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Cinnabar Media Hi-Def Azure Media
Inducers Stable Isotopic Labeling Media

Teknova offers both Complex and Defined protein expression media for small-scale and large-scale protein expression in E. coli.
Cinnabar is a Complex Growth Media that reaches optical densities of OD600: 40-65
Hi-Def Azure is a Defined Growth Media, reaches optical densities of OD600: 9-14
Note: In comparison, LB OD600: 3-5 and TB OD600: 15-25

Teknova formulated Cinnabar Media, a complex growth medium, based on Studier's original formulation developed for protein expression in E. coli with a protein expression protocol developed by Studier known as induction.
Cinnabar routinely reaches optical densities of OD600: 40-65
Compare to LB OD600: 3-5 and TB OD600: 15-25
Use of Cinnabar media can result in at least 3-8X more protein
Cinnabar media has been successfully used in E. colistrains BL21 and BL21(DE3).
We suggest supplementation of Cinnabar Media with 0.8% Glucose and 0.1mM IPTG.
Note: Cinnabar contains 0.7% Glycerol (so it is not necessary to supplement Cinnabar with Glycerol).

Teknova developed Hi-Def Azure Defined Growth Media based on Niedhardt’s original formulation for growth media for Enterobacteria
Hi-Def Azure can also be made in special formats for labeling and structural studies
Features of Hi-Def Azure Media :
1. All system components have a known chemical composition
2. Hi-Def Azure is Animal-Free
3. All components are tested in-house
4. Results in consistent performance
5. Eliminates lot-lot variability

A disadvantage of induction by IPTG is the need to monitor bacterial growth to achieve optimal induction conditions. This difficulty is especially apparent in experiments which seek to evaluate protein expression in a variety of clones. In this case, differences in growth rates will be observed and it will be unlikely to achieve optimal induction for all expression clones. The induction protocol provides a method to circumvent these difficulties and simplify the expression protocol.

William Studier developed induction as a method of protein expression in which cells are culture in media containing a mix of carbon sources, generally glucose, lactose and glycerol. Studier made a detailed study of induction and found that in addition to simplifying the method for protein expression by simply allowing for addition of the inducer (typically IPTG) at the beginning of the protocol, Studier also found that by utilizing induction, he was able to produce 5-20 times as much target protein per volume of culture as conventional IPTG induction.
In 2005, William Studier at Brookhaven introduced induction as a simplified protocol for high-level protein expression in E. coli in T7-pET vectors.

Components in the media allow for the introduction of the IPTG inducer at the beginning of the experiment, thus eliminating the need for continual monitoring of cell growth. Due to catabolite repression, the presence of glucose in the media will serve to repress the inducer until at least the mid-logarithmic phase of growth, the optimal stage for high-level protein expression. When the glucose in the media is depleted, lactose (via IPTG) serves to induce expression of the T7 RNA polymerase upon conversion to allo-lactose by beta-galactosidase (the LacZ gene product). The media is well-buffered at all times and T7 RNA polymerase is induced in logarithmic to late-logarithmic phase of growth, which results in greater cell mass and higher yield of recombinant protein.

Studier points out that induction should be performed only in strains encoding an intact lac operon. Studier's detailed study of induction found that cell mass and total soluble target protein yield are increased several-fold using induction when compared with conventional protein expression protocols.

1. No need to monitor cell growth (IPTG is added at the beginning). final OD600 is at least 3X higher than with conventional IPTG induction protocol.
2. Greater cell mass will often result in greater soluble protein yield.
3. Can induce numerous expression clones simultaneously. (amenable for highthroughput).
4. Typically, lower amounts of IPTG are needed for induction (lowering the cost of protein expression).
5. Compatible with cultures grown in flasks, culture tubes and deep-well plates.
6. Minimal sample handling.

1. Transform plasmid into strain of E. coli optimized for protein expression. We use BL21 or BL21(DE3).
2. Inoculate 2 mL pre-culture for overnight growth:
A. Pick a single colony (from a freshly transformed plate) or
B. Use a thawed glycerol stock of cells to a final 1% (1:100) total culture volume (We add 20ul thawed glycerol stock into 2 mL Hi-Def Azure or Cinnabar, with appropriate antibiotic).
C. Grow pre-culture for 14-18 hours, shaking at 250-300rpm at 30-37oC in container with 5X volume for sufficient aeration. For example, for a 2mL pre-culture, we use a 13ml (17x100mm) polypropylene culture tube.

Note: It is common practice to prepare a pre-culture in advance and freeze aliquots as glycerol stocks at -70-80oC. These stocks should be thawed only once. Use of re-thawed glycerol stocks can result in >50% loss of viability with commensurate loss of maximum cell density.

3. Supplement induction Hi-Def Azure or Cinnabar media (at this time add appropriate antibiotic to media for selection).
For Hi-Def Azure media: we recommend adding 0.8% Glucose, 0.5% Glycerol and 0.1mM IPTG. For Cinnabar media: we recommend adding 0.8% Glucose and 0.1mM IPTG (Do not supplement Cinnabar with Glycerol; Cinnabar media already contains 0.7% Glycerol).

4. Inoculate media for induction to 1% total culture volume (1:100).
Note: The size of the inoculum is a key variable to control for reproducibility. The size of the inoculum will affect the consistency of the results. We advise consistency in the size of the inoculum and consistency of the total inoculated volume to reduce variability between protein expression trials.

Induction will occur at an earlier time in the growth phase in 0.1% Glucose (than with 0.8% Glucose). We suggest supplementing with 0.8% Glucose to suppress the time of induction for toxic proteins or proteins that are known to reduce cell growth. For early induction: Use 0.1% Glucose supplemented media. For late induction: Use 0.8% Glucose supplemented media.

5. Grow culture at 16-30oC, shaking at 225-300 rpm for at least 22-28 hours for optimum protein expression.
We shake cultures at 300 rpm, (using Deutz microreactors) to increase aeration resulting in maximum cell density (OD600 = 50). For optimum protein expression, typically it is best to grow the cells well into stationary phase. Note: Cinnabar Media has a long lag-phase and will typically reach stationary phase at 26-28 hours.

6. Harvest Cells
Note: These are general guidelines. The best temperature for protein expression and the amount of time needed for optimum expression must be determined experimentally, for each protein. In general, for proper protein folding, for decreased production of inclusion bodies and for minimizing exposure to proteases, lower temperature of protein expression is suggested.

1. Carnes A.E., Hodgson C.P., Williams J.P. Inducible Escherichia coli Fermentation for increased plasmid DNA production. Biotechnol. Appl. Biochem.; 45(3): 155-66.(2006). PMID: 16819941

Protein Expression Articles: References for Protein Expression

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100X Glucose, Glycerol, IPTG Induction Supplement, Sterile. 1000mL Cat.No. G0565

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100X Glucose, Glycerol, IPTG Induction Supplement, Sterile. 100mL Cat.No. G0560

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Azure CF 50X Media for Fermentation Kit, Sterile. (3 part Kit - 1 Liter each) Cat.No. 3H5080

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Hi Def Azure Media Animal-Free, Sterile. 200mL in 1000mL Bottle Cat.No. 3H6200

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Hi Def Azure Media, Sterile. 200mL in 1000mL Bottle Cat.No. 3H5200

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TY Base for Induction. 1000mL, Sterile Cat.No. T5876

Our Price: $40.08

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