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Created page with "Vmax is derived from marine microorganism, Vibrio natriegens. This gram-negative, non-pathogenic bacterium exhibits the fastest growth rate of any known organism with a d..."

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Vmax is derived from marine microorganism, Vibrio natriegens. This gram-negative, non-pathogenic bacterium exhibits the fastest growth rate of any known organism with a doubling time of less than 14 minutes, a growth rate that is twice as fast as that of E. coli. Vmax™ Express competent cells are engineered to allow exogenous protein expression and have been demonstrated to produce high yields of recombinant proteins under a tightly controlled, inducible T7 promoter system. Similar to other historically used bacterial expression strains such as E. coli BL21(DE3), Vmax™ Express can be cultured with routine growth medium such as Luria Broth (LB) supplemented with salt, 2xYT, Terrific broth (TB) and other commercial auto-inducible media.

Antibiotics

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 
| Solid media 
|  Liquid culture  
| Temp
|-
| Ampicillin 
|  10–50 μg/mL 
| 50–100 μg/mL  
| 30 or 37 oC
|-
| Carbenicillin 
| 2–25 μg/mL* 
|   5–100 μg/mL 
| 30 or 37 oC 
|-
| Kanamycin  
| 100 μg/mL  
| 200 μg/mL ''' '''
| '''30''' oC
|-
| Tetracycline 
| 2.5 μg/mL 
| 2.5 μg/mL 
| 30 or 37 oC 
|-
| Chloramphenicol 
|  5–12.5 μg/mL 
| 12.5–25 μg/mL 
| 30 or 37 oC 
|}

Compatible origins: pMB1, ColE1, pUC, p15A

Vectors: pBR322, pET,  pCDNA3.0, pGEM5zf ,  pUC19, pACYC184

'''Compatible Medium''': LB-Miller, Terrific broth, Brain heart infusion broth + v2 salts, Enhanced 2xYT medium

V2 Salt: ‡v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2

'''Enhanced 2xYT medium is the best for protein expression: '''
20 g/L Yeast extract

32 g/L Tryptone

17 g/L NaCl

0.2% Glucose

7.6 mM Na2HPO4 adjusted to pH 7.4

== Transformation ==

1. Aliquot 5 ul cells into autoclaved microcentrifuge tubes, store at -80 oC.

2. Aliquot 70 ul Vmax™ Chemicompetent Cell Recovery Medium into autoclaved microcentrifuge tubes

3. Take 1 vial of cells out, thaw one vial of Vmax™ Express Chemically Competent Cells on ice for 5–10 minutes. Do not vortex or pipette up and down. If necessary, gently flick the tube to verify that the cells are thawed. Do not transfer cells to another tube; perform heat shock in the tube provided. Note: Use cells within 30 minutes of thawing.

4. As soon as the chemically competent cells are thawed, add 1–100 ng or 0.5 ul of plasmid DNA (in TE buffer or water only) to the competent cells on ice. Briefly mix by gently flicking tubes and immediately place the tubes back on ice. Avoid introducing bubbles and do not vortex or pipette up and down. Note: Add no more than 1 μL of plasmid DNA to the competent cells. Concentrate the plasmid DNA if necessary.

5. Incubate the DNA and cell mixture on ice for 1 hour. Note: DNA and cells can be incubated for as little as 20 minutes. However, we recommend the full hour incubation because transformation efficiency decreases by ~2-fold for every 15-minute incubation time reduction.

6. During incubation, warm up 1 vial of 70 uL Vmax™ Chemicompetent Cell Recovery Medium. Allow the medium to warm to 30°C or 37°C in a water bath or incubator until needed in Step 9.

Note: Vmax™ Chemicompetent Cell Recovery Medium is very rich and prone to contamination. Use good aseptic handling practices to avoid contamination. We recommend aseptically transferring an aliquot of media to a sterile tube if the entire bottle will not be used. Store remaining media at 4°C.

7. After incubating cells and DNA on ice, heat shock the Vmax™ Express transformation reactions by transferring them to a '''42°C''' water bath without shaking for '''30 '''seconds.

8. Immediately transfer the Vmax™ Express transformation reaction back to the ice bucket for 2 minutes. Do not mix.

9. While keeping the tube on ice, transfer the cells to a pre-warmed  tube with Chemicompetent Cell Recovery Medium 

10. Place the tubes in an orbital shaker (250 rpm) at 37°C for 2 hours to allow cells to recover and express the antibiotic resistance marker.

Note: Recovery can be performed at 37°C, but 30°C is also acceptable for all antibiotics.

'''Kanamycin vector-containing cells can be recovered at 37°C or 30°C, but plates must be incubated at 30°C or room temperature'''. It is important to allow the Vmax™ Express cells to recover for 2 hours. Shorter recovery times negatively impact transformation efficiency.

11. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature for your vector/antibiotic selection approach. 

12. Transfer all of the transformation reaction to the center of an LB-Miller plate supplemented with the appropriate antibiotic. Use a sterile spreader or sterile beads to distribute cells across the surface of the plate.

Note: If too few colonies are observed, use more  DNA  for  transformation reaction.

13. Incubate plates at 25°C, 30°C, or 37°C for at least 8 hours.

'''Note:''' For kanamycin plates, incubate at 25°C to 30°C. Do not incubate kanamycin plates at 37°C. 

Vmax™ Express has a naturally occurring resistance to kanamycin. Thus, the minimum inhibitory concentration (MIC) for kanamycin selection is higher than that of other laboratory bacteria. Using kanamycin selection with Vmax™ Express, positive clones appear larger and non-transformed naturally resistant colonies appear smaller. DO not use small colonies

'''Important notes'''

• Vmax™ cells on agar plates should be incubated at 25°C to 30°C overnight. If desired, plates can be incubated overnight at 37°C, except for those containing kanamycin, which must be incubated at 25°C to 30°C.

• Colonies typically appear after 12–16 hours at 25°C to 30°C and after ~8 hours at 37°C.

• Store agar plates with established Vmax™ Express at room temperature for up to 72 hours. Well-spaced single colonies will remain isolated at room temperature for several days.

• Do not store agar plates containing Vmax™ cells at 2°C to 8°C as this can adversely impact re-propagation and growth.

• For long-term storage, we recommend preparing glycerol stocks with 25% glycerol and storing at −80°C.

• Plasmid vectors isolated from E. coli tend to have a lower transformation efficiency than those isolated from Vmax™ cells. If needed, plate a larger volume of cells or transform with a larger amount of plasmid.

• Typical E. coli miniprep protocols are compatible with Vmax™ cells.

 

== Preparing seed cultures ==

Before starting

• Pre-warm growth media before use if it has been stored at 4°C. Inoculating cold media with Vmax™ Express will result in poor or no growth of the culture.

• For optimal results, we recommend using Vmax™ Enriched Growth Medium (SGI-DNA Cat. No. CL1500-1000) or enhanced 2xYT medium (see "Media recipes" on page 18). Refer to Table 2 on page 8 for additional information and media options.

• Add appropriate antibiotics at required concentrations to the media for maintaining plasmid selection (refer to Table 3 on page 9 for suggested concentrations).

• For selection with kanamycin, refer to the representative images in Figure 2 on page 16 for guidance before picking colonies.

=== Seed culture protocol ===

1. Pick the first/largest colonies to appear from the transformation plates.

2. Inoculate a culture tube containing 2–15 mL growth media that is at room temperature, 30°C, or 37°C.

Note: The volume of the seed culture medium should be at least 1/100th the volume of desired protein expression culture.

3. Culture cells at 30°C or 37°C at 250 rpm for ~3 hours for rapid protein expression or for 6–16 hours if the culture will be used as a standard seed culture or used to create glycerol stocks for long-term storage at –80°C.

Note: Refer to the following table for additional information.

Glycerol stocks

For critical clones, we recommend preparing multiple glycerol culture aliquots to avoid freeze/ thaw cycles.

1. To prepare a glycerol stock, choose from one of the following options:

• Add 250 µL of sterile 100% glycerol to a 1-mL cryopreservation vial or other −80°C compatible sterile tube. Add 750 µL of bacterial seed culture (prepared as described above) and mix for a final concentration of 25% glycerol.

• Alternatively, spin 1 mL of bacterial seed culture in a microcentrifuge tube for ~1 minute at 10 × g. Aspirate the media and resuspend the pellet in 1 mL sterile media supplemented with 25% glycerol.

2. Place vials in –80°C freezer for storage.

3. To recover culture from glycerol stock, use a sterile inoculation loop to scoop frozen culture and transfer to liquid or solid growth medium with appropriate antibiotics and incubate at room temperature, 30°C, or 37°C.

Note: Always incubate kanamycin-resistant strains grown on solid agar medium at 30°C or room temperature.

Protein expression protocol

The following protocol is a standard protocol for protein expression using Vmax™ Express cells. This protocol has been used to successfully express multiple proteins with high yields. For some proteins, optimization of expression temperature and/or expression times may improve yield, solubility, or activity. Vmax™ Express has been shown to grow and produce recombinant proteins at temperatures as low as 16°C. If expression at lower temperatures is required, we recommend evaluating expression at multiple temperatures.

For rapid expression

To perform a rapid expression assay, pick a single colony, inoculate a seed culture in selective media, and grow to an OD600 of 0.3 (~3 hours in 25 mL of media) before inoculating a growth flask (see "Preparing seed cultures" on page 14 for additional details).

Before starting

• To allow sufficient aeration, ensure the expression culture will not exceed ¼ of flask volume (e.g, for a 500-mL Fernbach baffled flask, grow a maximum of 125 mL of expression culture).

• Optimal expression media are Vmax™ Enriched Growth Medium or Enhanced 2xYT medium. Other liquid media listed in Table 2 on page 8 can also be used (see "Media recipes" on page 18).

Protocol

1. On the day of expression, inoculate a seed culture (OD600 of 2–7 for standard protocol, >0.3 for rapid protocol) into growth medium containing appropriate antibiotics. Use 1/100 volume of seed culture relative to the total amount of medium to inoculate the growth flask (e.g. for 125 mL of growth medium, add 1.25 mL of seed culture).

2. Grow culture at 30°C for ~1–2 hours on a rotating shaker incubator at 250 rpm until the OD600 reaches >0.5. Note: Expression can be performed at 37°C, but for controls and most proteins tested, expression is optimal when cells are grown at 30°C on a rotating shaker incubator at 250 rpm.

3. After reaching OD600 of ≥0.5, induce protein expression by adding IPTG to a final concentration of 1 mM. Note: Although induction is typically performed 1–2 hours after inoculation, Vmax™ Express cells can be induced within a wide range of time points during growth phase. The optical density at induction can be between 0.5–1 OD600 with no apparent difference in expression. If auto-induction medium is used, expression will initiate during overnight culture.

4. Incubate induced cells for 4–24 hours to express recombinant protein.

Note: For most proteins, longer post-induction incubation times will allow for increased biomass, which typically results in higher protein yields. Although some proteins may express high levels sooner, we recommend overnight induction to maximize yield. Vmax™ Express has been shown to maintain soluble protein expression after overnight culture.

5. Harvest cells by transferring to a centrifugation tube and spinning at 4000–5000 × g for 10 minutes at room temperature or 4°C. Carefully remove supernatant.

6. Store the cell pellet at −80°C or proceed to lysis and protein purification. Note: If you are using 96-well plates for expression, apply an adhesive plate seal prior to centrifugation or storage. 

[[Category:Protein Expression]][[Category:Protocols]]

Protein Expression using Vmax - Revision history

Paul.xu: Created page with "Vmax is derived from marine microorganism, Vibrio natriegens. This gram-negative, non-pathogenic bacterium exhibits the fastest growth rate of any known organism with a d..."