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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
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.
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.
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 range Cell growth Cell viability at 4 0C Cell stock Plasmid production Protein expression pH<5.5 Slow to stop 2 weeks Not recommended OK for most plasmids OK for some proteins pH 5.5 to 8.5 Optimal >90% for 1 month Ideal ideal for most plasmids Good for most proteins
pH>8.5 Stop to cell d <10% in 2 d Not usabl OK for some pl poor for most p
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.
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. Back to top ^ | Go to bottom
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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... >
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.
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. Back to top ^ | Go to bottom
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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 ProGroTM medium... >
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 InduXTM medium... >
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.
medium, high levels of recombinant protein can be expressed. SecProTM 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.
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).
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 LB Miller Broth LB Lennox Broth SOB SOC 2x YT Terrific Broth (TB) Applications E.coli growth and propagation; plasmid DNA and protein production E.coli growth and propagation; plasmid DNA and protein production Prepare high efficiency competent cells; plasmid DNA and protein production Plasmid transformation and growth of competent cells Phage DNA production High yield protein and plasmid DNA 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 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. Back to top ^ | Go to bottom
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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 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 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 Material Volume Test ml Baffled rpm OD600 Pyrex Glass 2L 500 No 450 30 Pyrex Glass 2L 500 Yes 350 30 Pyrex Glass 250 ml 50 No 450 50 Pyrex Glass 250 ml 50/100 Yes 350 40 Nalgene Polypropylene 2L 500 No 450 30 Nalgene Polypropylene 250 ml 50 No 450 50
Brand Corning Kimax Kimax Material Polycarbonate Glass Glass Volume 250 ml 250 ml 250 ml Test ml Baffled rpm OD600 50 No 450 50 50 No 450 50 50 Yes 350 40
Bellco UltraYield UltraYield Falcon Glass Polypropylene Polypropylene Polypropylene po 250 2.5L 250 ml 14 ml 50 Yes 350 40 500* Yes 350** 30 50 Yes 400 40 1.5 No 400 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
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 Volume* rpm Regular 1/8 to 1/4 400 t0 450 Baffled 1/4 to 1/2 350 to 400 tube 1/10 350 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 overgrowth. 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 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...^ Back to top ^
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 100C 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 121C 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 m 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 m. 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. coli 13.6g KH2PO4 - source of phosphate and a buffer 2.0g (NH4)2SO4 - source of nitrogen and sulfur 0.01g CaCl2 - source of calcium 0.0005g FeSO4(7H20) - source of iron 0.02g MgCl2(7H2O) - source of magnesium 1.0g glucose - source of carbon and energy deionized, 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 100C. How is that possible? What determines the upper temperature limit for survival?