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Bacterial E.coli Growth MediaSection list of bacterial E.coli growth media

Properties of Bacterial E.coli growth mediaCommonly used bacterial E.coli growth mediaHigh density bacterial E.coli growth media

Properties of Bacterial E.coli growth media

Solid   vs. liquid growth media E.coli cell growth curve and its biochemical applications E.coli OD600, wet weight, dry weight, cell volume, cell number and cell densityTrace metals, minerals, and vitamins in E.coli growth media Medium pH range and E.coli cell growth E.coli medium pH verses cell growth, viability, stock, plasmid production, and protein expression Aeration or oxygen O2 concentration in E.coli growth media Medium agitation and E.coli cell growth Temperature and E.coli growth Osmolarity and E.coli growth media

Solid vs. liquid growth media

Escherichia coli or E.coli cells may grow on a solid or in a liquid growth medium under a laboratory condition. Solid and liquid media may have exactly the same composition except that the solid medium contains an extra 1.5% agar. Different E.coli clones may have different properties. Colonies growing on solid media represent different clones. These colonies are used for clone selection in DNA cloning and protein expression in molecular biology studies. A selected E.coli clone can be grown in a large quantity in a liquid medium. A liquid medium is often used for plasmid DNA and recombinant protein production. E.coli cells have different growth properties on solid or in liquid media even though they have the same composition except agar. For example, E.coli cells can grow on a solid medium plate at pH 11, but they cannot grow in a liquid media with pH 9 or

higher. All growth conditions discussed below are related only to liquid media unless otherwise noted.

E.coli cell growth curve, growth phases and their biochemical applications

E.coli cells grow differently under different conditions. The E.coli cell density after inoculation can be graphically described as growth curves. The growth curve is comprised of four major phases: lag phase, log phase, stationary phase, and death phase. At the lag phase, the cell density increases slowly. The lag phase can be clearly observed if the cell culture is inoculated from a single colony on a old plate or from a small volume of a cell stock. The lag phase is not obvious if the inoculation is from a fresh culture with relatively large volumes at 1:50 or 1:100 inoculations. In these cases, E.coli cells grow directly into the log phase. The log phase is also called the exponential growth phase. At the log phase, E.coli cells grow rapidly or exponentially. The doubling time is often 20 to 30 minutes for most wild-type E.coli strains in a rich medium. The E.coli cells are healthy and are at the prime state to produce proteins at the early log phase. The E.coli cells are often harvested at the middle to late log phases for protein production. Harvesting cells later than the log phase may observe low protein yield and high protein degradation. E.coli cells at the early log phase are also used to make competent cells and cell stocks. The stationary phase is also called the saturation phase or steady phase. At the stationary phase, the medium's nutrients become limited and metabolic products accumulate to such a high level that they are inhibitory to the cell growth. E.coli cells are stressed at this phase. The cell density is highest at the stationary phase (see figure bellow) and most E.coli strains appear to contain high amount of plasmids. Therefore the E.coli cells at the stationary phase are mostly used for plasmid production, but they are generally not good for protein production or other purposes. The death phase is also known as the decline phase. At the death phase, nutrients become so limited and toxic metabolites are so high that the perished cells exceed newly formed cells and cell density decreases. E.coli cells at the death phase cannot be used to make competent cells or cell stocks. They are not good for plasmid or protein production. It is important not to grow the cells to the death phase for all biochemical applications.

Schematic E.coli cell growth curve

Under most laboratory conditions, E.coli cell growth often does not follow the growth curve above. For example, most scientists will not grow the cells to the death phase. This part of curve will not be observed for most studies. For protein productions, E.coli colonies are often inoculated into about 4 ml cultures, and these 4 ml cultures are freshly inoculated into larger cultures of 500 ml. Cell growth in these 500 ml cultures only presents log phase characters for protein production. The lag phase will not be observed in these cases.

E.coli cell growth curve(for most plasmid or protein preps in a shake flask container)

The growth curve above is also simplified because E.coli cells grow slower after protein induction. E.coli cells may also grow slower when harboring

some plasmids.

E.coli cell growth curve for protein prep(Example: a maxi prep of 500 ml in a shake flask container)

E.coli cells with most plasmids and some proteins will follow curve A. Sometimes E.coli cells expressing some proteins will follow curve B. In these cases, the recombinant proteins interfere with cell proliferation and/or differentiation. These recombinant proteins are toxic to E.coli cells when over-expressed. E.coli cells with extremely toxic proteins will follow growth curve C. In such cases, both plasmid and protein production will be very low. Different technologies should be used to produce these plasmids or proteins. The slope of the growth curve will be different for E.coli cells containing different plasmids or expressing different recombinant proteins.

E.coli OD600, wet weight, dry weight, cell volume, cell number and cell density

OD stands for optical density. The number following the OD indicates the wavelength of light. OD600, OD550, OD650 or other wave lengths may be used to estimate the E.coli cell density. OD600 is the most commonly used. OD600 measures the light absorbance of an E.coli cell culture sample. The OD600 value corresponds with the cell density or cell number in a given E.coli culture volume. Different cell strains may have different cell numbers at a given OD600 value, but OD600 = 1 usually means there are about 1 x 109 cells per ml culture. Different growth conditions will also give different OD600 values.

OD600 is used to measure the E.coli cell density. The OD600 value is also an

important indicator of the physiological condition of the E.coli cells in a given medium after a specific culture period. The OD600 value determines if the E.coli cells may be ready for making competent cells, cell stocks, induction, or for being harvested.

OD measurement is more accurate at values less than 0.5 for most spectrometers. For common medium this means 10 time dilution of the culture. For high density growth medium, this means 100 time dilution. Distilled or deionized water should be used as a blank and in dilution. Rich media often have color by themselves. OD measurement will not be accurate if a medium is used as a blank or in dilution.

OD600 may also be used to calculate E.coli cell weight. The E.coli cell wet weight is measured by the cell pellet weight after the centrifugation of a cell culture. Cell density at 1 x 109 per ml usually gives about 1 mg/ml cells or 1 gram/liter wet cell weight. The E.coli dry cell weight is usually 25% or 1/4 of its wet cell weight. the average E.coli cell mass is about 1 pg/cell or 1 x 10-12 g/cell wet weight. Different E.coli strains and growth conditions will present certain difference on these values. The differences in the values can be as much as a few times, but they are normally less than 10 times. The OD value of series dilution of E.coli cells may be plotted against the actual number of cells grown on a series of plates to generate a standard curve to get a relatively accurate estimation of cell density under normal growth conditions. However, accurate determinations of cell density may not be possible under extreme growth conditions since not all cells are viable under these conditions. Accurate determinations of cell numbers are not needed for most molecular biology experiments, such as determining inoculation volume, induction time, or harvesting time.

Scientists involved in large scale production often use wet or dry cell weights. Research scientists normally use OD600 or the cell number. The following equation may be used for a general estimation of cell density, number and weight.

OD600 = 1 ≈ 1 x 109 cells/ml ≈ 1 mg/ml or 1 g/liter wet cell weight ≈ 1/4 g/liter or 0.25 g/liter dry cell weight

Research scientists often grow a few milliliters or hundreds of milliliters of cell cultures. Few scientists will weigh the cell pellets after harvesting. However cell pellets are often centrifuged in test tubes. The cell volume can often be easily observed in a test tube.

OD600 = 1 ≈ 1 x 109 cells/ml culture ≈ 1 ul wet cell/ml culture or 1 ml wet cell/liter culture

The wet cell volume will be about 5 ml if the OD600 of a 500 ml culture is 10. A large volume of a cell pellet will indicate a high OD600 value of the culture. Wet cell volume can often be used to estimate the cell density without reading its OD600.

Trace metals, minerals, and vitamins in E.coli growth media

E.coli cells are what they eat and grow on. The E.coli growth medium is not only the food for E.coli cells but is also their habitat. The growth medium, medium container, aeration condition, and growth temperature form the E.coli cell growth ecosystem in a lab. One of the most important reasons that recombinant proteins are more soluble and active when expressed in insect cells and mammalian cells is their growth media. Insect and mammalian cells normally grow in serum-based media. Animal sera contain many nutrients needed for animal cell growth. These nutrients include many trace metals, minerals, and vitamins, which may serve as prosthetic groups, co-factors or ligands for recombinant proteins. E.coli cells can synthesize most of its nutrients and may not need them for their growth. However, only in the presence of these prosthetic groups, co-factors, or ligands will many recombinant proteins be expressed soluble, stable and functional in E.coli cells. The growth medium and culture conditions also determine the quantity of E.coli cells that may be obtained from a culture. Plasmid and protein yields are proportional to E.coli cell quantity. Therefore, the growth medium is one of the most important factors in determining the plasmid and protein yields. The growth medium may also determine the solubility, stability and activity of a recombinant protein.

Medium pH range and E.coli cell growth

In addition to nutrients, the pH of the growth medium is also important for E.coli growth rate and cell density. The optimal growth pH for E.coli is near neutral. E.coli cells can grow reasonably well over a range of three pH units (from pH 5.5 to 8.5). Extreme pH beyond this range will significantly decrease the cell growth rate and may sometimes even cause cell death. The minimum and maximum growth pHs for E.coli are pH 4.4 and 9.0 respectively. E.coli cells appear to tolerate a low pH better than a high pH. In fact, extended exposure of E.coli cells to a high pH causes cell lysis. At the saturation or stationary phase, the pH of the E.coli culture in commonly used media is near its pH limits. pH is another limiting factor for cell growth in addition to nutrition exhaustion and accumulation of toxic metabolites. The medium's pH is determined by medium compositions, buffers, cellular metabolites, and aeration conditions. Some common media such as TB use a phosphate buffer. Phosphate buffer capacity will be quickly exhausted at a high cell density (OD600 > 10). Organic salts such as sodium succinate and mono-sodium glutamate (MSG) and their acids may be used as buffers. After

exhaustion these organic buffers, the medium reaches the E.coli cells' maximum pH limit. E.coli cells can also use sugars such as glycerol and glucose as carbon or energy sources. When the E.coli cells use these sugars as carbon sources, they will produce acetic acid and therefore lower the medium pH. Carefully balancing the phosphate buffer, organic buffers, sugar contents, and aeration conditions can maintain the culture medium pH near E.coli optimum growth pH or within the range of the three pH units. Low aeration conditions lead the cells to produce acids. High aeration conditions allow the cells to use organic acids as carbon source and increase medium pH. Selected aeration conditions can also help cells maintain its medium pH.

E.coli medium pH verses cell growth, viability, stock, plasmid production, and protein expression

The medium's pH also affects the E.coli cells' viability, stock, plasmid production, and protein expression. The optimal pH is almost always near neutral pH 7. Acidic pH is often better tolerated by E.coli cells than basic pH. These pH ranges are summarized in the following table and diagram, but there is always an exception for a particular cell strain, plasmid, or protein.

pH rangepH<5.5 pH 5.5 to 8.5

Cell growth Slow to stop Optimal Stop to cell deathCell viability at 4 0C 2 weeks >90% for 1 monthCell stock Not recommended IdealPlasmid production OK for most plasmids ideal for most plasmids OK for some plasmidsProtein expression OK for some proteins Good for most proteins poor for most proteins

Aeration or oxygen O2 concentration in E.coli growth media

E.coli cells produce large quantities of acetic acid if the growth medium contains little or no oxygen causing the growth medium to reach pH 4 or lower. At this pH, E.coli cell growth slows down or even stops. Ampicillin will be Chemically degraded at low pH and the E.coli cells will partially or completely lose selection. The plasmid or protein production will be low because most cells do not contain the selected plasmid. Acetic acid is the major metabolic inhibitor under anaerobic growth condition. However, with proper aeration, E.coli cells will be able to use many organic acids as carbon sources and the pH of the growth medium will be maintained at near neutral or basic ranges. Aeration is another important factor in determining E.coli cell growth.

Medium agitation and E.coli cell growth

Agitation and aeration are closely related for most shake flasks under regular laboratory settings. Agitation is control by the shaking speed in revolutions per minute (rpm) of a shaker incubator. The higher the shaking speed (rpm),

the better the agitation and aeration, provided that the container permits sufficient air exchange. Tightly fitting of the container to the container holder is also important for good aeration. The shaker incubator and incubation room should also be sufficiently ventilated to allow good aeration. If the container cover does not allow sufficient air exchange, the ventilation fan of the shaker incubator is malfunctioning, or the incubation room is not ventilated, higher agitation will not result in higher aeration. In these cases, E.coli cells will grow anaerobically and the growth medium will soon become acidic. Agitation alone without increasing aeration will still lead higher growth rate for E.coli cells, presumably because agitation increases metabolic exchange of the cells with the medium.

Temperature and E.coli growth

The natural environment of E.coli cells is the lower intestine of a warm-blooded animal. Its optimal growth temperature is 37 0C. The doubling time or generation time for most E.coli strains in a rich medium at 37 0C is between 20 to 40 minutes. E.coli cells cannot grow well at temperatures higher than 42 0C. They can tolerate lower temperatures with lower growth rate. Protein synthesis slows at temperatures lower than 37 0C. It is also observed that inclusion body accumulation is decreased and more soluble recombinant proteins are produced at lower temperatures. Temperatures from 15 to 30 degrees are often used to produce soluble proteins. In most cases, the temperature set on the incubator is the E.coli growth temperature. The E.coli growth temperature will have some delay when the medium is kept at 4 0C or shifted from 37 0C to a lower temperature. This delay should be taken into consideration for short protein inductions.

Osmolarity and E.coli growth media

The concentrations of nutrients and chemicals in the medium determine the medium osmolarity. A careful balance of nutrient and chemical contents will maintain the medium osmolarity optimal for E.coli growth. E.coli cells can grow on wide range of nutrient concentrations. However, different E.coli cell strains do exhibit different optimal nutrient requirements. Commonly used medium components such as peptone, yeast extract, and sodium chloride, when reaching certain high concentrations, may inhibit E.coli cell growth. This is true for all other supplement nutrients, including sugars and buffers. In addition, high nutrient concentration may also cause precipitation.

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Commonly used bacterial E.coli growth media LB vs. SOB, SOC, 2x YT, Terrific Broth (TB), and Super Broth

(SB)

LB brothSOB mediumSOC medium2X YT brothTB mediumSB mediumNutrient contents in commonly used bacterial E.coli growth media Aeration and E.coli cell density in commonly used growth mediaMg2+ and E.coli cell density in commonly used growth media Applications of commonly used growth media LB, SOB, SOC, 2x YT, Terrific Broth (TB), and Super Broth (SB)Disadvantages of commonly used media and needs for High density growth media

LB broth (Luria broth, Luria-Bertani medium, or lysogeny broth) is the most commonly used medium in molecular biology for E.coli cultures. Easy to make, fast growth rate for most E.coli strains, readily available and simple compositions make LB the most popular medium. LB contains the enzymatic digestion product of casein commonly known as peptone (some vendors call it Tryptone), yeast extract, and sodium chloride. Peptone is casein digested by pepsin and Tryptone is casein digested by trypsin. We do not observe significant difference between these two casein digestions in E.coli cultures. Peptone is rich in amino acids and peptides. Its amino acid and peptide compositions reflect those of casein. In addition to amino acids and peptides, yeast extract also contains nucleic acids, lipids and other nutrients which are essential to E.coli cell growth. LB can support E.coli growth OD600 2 to 3 under normal shaking incubation conditions (250 rpm). LB Miller Broth and LB Lennox Broth contain higher and lower sodium chloride levels respectively. There are minimal differences between the two formulations in most molecular biology studies with commonly used E.coli strains. Different bacteria strains may require different salt concentrations. A low salt formula is more often used in salt-sensitive antibiotic selections. Buy LB broth... >

SOB medium (Super Optimal Broth or Hanahan's broth) is another commonly used rich medium. SOB has twice as much peptone as LB. SOB is richer in amino acids and peptides. In addition, SOB contains magnesium which is required for high density cell growth. SOB can support E.coli growth to OD600 3 to 5 under normal shake flask conditions (250 rpm). SOB can support cell density to OD600 10 to 15 with increased aeration (350 rpm). E.coli cells growing in SOB will have a higher transformation efficiency than those in LB medium. Buy SOB Medium... >

SOC medium (Super Optimal broth with Catabolic repressor) is the same as SOB with exception that it contains 20 mM glucose. Here, glucose serves as

a catabolic repressor. Cells prefer to use glucose as carbon or energy source. In the presence of glucose, cellular machineries using other sugars will be repressed. At a concentration of 20 mM or 0.36%, it provides sufficient transcription repression for lacI repressed promoters at low cell density. Expression of recombinant protein that may adversely affect host cell physiology is repressed. As a result, transformation reactions using SOC to grow the cells for one hour will have a higher efficiency than those using LB. When SOC is not available, adding 20 mM glucose to LB or other commonly used media will result in a similar transformation efficiency as that of using SOC. Buy SOC medium... >

2x YT medium (2x Yeast extract and Tryptone) is named in comparing its nutrition contents with that of LB broth. It does have 2x as much yeast extract compared to LB, but it only has 60% (not exactly 2x) more peptone. Basically 2x YT is a richer medium than LB and can support a higher cell density and longer growth period for E.coli. 2x YT was originally formulated for growth and maintenance of E.coli and its fibrous bacteriophages, such as M13 phage. It allows relatively a large quantity of phage production without exhausting the nutrients. Buy 2x YT medium... >

TB medium (Terrific Broth) is a phosphate buffered rich medium. In addition to 20% more peptone and 380% more yeast extract than in LB, TB also has 0.4% glycerol as an extra carbon source. All these nutrients in TB can support E.coli growth to OD600 5 to 8 under normal shaking incubation conditions. Buy TB medium... >

SB medium (Super Broth) has 220% more peptone and 300% more yeast extract than LB. Therefore SB is super rich in peptone and yeast extract. We estimated that the major nutrients in peptone and yeast extract are still in excess well after cells reach saturation while trace nutrients are depleted in the same culture. Many scientists use SB for plasmid or protein production. Buy SB medium... >

Nutrient contents in commonly used bacterial E.coli growth media

Peptone or Tryptone: Peptone is casein digested by pepsin and Tryptone is casein digested by trypsin. We do not observe significant difference between these two casein digestions in E.coli cultures. This nutrients is made from the enzymatic digestion of cow milk protein casein. Its amino acid composition reflects those of casein. Different preps from different vendors may contain different amounts of lactose. They are generally exchangeable in E.coli cell growth and plasmid production. They may exhibit differences in protein expression because of lactose contents.Yeast extract: Yeast extract is made from hydrolysate of yeast cells. It is rich in amino acids, lipids, and B vitamins. Trace metals and minerals

contained in yeast extract are often insufficient for recombinant protein over-expression.Sodium chloride: Sodium chloride is mainly used to maintain medium osmolarity.Magnesium Mg2+ (in SOB): Magnesium Mg2+ is required for cellular enzymatic reactions. Adding Mg2+ in the medium will increase the cell density with increased aeration.Potassium K+ (in SOB): Potassium K+ is used as a potassium source.Phosphate (in TB): Phosphate is used as a buffer and phosphate source.Glycerol (in TB): Glycerol is used as a carbon source.Glucose (in SOC): Glucose is the preferred carbon source for E.coli cells. Other sugars will not be used in the presence of glucose. Therefore protein induction by IPTG, lactose or any other sugars cannot be achieved in the presence of glucose.

Aeration and E.coli cell density in commonly used growth media

Cell densities in all common media will be enhanced by increasing aeration. In fact, increasing aeration alone will increase E.coli cell density significantly. For example, the E.coli cell density in LB can reach OD600 up to 8 at 400 rpm in a shake flask container. There are two problems associated with the E.coli cells when they reach OD600 > 8 in LB. The first problem is high medium pH at > 8.5. At this or a higher pH, the ability of E.coli cells to produce protein is poor. The second problem is that the cells grow very fast. E.coli cells reach the highest cell density in a short period of time. Induction for protein expression can be difficult to control at a fast cell growth rate.

Mg2+ and E.coli cell density in commonly used growth media

Mg2+ is required for E.coli cell growth. However, Mg2+ alone will not increase cell density. It must be combined with better aeration conditions at higher shaking speeds for shake flask containers. Final magnesium concentration of 10 mM will increase cell density at a higher shaking speed (at about 350 rpm). The SOB medium contains a sufficient amount of magnesium. Good aeration alone will increase cell density for the SOB medium. Mg2+ plus a high shaking speed will increase the E.coli cell density in all commonly used media.

Applications of commonly used growth media LB, SOB, SOC, 2x YT, Terrific Broth (TB), and Super Broth (SB)

LB broth is mostly used for E.coli cell growth and propagation. LB broth is also commonly used for plasmid DNA and protein production at a laboratory

scale. The yield and reproducibility of the LB broth is satisfactory for the mini prep, medium prep, and large prep of many plasmids and some proteins. LB agar (15%) is regularly used for E.coli colony selections. Some E.coli strains may grow better in low salt LB Lennox broth.

SOB medium is commonly used to make high-efficiency competent cells.

SOC medium is commonly used in the incubation after the heat-shock in the transformation reaction. When SOC is not available, adding 20 mM glucose to LB or other commonly used media will result in a similar transformation efficiency as compared to using SOC.

2x YT broth was originally formulated for the growth and maintenance of E.coli and its fibrous bacteriophages such as M13 phage. It allows a relatively large quantity of phage production without exhausting the host.

TB medium is mostly used for protein production in a laboratory scale. Some scientists also use TB for plasmid DNA production.

SB medium is mostly used for plasmid DNA production. Some scientists also use SB for protein production in a laboratory scale.

Disadvantages of commonly used media and needs for High density growth media

None of the general media can support E.coli growth to a cell density of OD600 to 10 or more in a shake flask under normal conditions (250 rpm). Therefore, the plasmid or protein yields in these media are relatively low. In addition, the buffers, if present in the medium, will be exhausted when cell growth reaches saturation. The pH of the cultures using these media will drop to 4 to 5 upon saturation under common conditions (250 rpm). At a low pH, antibiotics such as ampicillin will be chemically degraded. Degradation of ampicillin will cause partial or no selection of the cells. Under these conditions, 80% of the total cell population may not contain the intended plasmid. The plasmid yield is significantly lower than it should be. Cells cannot be induced to express the target protein when their densities are near saturation. Furthermore, nutrients, especially critical ones, are exhausted or depleted at or near cell saturation. Nutrition exhaustion can force the cells to recycle some of the cellular components (such as proteins) in order to survive. As a result, the induced protein may be severely degraded after the medium reaches saturation.

Commonly used growth media cannot reach a cell density of OD600 >20, even with added magnesium and increased aeration. Relatively low cell density in commonly used media limits plasmid and protein yields. Some

commonly used media, such as TB, can reach OD600 = 15 with added magnesium and increased aeration (OD600 = 8 for LB). However the time window to express proteins for TB at these cell density is short. Some medium nutrients and buffer capacities are quickly exhausted at this cell density. We estimated that the time window for expressing protein in TB is less than three hours and that in LB is less than one hour at their respective highest cell density. This short time window is not useful for most recombinant protein expression. In addition, recombinant proteins may have solubility, stability, and activity problems in common media because they lack a sufficient amount of required trace metals, minerals, or vitamins. It will be useful if the proprietarily formulated media can reach a cell density of OD600 = 30 to 50, similar to that which can be reached in a fed-batch fermentor. At the same time, the time window to express protein can be extended. The plasmid and protein yields should be increased accordingly. In addition, all known trace metals, minerals, and vitamins as well as unknown factors in animal serum supplemented in the high density growth media should also help the solubility, stability and activity of expressed recombinant proteins.

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High density bacterial E.coli growth media DNAGroTM, ProGroTM, AutoXTM, DetoXTM, InduXTM, and SecProTM

media

DNAGro TM growth medium ProGro TM growth medium AutoX TM growth medium DetoX TM growth medium InduX TM growth medium SecPro TM growth medium Nutrient contents in high density growth mediaAeration and E.coli cell density in high density growth mediaApplications of high density growth mediaFeatures of high density growth mediaBenefits of high density growth mediaExamples of using high density growth media

DNAGroTM growth medium

For any given plasmid (copy number of the plasmid is determined), there are two ways to increase plasmid yield. The first way is to increase the cell density or the number of cells. The other way is to increase the percentage of cells containing the plasmid. Adding chloramphenicol to the medium may amplify some plasmids but not others. E.coli growth medium DNAGroTM is

proprietarily formulated for plasmid DNA and phage production. Most commonly-used E.coli strains can reach OD600 = 30 in a shake flask container in this  medium. Recombinant protein cannot be expressed in this medium because the medium composition inhibits protein induction by IPTG, arabinose, or any sugar-based inductions. Protein induction may be achieved in this medium if heat or salt inducible promoters are used. Ten times or higher cell density may be obtained from this medium when compared to that from LB broth when growth conditions are closely followed. Plasmid or phage DNA yields will increase accordingly if there is sufficient selection. Buy DNAGro TM medium... >

ProGroTM growth medium

E.coli growth medium ProGroTM is proprietarily formulated for recombinant protein production. Most commonly used E.coli strains can reach OD600 = 30 in a shake flask container in this  medium. Recombinant proteins can be expressed in this medium regardless whether they are induced by IPTG, sugar, heat or salt. Ten times or higher cell density may be obtained in this medium than that from LB broth when growth conditions are closely followed. Recombinant protein yield will increase accordingly if there is sufficient selection. This growth medium contains trace metals, mineral, and vitamins that may be needed for solubility, stability, and activity of a recombinant protein. Buy ProGro TM medium... >

AutoXTM growth medium

E.coli growth medium AutoXTM is proprietarily formulated for recombinant protein production. Most commonly used E.coli strains can reach OD600 = 30 in a shake flask container in this  medium. Recombinant proteins can be automatically expressed in this medium if they are induced by IPTG. Induction occurs after cell density reaches OD600 = 10. A specific inducer must be added if the recombinant protein is not induced by IPTG or lactose. Cell density ten times or higher may be obtained in this medium when compared to that from LB broth when growth conditions are closely followed. Recombinant protein yield will increased accordingly if there is sufficient selection. This growth medium contains trace metals, mineral, and vitamins that may be needed for solubility, stability, and activity of a recombinant protein. Buy AutoX TM medium... >

DetoXTM growth medium

DetoXTM E.coli growth medium is proprietarily formulated for toxic protein production. Most commonly used E.coli strains can reach OD600 = 30 in a shake flask container in this  medium. This medium is designed for E.coli cell

growth and propagation, but not for recombinant protein expression or production. The inhibitors in this medium will prohibit protein expression induced by IPTG, lactose, arabinose, or any other sugars. After cells reach inducible OD (5 to 20 for most cell strains or recombinant proteins), other high density growth media such as InduXTM or ProGroTM should be transferred to the cell pellet and induction may be performed in these media instead. Using common bacterial growth medium such as LB will result in lower yield. The medium has no effect on protein expression induced by heat or salt. This growth medium contains trace metals, mineral, and vitamins that may be needed for solubility, stability, and activity of a recombinant protein. Buy DetoX TM medium... >

InduXTM growth medium

InduXTM E.coli growth medium is proprietarily formulated for recombinant protein production. Most commonly used E.coli strains can reach OD600 = 30 in a shake flask container in this medium. Recombinant proteins can be expressed in this medium regardless whether they are induced by IPTG, any other sugars, heat or salt. Ten times or higher cell density may be obtained in this medium than that from LB broth when growth conditions are closely followed. Recombinant protein yield will increase accordingly if there is sufficient selection. No IPTG is needed if the recombinant protein is induced by IPTG or lactose. A specific inducer must be added at induction if the protein is not induced by IPTG or lactose. This growth medium contains trace metals, mineral, and vitamins that may be needed for solubility, stability, and activity of a recombinant protein. Buy InduX TM medium... >

SecProTM growth medium

SecProTM E.coli growth medium is proprietarily formulated for secretory protein expression. Most commonly used E.coli strains can reach OD600 = 20 in a shake flask container in this  medium. Recombinant proteins can be expressed in this medium regardless whether they are induced by IPTG, any other sugars, heat or salt. Ten times or higher cell density may be obtained in this medium when compared to that from LB broth when growth conditions are closely followed. Secretory protein expression will increase one hundred times or more. This growth medium contains trace metals, mineral, and vitamins that may be needed for solubility, stability, and activity of a recombinant protein. Some mutant cell strains or strains with toxic proteins do not grow well in this medium. In these cases, these cell strains should be grown in DetoXTM or ProGroTM medium first to reach inducible OD. Then change the medium to SecProTM for induction. Buy SecPro TM medium... >

Nutrient contents in high density growth media

The exact formula of high density growth media are proprietary. In general, the high density growth media contain all the nutrients in commonly-used growth media. In addition, they contain trace metals, minerals, vitamins, and animal serum. These chemicals may serve as prosthetic groups, co-factors or ligands for recombinant proteins to increase protein solubility, stability, and activity. The high density growth media also contain phosphate and organic buffers which maintain the medium pH near optimal at high cell density for E.coli growth. Furthermore the high density growth media contain different sugars as carbon or energy sources for high density cell growth.

Aeration and E.coli cell density in high density growth media

These media are designed for high density E.coli cell growth with good aeration. E.coli cells need oxygen to grow to high cell density. Without oxygen, E.coli cells will produce large quantity of acids which will inhibit cell growth. The oxygen needs for high density growth media are more than those of commonly used media. We recommend the shaking speeds of 350 to 400 rpm for baffled flasks, 400 to 450 rpm for regular flasks and tubes. All containers should be fixed on the platform, otherwise medium agitation will not increase with shaking speed. All high density growth media can reach OD600 = 30 to 50 at recommended shaking speeds with sufficient ventilation of the incubator and incubation room. At normal shaking speed of 250 rpm, high density growth media can reach up to OD600 = 10 which is still higher than commonly used media.

Aeration is directly related to the medium/container volume ratio. The lower the medium/container volume ratio, the better aeration. We recommend 1/4 medium/container volume ratio or less for shake flasks and 1/10 medium/tube volume ratio for round-bottom tubes. For example, 500 ml or less medium should be used for a 2 liter flask and 1.5 ml or less medium should be used for a 14 ml tube. higher medium/container volume ratios will result in lower cell densities. Conical tubes should not be used for bacterial cultures because of bad aeration.

Incubator and incubation room should also be sufficiently ventilated especially when 500 ml or more medium is used. Many incubators require temperature setting to turn on the ventilation fan. Room temperature incubation still needs temperature setting of 25 degree Celsius to turn the fan on.

Applications of high density growth media

High yield plasmid production, 10 times over LBHigh yield phage production, 10 times over LBHigh yield protein production, 10 times over LBHigh yield secretory expression, 100 times over LB or TBToxic protein expressionIncrease protein solubilityIncrease protein stabilityIncrease protein activity

Medium NameApplications

DNAGroTM Increases plasmid and phage DNA yields 10 times over LBProGroTM Increases protein yield, solubility, stability and activity

AutoXTM Increases protein yield, solubility, stability and activity; automatic protein expression  without adding IPTG

DetoXTM Reduces protein toxicity and support E.coli growth to OD600 = 30InduXTM Increases protein yield, solubility, stability and activitySecProTM Increases secretary protein expression over 100 times than LB or TBFeatures of high density growth media

High density growth media support high density E.coli growth. In a shake flask under normal aeration conditions, common media such as LB can support E.coli growth up to a cell density of OD600 = 2 to 3. Richer media such as TB can grow E.coli to OD600 = 5 to 8. By contrast,  all of our proprietary high density bacterial growth media can grow E.coli to a cell density of OD600 = 30 to 50. This is over ten times higher than LB and over five times higher than TB. As a result, 5 to 10 times more plasmid DNA or protein can be produced in our high density growth media. Paired media DetoXTM and InduXTM can also reduce the toxicity of recombinant proteins. DetoXTM medium contains a high level of inhibitors to inhibit the non-induced expression. When switched to InduXTM medium, high levels of recombinant protein can be expressed. SecPro TM medium can increase secretory expression 100 times over TB or SB.

In addition to supporting high density growth, our special media also contain trace metals, trace minerals, various vitamins, and unknown nutrients from animal serum. These chemicals will serve as prosthetic groups, co-factors or ligands for recombinant proteins. Therefore increased protein solubility, stability and activity are obtained from using these special media.

Time window to express protein is significantly extended in high density growth media. Protein expression is induced at early log phase which is OD600 = 5 to 10 for high density growth media. Protein expression may also be induced at OD600 = 20 to 30 if the recombinant protein is toxic to the host. In this case, the induction temperature may be lowered to 15 to 25 degree Celsius and shaking speed should be changed to 250 rpm. the time

window to express protein can be as long as overnight or longer depending on the induction temperatures.

Plasmid and protein yields in these media are significantly higher than those that can be obtained from the general media. Our high density growth media are buffered chemically and metabolically. In addition to common buffers such as the phosphate buffer, our media are also buffered with balanced amount of sugars and organic buffers. Carefully balanced amounts of both sugars and organic buffers will maintain the pH of the culture in the E.coli growth pH units. For example, the pH of E.coli cultures of AutoXTM media at OD600 20 is about 7. Close to neutral pH, antibiotics such as ampicillin will not be chemically degraded. As a result, over 90% of the cell population contains the plasmid. Both higher cell densities and higher proportions of the population containing the plasmid contribute 10 times or higher plasmid yield than LB. Recombinant proteins may also be induced at this cell density. A 10 to 20 times higher recombinant protein yield is also obtained from our special media than LB. In addition to casein peptone, yeast extract, sodium and potassium salts, and balanced buffer systems, our special media also contain vitamins, minerals, and trace metals. Some vitamins, minerals, and trace metals are needed for high density cell growth. Others are not needed by E.coli growth, but these additives and their metabolic products may serve as prosthetic groups, co-factors or ligands for recombinant proteins and therefore are critical to protein solubility, stability, and folding. In addition to higher yield resulting from using our high density growth media, many proteins are more soluble, stable, and functional when expressed in these media.

Benefits of high density growth media

Yield: High density growth media can increase DNA and protein yields by increasing cell density. They can also increase protein yield by decreasing protein toxicity. A yield of at least 10 times that of LB can be obtained with the special media if the induction is performed at OD600 = 5 or higher. They may increase the yield over 100 times if pre-induction toxicity is the limiting factor and for secretory expression. Protein yield will not increase if there is no expression determined by Western analysis. Solubility: If recombinant proteins require prosthetic groups, co-factors, or ligands, the proteins may be soluble in high density growth media. Otherwise cell strains with molecular chaperones may be needed. Some proteins may require both high density growth media and chaperones for soluble expression. Stability: The most important factor for protein stability is the correct folding of an intact structure (domain). These may require prosthetic groups, co-factors, or ligands in the culture medium, molecular chaperones in the host cells, and intact expression of a protein structure (domain).

Medium pH is also important for many protein stability. Most proteins are stable at neutral pH. Some proteins are stable at acidic or basic pHs. The pH ranges of high density growth media are pH 5 to 6 at OD600 < 10, pH 6 to 7 at OD600 = 10 to 30, and pH 8 to 8.5 at OD600 >30, which are within the E.coli growth pH ranges.  Inducing the protein at different OD will also meet different protein pH needs. There will be no protein degradation caused by nutrition exhaustion in high density growth media.Activity: Only soluble proteins are functional. Factors important to protein solubility are important to protein activity as well. Therefore, proteins may require prosthetic groups, co-factors, or ligands in the culture medium to be active. In addition, they may also require chaperones in the host cells and the intact expression of a structure (domain).

Examples of using high density growth media

InduX TM increases protein yield. ProGro TM enhance protein solubility and activity. AutoX TM improve protein yield and stability.

Related literatures of growth media

Growth media FAQsPlasmid DNA yieldProtein yieldProtein solubilityToxic protein cloning and expression

Related products of growth media

Bacterial E.coli growth mediaDNA ladders or DNA markersExpression vectorsCompetent cells for cloning and expressionE.coli cell strains for protein expression

We appreciate your comments and feedback at info@exptec.com.

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Growth Medium FAQs

Commonly used bacterial E.coli growth media FAQs

1. How do you   select a commonly used growth medium for a research project?

2. Why is LB broth the most used medium in many academic labs? 3. What is the difference between the LB Miller and LB Lennox broths? 4. Will magnesium (Mg2+) increase the cell densities for commonly-used

bacterial E.coli growth media?5. Will agitation increase the cell density for commonly used bacterial

E.coli growth media?6. What is the best commonly used E.coli growth medium for plasmid

production?7. What is the best commonly used E.coli growth medium for protein

production?8. What is the best commonly used E.coli growth medium for phage

production?9. What is the best commonly used E.coli growth medium for making

competent cells?10. What is the highest cell density obtainable from commonly used

bacterial E.coli growth media?

High density bacterial E.coli growth media FAQs...>

FAQ 1. How do you select a commonly used growth medium for a research project?

It will depend on the research project. The following table lists the commonly used media and their applications

Medium Name ApplicationsLB Miller Broth E.coli growth and propagation; plasmid DNA and protein productionLB Lennox Broth E.coli growth and propagation; plasmid DNA and protein production

SOBPrepare high efficiency competent cells; plasmid DNA and protein production

SOC Plasmid transformation and growth of competent cells2x YT Phage DNA productionTerrific Broth (TB) High yield protein and plasmid DNA production

Super Broth (SB) High yield plasmid DNA and protein production

FAQ 2. Why is LB broth the most used medium in many

academic labs?

LB broth is the most commonly used medium in molecular biology labs for E.coli cell culture. Easy to make, readily available and simple compositions contribute the popularity Of LB broth. In addition, LB broth also has rich nutrient contents. Its osmolarity is close to optimal for E.coli cell growth at early log phase. E.coli strains often grow reasonably fast in LB broth at early log phase. Furthermore, LB broth is sufficient for most molecular biology applications at laboratory scale such as E.coli cell growth and propagation. It is also sufficient for most plasmid, phage, and protein productions at laboratory scales.

FAQ 3. What is the difference between LB Miller and LB Lennox broths?

The LB Miller Broth and LB Lennox Broth contain higher and lower sodium chloride levels respectively. There are minimal differences between the two formulations in most molecular biology studies with commonly used E.coli strains. Different bacteria strains may require different salt concentrations. The low salt formula is more often used in salt-sensitive antibiotic selections. The LB broth used in most labs is LB Miller broth which contains 10 grams NaCl per liter of medium.

FAQ 4. Will magnesium (Mg2+) increase the cell densities for commonly used bacterial E.coli growth media?

Yes. Mg2+ can increase the cell density for LB, 2x YT, TB, and SB. The commonly used Mg2+ concentration is 10 to 20 mM. Either MgCl2, MgSO4, or both of them may be used. In addition, the aeration or the shaking speed of the incubator has to be increased at the same time. The normal shaking speed range of 150 - 250 rpm should be increased to a range of 350 - 400 rpm to obtain significant cell density increase.

SOB and SOC contain sufficient amounts of Mg2+. Adding Mg2+ to these media will not further increase cell density.

FAQ 5. Will agitation increase the cell density for commonly used bacterial E.coli growth media?

Yes. Agitation will increase the aeration of the E.coli cell growth. Oxygen is required for high density growth of E.coli cells. Agitation is controlled by the shaking speed of a shaker incubator. Agitation alone can increase the cell density for all commonly used bacterial E.coli growth media. Cell density will be further increased with combination of increased agitation and added

Mg2+ in the common media.

FAQ 6. What is the best commonly used E.coli growth medium for plasmid production?

Super Broth (SB) is the best commonly used E.coli growth medium for plasmid production.

FAQ 7. What is the best commonly used E.coli growth medium for protein production?

Terrific Broth is the best commonly used E.coli growth medium for protein production.

FAQ 8. What is the best commonly used E.coli growth medium for phage production?

Most scientists use 2x YT for phage production.

FAQ 9. What is the best commonly used E.coli growth medium for making competent cells?

SOB is the best commonly used E.coli growth medium for making competent cells.

FAQ 10. What is the highest cell density obtainable from commonly-used bacterial E.coli growth media?

TB can support the highest E.coli cell density when Mg2+ is added and agitation is increased. The highest cell density may be obtained from TB is about OD600 = 18. Some cell strains may reach OD600 = 20, but this result is often inconsistent and cells lose protein production ability at this density or higher.

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High density bacterial E.coli growth media FAQs

1. Why should a high density growth medium be used for a project? 2. How do you select a high density growth medium? 3. Can DNAGro TM growth medium increase the yield for any plasmid? 4. Can DNAGro TM growth medium be used for protein expression?

5. Can chloramphenicol be used in DNAGro TM growth medium? 6. Can ProGro TM , AutoX TM , and InduX TM growth media increase the yield for

any recombinant protein?7. Can DetoX TM growth medium reduce the toxicity of any toxic protein to

the host cells?8. Why can these high density growth media increase the solubility of a

recombinant protein?9. Will these high density growth media increase the solubility for any

insoluble protein?10. Can these high density growth media increase protein stability?

How about protein activity?11. What is the typical conditions used for the high density growth

media?12. What are the highest OD600 values from different shake flask

containers?13. What are the OD600 values at the normal shaking speed? 14. Can high density growth media be used at low temperatures? 15. What are the differences between glass and plastic flasks? How

about different plastics?16. What are the differences between baffled and regular flasks? 17. What are the differences among conical, round- bottom, and

square-bottom tubes?18. Why can't I reach OD600 = 30 to 50 even though the high

density media were shaken at 350 to 450 rpm?19. Can we use the high density media in deep-well plates? What will

be the OD?20. Why do I sometimes get OD higher than 50? 21. Can all high density growth media reach OD600 = 30 to 50? 22. How do these high density growth media compare to other

special media?23. What are the benefits of using high density growth media?

FAQ 1. Why should a high density growth medium be used for a project?

Common media cannot support sustained high density cell growth. With increase aeration and added Mg2+, LB broth can support E.coli cell density to OD600 = 10. Since LB broth contains no buffer, the pH of the medium will quickly change to pH 9 or higher. At this pH, E.coli cells will be unable to express protein. TB can support E.coli cell density to OD600 = 18 with increased aeration and added Mg2+. The medium pH also quickly changes to pH 9 or higher. The time window that allows protein expression is still short. It often does not allow induction of three hours or longer.

All high density growth media can support E.coli cell density OD600 = 30 to 50 depending on the containers used. The high density growth media are

buffered by phosphates and organic acids/salts. At OD600 = 30, the pH of all high density growth media is at about 7, which is optimal for most protein expressions. The time windows allowing protein expression in these media are sufficient for most protein productions.

High density growth media allow the yield of plasmid and protein productions to reach the levels of fed-batch fermentation in the shake flask containers. This increase plasmid and protein yields 10 times more than common media such as LB broth. The amounts of mini-prep plasmid and protein will be sufficient for many biochemical analyses. The amounts of large prep plasmid and protein will be comparable with those that may be obtained from a fed-batch fermentor. High density growth media increase efficiency and save time.

High density growth media contains trace metals, minerals, and vitamins which may serve as prosthetic groups, co-factors, or ligands for recombinant proteins. These prosthetic groups, co-factors, or ligands are often critical for protein folding, solubility, stability, and activity. Therefore, many proteins are more soluble, stable, and functional when expressed in these media.

FAQ 2. How do you select a high density growth medium?

The following table lists the high density growth media and their applications.

Medium NameApplications

DNAGroTM Increases plasmid DNA yield 10 times over LBProGroTM Increases protein yield, solubility, stability and activity

AutoXTM Increases protein yield, solubility, stability and activity; automatic protein expression without adding IPTG

DetoXTM Reduces protein toxicity and support E.coli growth to OD600=30InduXTM Increases protein yield, solubility, stability and activity

SecProTM Increases secretary protein expression over 100 times than LB or TB

FAQ 3. Can DNAGroTM growth medium increase the yield for any plasmid?

Yes. DNAGroTM growth medium will support E.coli cell density to OD600 = 30 or higher. The results apply to all commonly used E.coli strains such as DH5a, XL1blue, and Top10 cells. It is important to make sure that the amount of appropriate antibiotic is sufficient for plasmid selection at higher cell density.

FAQ 4. Can DNAGroTM growth medium be used for protein expression?

No. DNAGroTM growth medium contains inhibitors for protein expression.

FAQ 5. Can chloramphenicol be used in DNAGroTM growth medium?

Yes. Chloramphenicol can be used to amplify relaxed plasmid copy number in E.coli cells, but not the stringent plasmids. This applies to common media such as LB as well as high density growth medium DNAGroTM.

FAQ 6. Can ProGroTM, AutoXTM, and InduXTM growth media increase the yield for any recombinant protein?

Yes. ProGroTM and AutoXTM growth media increase the cell density for all bacterial strains such as BL21. Protein expression will increase proportionally with the cell density. It is important to make sure that the amount of appropriate antibiotic is sufficient for plasmid selection at higher cell density.

FAQ 7. Can DetoXTM growth medium reduce the toxicity of any toxic protein to the host cells?

No. DetoXTM growth medium can only reduce the toxicity of proteins induced by sugars such as lactose, IPTG, and arabinose. It cannot reduce the toxicity of proteins induced by heat, salts, or anything other than sugars. In addition, it can only reduce the pre-induction toxicity. It cannot reduce the post-induction toxicity.

FAQ 8. Why can these high density growth media increase the solubility of a recombinant protein?

High density growth media contains trace metals, minerals, and vitamins which may serve as prosthetic groups, co-factors, or ligands for recombinant proteins. These prosthetic groups, co-factors, or ligands are often critical for protein folding and solubility. Therefore, many proteins are more soluble when expressed in these media.

FAQ 9. Will these high density growth media increase the solubility for any insoluble protein?

No. The high density growth media will only increase the solubility of the proteins which required trace metals, minerals, and vitamins.

FAQ 10. Can these high density growth media increase protein stability? And how about protein activity?

High density growth media contain trace metals, minerals, and vitamins which may serve as prosthetic groups, co-factors, or ligands for recombinant proteins. These prosthetic groups, co-factors, or ligands are often critical for protein stability and activity. Therefore, many proteins are more stable and functional when expressed in these media.

FAQ 11. What are the typical conditions used for the high density growth media?

High density growth media can be used in 14 ml tubes or in any flasks with or without baffles. Both glass and plastic containers may be used. these growth media are designed to grow bacterial cells at 370C. Temperatures lower than 370C may be used for protein expression. Typical shaking speeds for high density growth media in a baffled flask is 350 rpm and 450 rpm in a regular (non-baffled) flask. All containers should be balanced and securely fixed in a shaker incubator. In addition, the incubator and incubation room must be sufficiently ventilated for cultures of 500 ml or more. High density growth media may be used the same as common media except that they need better aeration and will support higher cell density.

FAQ 12. What are the highest OD600 values from different shake flask containers?

The highest OD600 values from the different shake flask containers are listed in the following tables.

Brand Pyrex Pyrex Pyrex Pyrex Nalgene Nalgene NalgeneMateri

alGlass Glass Glass Glass

Polypropylene

Polypropylene Polycarbonate

Volume 2L 2L 250 ml 250 ml 2L 250 ml

Test ml 500 500 50 50/100 500 50

Baffled No Yes No Yes No No Yes

rpm 450 350 450 350 450 450 350OD600 30 30 50 40 30 50

 

Brand Corning Kimax Kimax Bellco UltraYield UltraYield FalconMateri

alPolycarbonat

eGlass Glass Glass

Polypropylene

Polypropylene

Polypropylen

Volume

250 ml 250 ml 250 ml 250 2.5L 250 ml 14 ml

Test ml

50 50 50 50 500* 50

Baffled No No Yes Yes Yes Yes

rpm 450 450 350 350 350** 400OD600 50 50 40 40 30 40

* Larger volume will result in medium spilling out.** Higher shaking speed will result in medium spilling out.

FAQ 13. What are the OD600 values at the normal shaking speed?

The normally-used shaking speed for E.coli growth is 250 rpm in most labs. At 250 rpm, the high density growth media can only reach about OD600 = 10. The OD values will be lower at speeds slower than 250 rpm. At low shaking speeds (<350 rpm), the oxygen concentration is limited in the medium and the cells will produce large quantity of acids which will make the medium acidic. Therefore, it is not recommended to use high density growth media at low shaking speeds.

FAQ 14. Can high density growth media be used at low temperatures?

Yes. Temperatures lower than 37 0C are often used to express insoluble or toxic proteins. In these cases, the E.coli cells should be grown at 37 0C to reach inducible OD and the temperature should be changed to induce the protein expression rate.

FAQ 15. What are the differences between glass and plastic flasks? How about different plastics?

There are no significant differences between glass and plastic flasks in E.coli cell cultures (see tables in FAQ 12). The differences between polypropylene and polycarbonate are also minimal. Different materials exhibit little difference in E.coli cell culture. The designs of the containers are important for E.coli growth and cell density. Flasks generally give better aeration than the tubes. Smaller flasks allow better aeration than larger ones.

FAQ 16. What are the differences between baffled and regular flasks?

Baffled flasks will generate more agitation. More agitation means better aeration. More agitation also produces more foam. Foams act as oxygen

barriers. Foams produced at high shaking speeds offset the benefits of agitation. We found that shaking speed range of 350 - 400 rpm is optimal for E.coli growth in baffled shake flask containers. For regular flasks, the optimal shaking speed range is 400 - 450 rpm. Regular flasks (non-baffled) generate less agitation. Less agitation also produces less foam. These flasks produce no foam with our high density growth media with antifoaming agents. With no foam produced, aeration increases with higher shaking speed. In general, the cell densities in regular flasks are higher than those in baffled flasks, but they require higher shaking speed.

Baffled flasks should be used if the shaker incubator cannot reach a high speed. Regular shake flasks should be chosen if the shaker incubator can reach a high speed. Both regular and baffled flasks can achieve OD600 = 30 to 50 with high density growth media.

FAQ 17. What are the differences among conical, round- bottom, and square-bottom tubes?

Conical tubes allow least aeration. They should be avoided in E.coli cell culture. Round-bottom tubes are most commonly used in E.coli cell cultures. They allow less aeration than flasks. The medium and container volume ratio for round-bottom tubes should be at about 1/10. Square-bottom tubes give best aeration at a given shaking speed. They can be easily fixed on the shaking platform too. Containers tightly fixed on the shaking platform give better aeration than those loosely placed on the holders. Similar as the flasks, different materials do not make significant difference on E.coli growth and cell density.

FAQ 18. Why can't I reach OD600 = 30 to 50 even though the high density media were shaken at 350 to 450 rpm?

1. Glycerol concentration has to be correct when preparing the medium from powder. Glycerol is viscous. Measurement will be accurate if it is made into 50% solution in water.2. Additives must be added just before use with antibiotics. Adding Additives when making the medium will cause precipitation. Medium with precipitation will only reach OD600 = 20.3. Medium volume should not exceed the recommended volume:

Flask Regular Baffled tubeVolume* 1/8 to 1/4 1/4 to 1/2 1/10

rpm 400 t0 450 350 to 400 350 to 400*The recommended volume is the medium volume divided by container volume. For example, the recommended volume is 250 to 500 ml if the

container volume is 2 liters and it does not have baffles. 500 ml is recommended for the containers with volume larger than 2 liters.4. All containers MUST be securely fixed on the shaker incubator. This is especially critical for when the tubes are used in the culture. If the tubes are loosely placed on a rack with large holders, the tubes will rotate in the holders and the media inside the tubes will not be sufficiently agitated. Racks with small holders should be used. Rubber bands may be used to fix the tubes onto the rack. Balanced loading is also important when the medium volumes are greater than 50 ml. Fixing containers securely is not only for safety but also for aeration.5. The container cover should allow the best aeration possible. In no case should the container cover be completely closed. Use the cover that allows the best ventilation possible. After the OD600 reaches 10, the container cover may be removed to maximize aeration. We never encountered any cross-contamination at this or higher cell density.6. The inoculation ratio is incorrect. The recommended inoculation ration is 1:100 with the seed culture prepared in a high density growth medium. With this inoculation ratio, the E.coli cells will reach OD600 = 30 or higher in 12 to 16 hours (overnight). Any ratio lower than the recommended inoculation ratio will require longer growth time. Any ratio higher than the recommended inoculation ration will result in over-growth. 7. OD measurement is more accurate at values less than 0.5 for most spectrometers. For common media, this means to dilute the culture 10 times. For high density growth media, this means a 100-time dilution. Distilled or deionized water should be used as a blank and in dilution. Rich media often have color by themselves. OD measurement will not be accurate if a medium is used as a blank or in dilution.

The high density growth media cannot reach expected cell density if any of the recommended operations above cannot be followed. However the cell density in the high density growth media will be significantly higher than what may be obtained from common media such as LB or TB.

FAQ 19. Can we use the high density media in deep-well plates? What will be the OD?

Yes. The OD600 will be 50 or higher. Use caution to prevent the medium from desiccating or dry out.

FAQ 20. Why do I sometimes get OD higher than 50?

1. The medium may desiccate during incubation. Open water may be placed in the incubator to increase the humidity and to avoid medium desiccation.2. Incubation  longer than recommended time may result in OD600 higher than 50.

3. OD600 up to 200 may be reached with our high density growth media in a fed-batch fermentor. In these cases, extra oxygen should be supplied and the medium pH should be adjusted. The cells must be diluted 500 to 1000 times to get an accurate OD reading. Only water should be used both as a blank and in dilution for OD reading.

FAQ 21. Can all high density growth media reach OD600 = 30 to 50?

Yes. All these high density growth media can reach OD600 = 30 to 50 in a shake flask container. Most wild-type cell strains can reach OD600 = 20 to 30 in SecProTM medium. Some cell strains with some toxic proteins do not grow well in SecProTM medium. In these cases, the cells may be grown in DetoXTM or ProGroTM medium to reach inducible OD and induce the cells in SecProTM medium to get high yield secretary expression.

FAQ 22. How do these high density growth media compare to other special media?

We tested ON Express, Circular Grow, and Magic media. The highest cell density may be reach in these media is about OD600 = 20. The plasmid and protein yields are lower in these media than those in high density growth media. Some proteins are not soluble and functional when expressed in ON Express and Magic media indicating these media lack required trace metals, minerals, and vitamins for protein solubility and activity.

FAQ 23. What are the benefits of using high density growth media?

1. High density growth media will increase the yields of both plasmid DNA and recombinant proteins. The yields are often increased 10 times or more. The yields will increase over 100 times in secretary and toxic protein expression. They will save time and labor and therefore increase the efficiency of your work.2. Since the high density growth media contain trace metals, minerals, and vitamins, they may serve as prosthetic groups, co-factors or ligands for recombinant proteins and therefore increase the solubility, stability, and activity of a recombinant protein.

High density bacterial E.coli growth media FAQs...^

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Go to growth media literatures...>Back to bacterial E.coli growth media ...<

Copyright © 2003 Expression Technologies Inc.

E. coli is a chemoautotroph; it can synthesize all it needs to grow from water, simple inorganic chemicals and an energy source. It can extract the energy it needs through the oxidation of organic compounds.  

Organisms that capture the energy of light are known as photoauxotrophs, those that extract energy from inorganic compounds, known as chemolithoauxotrophs.  

Starting sterile: If you want to study the behavior of a single organism, you need to know that there are no other organisms in your system.  A sterile system contains no living organisms or viruses. 

To do that you must sterilize all of the instruments, containers and materials you plan to use.  

Laboratory containers used to be made of glass.  Glass containers can be sterilized by baking them at high temperatures;

In modern labs, plastic containers are more common.  Nevertheless, we still refer to organisms grown in the lab as grown in vitro, which means in glass.

Plastic containers are sterilized using chemicals or radiation.  Most "plastic ware" comes from the manufacturer sterile and is discarded after use Growth media: Early on, scientists such as Redi, Spallanzani and Pasteur used simple solutions, such as beef broth or green infusions (tea), to grow microbes.  

They boiled their media (the plural of medium) to sterilize it.

 

 Boiling does not kill all organisms, however – some thermophilic bacteria flourish at temperatures well above 100°C

Moreover, many organism generate spores, which can survive boiling, as well as drying, high levels of radiation, and other rather nasty conditions.  Spores are so hardy that it has been suggested that they can travel through interstellar space. 

A more effective method for sterilizing media is the use an autoclave, essentially a pressure cooker.  

Increasing the pressure increases the temperatures that can be attained, without boiling.

In an autoclave, media is heated to 121°C at a pressure of 20 atmospheres.

Media that has been autoclaved will generally remain sterile as long as the container in which it is place is itself sterile and airtight. 

 

 For simple media, autoclaving is the method of choice.   Some more complex nutrient broths contain molecules that are destroyed at the temperatures used in an autoclave.  

These liquids can be sterilized by passing them through using special filters.  These filters have pore sizes so small (> 0.2 µm in diameter) that organisms and spores cannot pass through them.

These filters will not remove all bacterial viruses however, many of which are smaller than 0.2 µm. In fact viruses were first classified based on the fact that they can pass through such filters!

 

 

Growing coli:  E. coli is a generalist. It can grow under a wide range of conditions.

An E. coli can synthesize everything it needs from a single simple carbon and energy source and inorganic salts. This type of media is referred to as minimal media

 

A minimal media for E. coli13.6g KH2PO4 - source of phosphate and a buffer

2.0g (NH4)2SO4 - source of nitrogen and sulfur0.01g CaCl2 - source of calcium

0.0005g FeSO4(7H20) - source of iron0.02g MgCl2(7H2O) - source of magnesium1.0g glucose - source of carbon and energydeionized, distilled (i.e. pure) water to 1 liter

pH to 7.2 to 7.4 with 1M NaOH.

E. coli grows slowly in minimal media.   Why? because it must synthesize all of the complex molecules it needs to build a copy of itself - amino acids, sugars, lipids, nucleotides, vitamins, etc.  For most routine studies, E. coli are grown in a rich broth, usually Luria-Bertani medium or LB for short.   In LB, it grows quite fast.

Luria Bertani Medium (LB):

10.0g Tryptone (enzymatically digested milk protein casein - supplies amino acids)

5.0 g of Yeast Extract (supplies lots of nutrients) 1g glucose 10.0g NaCl pH ~7.2 deionized, distilled water to 1 liter

 

 The growth of a bacterial culture is typically divided into three phases, known as lag, exponential and stationary phases.

During lag phase, the bacteria are adapting to their new environment.  In exponential phase, they grow as fast as possible.  As resources are exhausted and waste produces accumulate, growth slows and the culture enters stationary phase.  

If two organism depend upon one another to grow, how would you approach the problem of growing them as monocultures?

Which molecules does E. coli not have to synthesize when grown in LB, compared to minimal media?

Both types of media contain glucose, what is is used for? Why would the presence of other (unknown) organisms and viruses confuse

studies on the growth a specific organism? Why did you let the autoclaved media cool before you add the organism you

what to study to it? Some organisms, known as hyperthermophiles, grow at temperatures above

100ºC.  How is that possible?   What determines the upper temperature limit for survival?