Paul.xu at 19:51, 7 March 2017

2017-03-07T19:51:41Z

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== Medium ==

'''Standardized growth media''' for Vibrio natriegens '''LB3''' Lysogeny Broth with 3% (w/v) final NaCl. 

Add 20 grams of NaCl to 25 grams of LB Broth Miller (Fisher BP9723500).

'''Rich media '''  Add 1.5% final Ocean Salts (Aquarium System, Inc.) (w/v) to make high salt versions of Brain Heart Infusion (BHIO), Nutrient Broth (NBO), and Lysogeny Broth (LBO). No additional salts is added to Marine Broth (MB). 

'''Minimal M9 medi'''a .

Add 2% (w/v) final sodium chloride  M9.

Add 0.4% Carbon sources. 

'''SOC3 media'''

5 grams of yeast extract

20 grams tryptone

30 grams sodium chloride,

2.4 grams magnesium sulfate

0.4% (v/v) final glucose.

'''Enhanced 2xYT medium: preferred medium for high mass protein expression in liquid'''

20 g/L Yeast Extract

32 g/L Tryptone

17 g/L NaCl

2% Glucose

17.6 mM Na2HPO4,  pH 7.4

'''To make 20 L''':

400 g Yeast Extract

640 g Tryptone

340 g/L NaCl

400 g Glucose

50 g Na2HPO4

'''2xYT media:  for routine cell culture'''

16 g/L Tryptone

10 g/L Yeast Extract

5 g/L NaCl

'''Terrific broth'''

12 g/L Tryptone

24 g/L Yeast Extract

4 g/L Glycerol

1X Phosphate Buffer (17 mM KH2PO4, 72 mM K2HPO4)

== Storage ==

Store agar plates with established Vmax™ Express cells'''at room temperature.'''

'''Do not store agar plates at 2–8°C '''as this can adversely impact re-proliferation. •

You may leave agar plates at room temperature for '''up to 72 hours.'''

Single colonies will remain well isolated since Vmax™ Express cells exhibit contact inhibition. •

For long-term storage,  prepare glycerol stocks with 25% glycerol and storing at −80°C.

== '''Overnight culturing''' ==

An inoculation of 80℃ frozen stock of Vibrio natriegens can reach stationary phase after 5 hours when incubated at 37℃. Prolonged overnight culturing (>15 hours) at 37℃ can lead to an extended lag phase upon subculturing. Routine overnight culturing of Vibrio natriegens is performed for 815 hours at 37℃ or 1224 hours at room temperature. Unless otherwise indicated, Escherichia coli cells used in this study were K12 subtype MG1655 unless otherwise indicated and cultured overnight (>10 hours) at 37℃. Vibrio cholerae O395 was cultured overnight (>10 hours) in LB at 30℃ or 37℃ in a rotator drum at 150rpm.

== Glycerol Stock ==

To prepare Vibrio natriegens cells for 80℃ storage,'''an overnight culture of cells must be washed in fresh media before storing in glycero'''l. A culture was centrifuged for 1 minute at 20,000 rcf and the supernatant is removed. The cell pellet is resuspended in fresh LB3 media and glycerol was added to '''20-25% final concentration'''. The stock is quickly vortexed and stored at '''80℃'''. Note: unlike glycerol stocks of Escherichia coli for 80℃ storage, '''neglecting the washing step prior to storing Vibrio natriegens cultures at 80℃ can lead to an inability''' to revive the culture.

== Preparation of electrocompetent Vibrio natriegens ==

Grow Vibrio natriegens overnight.

Prepare a subculture  by inoculating an overnight culture, washed once in fresh LB3, at 1:100 dilution into fresh media. For example, spin down 500μL of the overnight culture  (centrifugation for 1 minute at 20,000rcf), resuspendein 500μL fresh LB3 media and inoculated in 50mL of LB3 media.

Incubate the culture  at 37℃ at 225rpm for 1 hour reaching OD ~0.4.

Spin down cells (centrifugation at 3500rpm for 5min at 4℃)

Wash by resuspension in 1ml of cold 1M sorbitol followed by centrifugation at 20,000rcf for 1 minute at 4℃.

Repeat wash for a total of three times.

Resuspend the  cell pellet in 250μL of 1M sorbitol.

Use 50μL of concentrated cells for each transformation.

For long term storage, aliquot the concentrated cells  in 50μL shots in chilled tubes, snap frozen in dry ice and ethanol, and stored in 80℃ for future use.

== Transformation ==

Add 50ng of plasmid DNA to the cells in 0.1mm cuvettes and electroporated using Biorad Gene Pulser electroporator at 400 V, 1 k Ω , 25μF.

Recover cells in 1mL LB3 or SOC3 media for 45 minutes at 37℃ at 225rpm

Plate on selective media.

Incubate plates at least 6 hours at 37℃ or at least 12 hours at room temperature. 

=== '''Protocol 2''' ===

1. For each transformation reaction,

• Thaw one vial of Vmax™ Express Electrocompetent cells on ice for 5 minutes.

• Warm one vial of Vmax™ Recovery Media to room temperature (20°C to 24°C).

• Pre-chill a 1mm electroporation cuvette on ice.

2. Transfer 500 µL Vmax™ Recovery Media to a Falcon® 14-mL Round-Bottom Tube. Allow media to sit at room temperature until needed in Step 8.

Note: Vmax™ recovery medium is rich and prone to contamination. Discard any remaining medium or return it to −20°C for storage.

3. As soon as the electrocompetent cells are thawed, add 500 pg – 5 ng of plasmid DNA to the competent cells on ice. Briefly mix by flicking tubes.

4. Incubate the DNA and cell mixture on ice for 3–5 minutes.

5. During incubation, verify the Gene Pulser® Xcell System is set to the following electroporation parameters:'''700 V, 25 uF, 200 Ohms, 1mm cuvette.'''

Note: These are the optimized electroporation settings for the Gene Pulser® Xcell™ Microbial System using a cuvette with a 0.1-cm gap. If other instruments and/or cuvettes are used, you may need to optimize the conditions for best results.

6. Transfer cells to a chilled cuvette using a pipette. Verify that the cells completely occupy the bottom of the cuvette and that no air remains between walls of the cuvette and the cells.

7. Transfer the cuvette to the shock pod and perform electroporation. Note: '''Time constants''' typically range from '''3.3–3.7 ms'''.

8. Transfer cells to a room temperature Falcon® 14-mL Round-Bottom Tube containing recovery media immediately after pulse.

9. To transfer any remaining cells, add 200 μL of Vmax™ Recovery Media to the cuvette. Dispense the media to the same round-bottom tube from Step 8.

10. Place the Falcon® 14-mL Round-Bottom Tubes in a tube rack in an orbital shaker (250 rpm) at 30°C for 2 hours at to allow recovery of cells and expression of antibiotic resistance marker. Note: Recovery can be performed at 37°C unless kanamycin is used for selection.

11. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature.

The optimal recovery temperature can vary with different combinations of origins of replication and antibiotic selection markers used. As shown in the following table, most combinations of post-transformation growth on agar plates can be performed at either 30°C or 37°C. However, for cells with a kanamycin resistance marker, optimal recovery occurs solely at 30°C. The following table shows different plasmid origins of replications tested. If the origin of replication for the plasmid is unknown, do duplicate reactions and recovering at both 30°C and 37°C

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Antibiotic Marker 
| Solid Media 
| Liquid Culture 
|-
| Amp/Carbenicillin
| 50 μg/mL 
| 50 μg/mL 
|-
| Kanamycin 
| 100 μg/mL 
| 50-100 μg/mL 
|-
| Tetracycline 
| 1 μg/mL 
| 1 μg/mL 
|-
| Chloramphenicol 
| 5–12.5 μg/mL 
| 12.5 -25 μg/mL 
|}

Vmax™ Express has natural resistance to Kanamycin. Perform selection of Kanamycin-resistant (KanR) colonies on plates with 100 μg/mL Kanamycin. This concentration slows the growth of cells lacking a plasmid with the Kanamycin-resistance gene. Using a lower concentration of Kanamycin will result in an increase of background colonies, making it difficult to identify transformed cells.

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Plasmid Origin of Replication 
| style="width: 97px;" | Plasmid Backbone 
| style="width: 67px;" | Compatible? 
| style="width: 25px;" | Growth Plate Recommendation 
| 
|-
| pMB1 
| style="width: 97px;" | pBR322 pET vectors 
| style="width: 67px;" | Yes 
| style="width: 25px;" | 37°C recommended (30°C acceptable) 
| 
|-
| ColE1 
| style="width: 97px;" | pCDNA3.0 pGEM5zf 
| style="width: 67px;" | Yes 
| style="width: 25px;" | 
| 
|-
| pUC 
| style="width: 97px;" | pUC19 Selection with Kanamycin 
| style="width: 67px;" | Yes 
| style="width: 25px;" | Grow plates at 30°C only 
| 
|-
| pUC 
| style="width: 97px;" | pUC19 Selection with all other antibiotics 
| style="width: 67px;" | Yes 
| style="width: 25px;" | 37°C recommended (30°C acceptable) 
| 
|-
| p15A 
| style="width: 97px;" | pACYC184 
| style="width: 67px;" | Yes 
| style="width: 25px;" | 37°C recommended (30°C acceptable) 
| 
|-
| RK2 
| style="width: 97px;" | N/A 
| style="width: 67px;" | Maybe 
| style="width: 25px;" | Incompatible with electroporation. Can be introduced by conjugation. 
| 
|}

{|
|-
| '''Common Vectors'''
| '''Copy Number'''
| '''ORI'''
|-
| pUC
| ~500-700
| pMB1 (derivative)
|-
| pBR322
| ~15-20
| pMB1
|-
| pET
| ~15-20
| pBR322
|-
| pGEX
| ~15-20
| pBR322
|-
| pColE1
| ~15-20
| ColE1
|-
| pR6K
| ~15-20
| R6K*
|-
| pACYC
| ~10
| p15A
|-
| pSC101
| ~5
| pSC101
|-
| pBluescript
| ~300-500
| ColE1 (derivative) and F1**
|-
| pGEM
| ~300-500
| pUC and F1**
|}

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. For a rapid expression assay, pick a single colony, inoculate 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 13 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 Media or Enhanced 2xYT media. Other liquid media listed in Table 2 on page 8 can also be used (see "Media recipes" on page 19).

Protocol 1. On the day of expression, inoculate a seed culture (OD600 of 2–7) into growth media containing appropriate antibiotics. Use 1/100 volume of seed culture relative to the total amount of media to inoculate the growth flask (e.g. for 100 mL of growth media, add 1 mL of seed culture).

2. Grow expression culture at 30°C for ~2 hours.

Note: Expression can be performed at 37°C, but protein expression is optimal when cells are grown at 30°C on a rotating shaker incubator at 250 rpm.

3. After ~2 hours, induce protein expression by adding IPTG to a final concentration of 1 mM.

Note: Although induction is typically performed 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.

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, overnight induction is recommended 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 4°C. Remove supernatant.

6. Store the cell pellet at −80°C or proceed to protein purification or lysis and protein purification.

Note: For 96-well plates, apply an adhesive plate seal.
[[Category:Protein Expression]]

Protein Expression Vibrio natriegens - Revision history

Paul.xu at 19:51, 7 March 2017