of the language of life a DNA, RNA or protein sequence can more or less informs the sequence of related sequences. This knowledge is the basis of molecular biology.

[edit] Systems biology and the omes

All life on earth is cellular (occurs in cells) and by understanding the different levels of action occurring in cells a comprehensive picture of the physical processes of life can be described. Massive amounts of biological data has been gathered over recent decades due to improvements in molecular techniques and an increase in computational power and tools. New system wide information and approaches are creating a new understanding of life and allowing manipulation of the process on a rational level.

The genome is the entire genetic (DNA) sequence of an organism and since the mid 1990s organisms' complete genomes have been assembled by researchers. Now hundreds of organisms representative genomes have been assembled and are available to the scientific community. Well equipped universities and companies have the technology to sequence whole genomes on-site in a matter of weeks. The transcriptome is the complete set of RNA transcripts that exist in a cell. The RNA transcripts of an organism will change in response to environment and internal controls, making a transcriptome only representative of a specific external and internal cellular environment. The proteome is a complete set of proteins that exist in cell and is derived from the transcriptome and other levels of regulation. A proteome is also only representative of a cell as it exists in a specific external and internal environment. In multicellular organisms the genome is the same in all cells, while regulation of the transcriptome, proteome, and higher levels are what allow different cells to specialize and handle different functions.

A systems biology approach uses the complete descriptions of the actions of cells to understand how each level and their interactions contribute to specific metabolic processes and the continuation of life. A systems approach is key to extracting utility from (micro)organisms and increasing the efficiency with which it is possible. Rational manipulation of the "omes" and their regulation in a living cell can be used compel organisms to take on desirable actions and desist from undesirable action. The sharing of this information and the tools of manipulation has the potential to create better agricultural feedstocks and microorganismal microfactories.

[edit] DNA

[edit] Genetic Engineering

Genetic engineering is the alteration of the genetic code in an organism. Genetic engineering includes the insertion of standalone DNA fragments (plasmids) that are capable of action in an organism or alteration to an organisms genome. Through successful genetic engineering alterations it is possible to control any aspect or action of a cell.

[edit] PCR

Polymerase chain reaction is a workhouse technique of molecular biology that produces a many fold replication of a DNA sequence. The technique is based upon the action of thermostable DNA polymerase, a DNA template containing the DNA sequence of interest, primers a set of dual short sequences (~20 base pairs) of DNA that are complementary to the flanking regions of the sequence of interest, and nucleotide triphosphates (NTPs) which are the components of DNA (additional components enhance DNA polymerase action and include buffering agents and magnesium as a cofactor). A PCR is conducted in a thermal cycler which holds vials of the reaction and accurately controls the temperature. The reaction starts at an elevated temperature ~95 C which denatures or separates the double stranded DNA. The temperature is lowered for the annealing phase during which the primers bind to their complementary sequences in the template strand, the temperature is determined by the thermodynamic annealing temperature (Tm) of the primers. Determining the annealing temperature has a shorthand equation of adding up the number of hydrogen bonds that will be formed between the primers and template, 3(C+G)+2(A+T). Both primers should be similar between ~40-60 C. The temperature is raised to the DNA polymerase temperature of action (~68 C), and is known as the elongation phase because the DNA polymerase binds to the primers and elongates according to the template sequence. These steps, collectively known as a cycle, are repeated typically 20-40 time. There is an exponential increase in the desired sequence as each newly created DNA strand acts as a template in the following cycle.

The thermal cycling devices required for PCR tend to be quite expensive, frequently costing tens of thousands of U.S. dollars. However, OpenPCR has recently begun shipping a thermal cycler for only $512, and instructions for building your own machine for less than $85 can be found here

[edit] Gel electrophoresis

DNA has a negative charge due to its phosphate backbone and can be drawn to a positive electrode when placed in a current. When placed in a gel matrix (material containing pores) and a current, the migration of a DNA strand will be controlled by its size and ability to move through the pores. The polysaccharide, agarose, is typically used as a matrix and DNA size is determined by comparison to a series of known standard length DNA strands (called a ladder) that is included in the run. Ethidium bromide is included in the matrix and binds to the DNA; EtBr is visible under UV light allowing visualization of the DNA in the matrix.

Gels require buffering solution to run properly; these can be can be made of several different materials. Traditionally, TAE, a combination of Tris base, ascetic acid, and EDTA (a polyamino carboxylic acid) was used. However, research has since shown that a buffering solution using sodium boric acid (derived from sodium borate, better known as Borax) can be used. In addition to be considerably cheaper and easier to manufacture, the sodium boric acid gel is able to tolerate higher voltages, reducing the amount of time the gel needs to "run". More information can be found here and here.

[edit] DNA sequencing

[edit] RNA

[edit] RNA isolation

[edit] Reverse transcription

[edit] Proteins

[edit] Native versus heterologous expression

[edit] Purification

[edit] HPLC/FPLC

[edit] Assays

[edit] Plant Tissue Culture

Theoretically, any plant tissue can give rise to an entire plant. In practice, cultivating plant tissue culture is dependent on variables such as type of plant, stage of plant growth, which tissue was selected, and what hormones and environmental chemicals are present.

It's very important to sterilize your plant tissue before cultivating it. MS0 medium is popular for cultivating plant tissue, it is a mixture of macronutrients, micronutrients and additives such as sucrose and agar.

Resources: http://aggie-horticulture.tamu.edu/tisscult/microprop/microprop.html

[edit] Animal Tissue Culture

This section presents a potential health hazard and should be carefully considered. It also needs work.

[edit] Reagents: Introduction

After glassware and equipment, a lab requires reagents. This very broad heading comprises acids and alkalis, alcohols, dyes, polymers and enzymes. To a certain extent, there is a feedback effect whereby an existing lab can produce many of its own requirements in-house for continued work or for setting up a new lab. Also, many of these reagents may be considered outputs if desired by the community; alcohols, dyes, polymers and enzymes all have valuable uses in a community for sanitation, textiles, food production and waste degradation, among other things.

Acids and Alkali are needed for their own sake and to produce important salts by reaction with minerals and each other. Alcohols are needed as sterilants and as precipitants for purifying proteins, enzymes, DNA, and other compounds. Dyes are needed for diagnostic differentiation between bacterial species, and for staining DNA in molecular work. Polymers are needed for producing bacterial growth plates, electrophoretic gels for DNA, and for sterilising heat-labile reagents. Enzymes are needed to catalyse reactions such as PCR, to degrade contaminants, and perhaps as an end in themselves (as many industrially significant enzymes may be of use to local communities in green cleaning and food production).

Many chemical needs can be satisfied locally by intelligent substitution, whereas others may present a problem that will need to be addressed over time. Enzyme needs are a problem for which an immediate solution is foreseeable but will be expensive; transgenic strains of laboratory bacteria can be engineered to produce as much enzyme as required for a given application. Polymers may be extracted from locally sourced wild flora such as seaweeds and purified chemically (agars), or might be prepared in like manner to enzymes with transgenic strains of bacteria.

[edit] Present Strengths

Requirements for a local microbiology lab, which could be used for diagnostic purposes, are achievable today. Methods such as pressure-sterilisation, oven sterilisation and tyndallisation are required to produce sterile growth media for microbes, but can be learned easily once equipment is available. Rich growth media are easily produced using ingredients that can be locally produced or sourced; a simple diagnostic medium such as blood agar can be produced using byproducts from a meat processing facility or butcher.

[edit] Present Limitations

To produce enzymes and other limiting compounds locally, transgenic strains of laboratory-strain bacteria may need to be developed and protocols for easy extraction will need to be tested.

For example, for production of PCR enzymes for use in PCR diagnostics of locally relevant diseases, it should be feasible to produce the thermostable enzymes used in PCR using a laboratory-domesticated strain of either E.coli or B.subtilis. The enzyme can then be easily purified by boiling cells and filtering the result; the crude lysate will contain the enzyme, which should outlast contaminating enzymes under heat treatment. However, it is not feasible to locally produce such a strain as required, because the gene needed to produce the thermostable enzyme is found in wild cultures of deep-sea, thermophilic bacteria which are practically impossible to locally culture. However, once produced, such a strain can be transferred with trivial ease between AT-biolabs and constitutes a landmark development in sustainable biotechnology.

[edit] Existing Methods

[edit] Acids / Alkali / Feedstocks

  • Acetic Acid - Distillation or Recrystallisation from Vinegar/Kombucha - Acetate salts are used for a wide variety of protocols.
  • Acetone - Can be produced via the ABE Process (or transgenic bacteria). Can also be distilled from acetates, for example calcium acetate formed from egg shells and acetic acid from vinegar.
  • ATP - Adenosine Triphosphate. Molecular energy unit of most living cells. Could probably be extracted from living cells but is highly unstable owing to its high energy content. Required for many enzyme-catalysed reactions, such as the use of Ligase (below).
  • Benzoic Acid may be distilled from the injury-induced resin of Styrax family trees. The resin may be 20% Benzoic Acid. It may alternately be chemically produced from benzyl alcohol, which can be extracted from essential oils or fruits, though likely not in the same quantity as Styrax resin.
  • Calcium Carbonate - Egg Shells, DE, Sea Shells, Mineral Deposits
  • Citric Acid - Fermentation of glucose by Aspergillus niger yields citric acid which can be recrystallised and purified.
  • Formic Acid - Distillation from ant bodies - Can be used for making salts, also has output applications in beekeeping.
  • Potassium Hydroxide - Purification from Lye from Hardwood Ash - Provides ~90% Potassium Hydroxide, but presents hazards.
  • Sodium Carbonate - Can be produced in low quality from burned Kombu/Kelp but is also produced via the Solvay Process.
  • Sodium Hydroxide - Produced from Calcium Hydroxide and Sodium Carbonate, both outputs of the Solvay Process.

[edit] Alcohols

  • Benzyl Alcohol - Can be extracted from fruit or some essential oils, though probably not in quantity.
  • Butanol - Can be produced via the ABE Process (or transgenic bacteria).
  • Ethanol - Produced during yeast fermentation or ABE Process. Can be distilled from fermentation medium, although high-grade ethanol will require more than a pot still.
  • Methanol - Can be distilled from wood.
  • Phenol - Possibly isolated from lignin

[edit] Polymers

  • Cellulose - Glucose polymer, most common biological compound on earth but usually highly impure. Easily produced as pure polymer by Kombucha fermentation, potentially useful as alternative to agarose DNA gels.
  • Agar - Extracted from some seaweeds. In principal possible to produce via transgenic bacteria/yeast in-house. Useful for food production as an output.
  • Agarose - Highly purified galactose polymer from Agar, requiring solvent or enzyme treatment to produce. Also in principal possible to produce with transgenic bacteria/yeast in-house. Supersedes need for agar if produced as pure agarose for lab or culinary applications.
  • Gelatine - Easily boiled from bones and collagenous animal matter. Has limited uses in the lab due to being readily digested by many bacteria during growth.
  • Alginates - Boiled as with Agar from certain species of seaweed/alga. Has food applications and can be processed to form a powder that, when dissolved in water, forms a gel upon exposure to calcium. Useful for encapsulating cells for ease of extraction from fermentations. Also has culinary applications and can be used to produce a "spray on bandage" to rapidly stanch bleeding as a medical application.
  • DNA Monomers - Generally called "NTPs". Extracted industrially from salmon sperm DNA. Necessary for PCR and some other DNA manipulation reactions to produce or extend DNA.

[edit] Dyes

Dyes actually pose a strong problem for community biolabs. Although many natural dyes can be easily prepared from indigenous species or by fermentation of transgenic strains, most dyes used in a modern lab for essential techniques like DNA visualisation are synthetic and/or present a mutagenic hazard. Substitution with natural stains and dyes may be a matter of trial and error.

  • Indigo may have potential lab applications and can be grown easily or fermented by transgenic cultures. Also used as a clothing dye.
  • Iodine can be extracted from Kelp/Kombu using Sulfuric Acids, and probably other acids more easily attained such as Acetic acid. Iodine is used in the gram staining method that helps identify microbes in medical samples.
  • Lawsone from Henna could be used as a protein stain.
  • Hematoxylin is extracted from log heartwood. It is used for a medically important staining procedure. As a biosynthesised dye, it could in principal be fermented by transgenic bacteria.
  • Carmine/Cochineal is a traditional foodstuff dye produced from scale insects, and may have biolab applications.
  • Turmeric is a traditional foodstuff and clothing dye and may have laboratory dye applications.

[edit] Enzymes

Many degradative enzymes can be produced by fermentation of saprophyte species such as B.subtilis, which possesses a host of useful enzymes for breaking down dead plant matter. These enzymes can be used for degrading waste and quickening composting or disposing of awkward wastes such as rancidified oils.

In a biolab, enzymes are the molecular machinery that perform many essential tasks such as copying, modifying and pasting DNA into desired sites, degrading contaminants, binding and purifying specific desired components of mixed samples, or cell-free production of proteins for advanced medical applications.

The below enzymes mostly do not come with instructions or suggestions for sources; the probable route to production in a community lab would be to acquire transgenic strains of B.subtilis or E.coli producing the desired enzyme, from which the enzyme can be extracted after a scaled-to-order fermentation. These strains generally do not exist in a form that is suitable or available to the community lab, but will surely be designed in coming years and disseminated where possible and required.

Essential Lab Enzymes:

  • Restriction Enzymes - The more the merrier. Less necessary where synthesised DNA is available on demand..i.e. not in a community biolab, yet.
  • DNA Polymerase(s) - Generally heat-stable enzymes extracted originally from deep sea bacteria, now produced from transgenic E.coli. Essential for the PCR reaction, easily produced and purified from lab strains such as E.coli or B.subtilis provided the correct genes are available in-house.
  • Ligase - Used to "paste" DNA together, can be extracted in some form from probably any living cell but is generally extracted specially from transgenic E.coli. Could be produced in house from natural species with some difficulty, probably easier to produce with transgenic, tailor-made strains.
  • Exonucleases - For degrading RNA or DNA, and for modern DNA cloning methods such as the Gibson method.
  • Cellulase - For degrading cellulose, whether for biofuel production (probably inefficient to use enzyme for this) or to prepare plant cells for further manipulations.

Mostly culinary outputs:

  • Invertase - Produced by Bacilli such as B.subtilis. Catalyses Sucrose -> Glucose + Fructose.
  • Lipase - May assist in purification procedures. Can be used to degrade fats and remove fatty deposits. Can also be used to produce biofuel from oils/fats.
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