Difference: ProtocolsWorkingWithVibrioNatriegens (13 vs. 14)

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Working with Vibrio natriegens (Vmax)

Vibrio natriegens has the fastest doubling time of any known organism and has the potential to shorten experimental timelines while still using methods that are similar to those used in E. coli. In addition, it is a naturally competent organism, which can be used to make genome edits more easily than is possible in E. coli.

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 There are three primary references for Vmax methods:
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SAD613 Sm10 lambda pir (E. coli mating strain) w/ pMMB-tfoX (Vc)
CAH602 S17 (E. coli mating strain) w/ pMMB-tfoX (Vn)
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Vibrio natriegens is meant to largely function using E. coli laboratory methods but there are some VERY IMPORTANT differences between the two:
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Vibrio natriegens is meant to largely function using E. coli laboratory methods but there are some VERY IMPORTANT differences between the two: media type and temperature conditions


  Media
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  Vmax also grows exceptionally well in BHIv2 (BHI supplemented with v2 salts) as recommended by SGI.
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 Temperature Conditions:
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Vmax grows rapidly at 37C and grows well at 30C also.
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Vmax grows rapidly at 37C and grows well at 32, and 30C as well.
  Plates or liquid cultures containing Vmaxshould not be stored at 4C!
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Plates can be stored at room temperature for 2-3 days and the colonies should exhibit contact growth inhibition to keep the colonies separate.

For long-term storage Vmax can be stored in normal glycerol stocks (~15%-20% w/v final) at 80C.

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  • Wash the culture in fresh LB before adding glycerol, to avoid problems with reviving the culture later [1]


Growth

Vmax cells proliferate at a rapid rate, which makes overnight incubation/inoculation unnecessary if the temperature is high enough (37C)

At 37C:

  1. Liquid cultures that are inoculated from frozen stocks typically reach stationary phase within 5-6 hours. [1]
  2. Regular overnight cultures are ready within ~8-12 hours
  3. A substantial amount of colonies will form on a plate within 6 hours
  4. Growth rate is high, even without supplemented LB

If cells will be left to grow overnight (~12 hours or more), grow them at a lower temperature (risk of extended lag phase if cells are grown at 37C for longer periods:

  • room temperature
  • 32C
  • 30C


  Antibiotic Resistance:
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  Chloramphenicol = 12.5-25 ug/mL : 12.5-25 ug/mL
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 Plasmid Transformation:
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 Creation of Vibrio natriegens Electrocompetent Cells:

1. Grow a 10mL culture of V.nat cells in BHIv2 overnight.
2. Inoculate 100mL BHIv2 with 1mL of overnight culture.
3. Place in orbital shaker at 37C at 200 rpm until OD600=0.5.
4. Separate culture into two 50mL falcon tubes and place on ice for 15 min.
5. Pellet cells at 6500 rpm for 20 min at 4C.
6. Resuspend cells in 5 mL(each tube, 10 mL total) of electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7).
7. Fill tube to ~35mL of electroporation buffer and invert gently to mix.
8. Centrifuge cells at 6500 rpm for 15 min at 4C.
9. Decant supernatant with a pipette.
10. Wash the cells with electroporation buffer as described above a total of three times.
11. After the final wash, resuspend cells in electroporation buffer as to have OD600=16.
12. Aliquot cells at 100 uL each tube and store in -80C.

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  Note on spectinomycin selection: Recommended spec concentration is at least 250ug/mL. Yet, even at this high concentration we've observed a thin layer of growth or lawn on noDNA control plates incubated overnight, although these cells die off (a scrape of it is not culturable in regular LB after 24hrs) which would allow picking/visualizing colonies of the desired transformants.
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ROS
The Vibrio species in unable to detoxify reactive oxygen species at colder temperatures...




References
  1. https://www.biorxiv.org/content/biorxiv/early/2016/06/12/058487.full.pdf
 
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