ProtocolsWorkingWithVibrioNatriegens < Lab

---+ Working with <i>Vibrio natriegens</i> (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:
   * http://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00116
   * http://www.nature.com/articles/nmeth.3970
   * https://www.biorxiv.org/content/early/2016/06/12/058487

| SAD1302 | V. natriegens ATCC14048 |
| SAD1304 | V. natriegens ATCC14048 spontaneous Rif100 resistant colony |
| SAD1306 |V natriegens ATCC14048 spontaneous Rif100 resistant colony w/ pMMB-tfoX(Vc) (Amp100ug/mL or Carbenicillin 100ug/mL) (exhibits the highest transformation frequencies; Dalia et al., 2017) |
| SAD1495 | V natriegens ATCC14048 spontaneous Rif100 resistant colony w/ pMMB-tfoX(Vn) (Amp100ug/mL or Carbenicillin 100ug/mL) |
| 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: media type and temperature conditions

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*Media*

Vmax can grow in LB or BHI for liquid cultures, but it grows faster in liquid media that have been supplemented with salt. You can either use LBv2 or LB3 formulations.

LBv2 is LB-Miller supplemented with v2 salts (v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl<sub>2</sub>). You can make a 10x v2 salt solution to achieve this or (better) add these salts when making the media. (Yes: The NaCl is *in addition to* the normal amount in LB-Miller medium). This is the method used by SGI and Dalia labs.

LB3 is LB-Miller supplemented with 2% NaCl (bringing the total NaCl concentration to 3%). This is the method used by the Church lab.

Vmax also grows exceptionally well in BHIv2 (BHI supplemented with v2 salts) as recommended by SGI.

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*Temperature Conditions:*

Vmax grows rapidly at 37°C and grows well at 32°, and 30°C as well.

%RED% *Plates or liquid cultures containing Vmax should not be stored at 4°C!* %ENDCOLOR%
 <ul>
<li><a href="#C4"> ROS is behind this </a></li>
</ul>
%BR%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 –80°C.
<ul>
<li>Wash the culture in fresh LB *before* adding glycerol, to avoid problems with reviving the culture later <sup>[1] <sup></li>
</ul>

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*Growth*

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

At 37°C: 
<ol>
 <li>Liquid cultures that are inoculated from frozen stocks typically reach stationary phase within 5-6 hours. <sup>[1] <sup> 
 <li>Regular overnight cultures are ready within ~8-12 hours
 <li>A substantial amount of colonies will form on a plate within 6 hours 
<li>Growth rate is high, even without supplemented LB  

</ol>

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 37°C for longer periods:  
 <ul>
<li>room temperature</li>
<li>32°C</li>
<li>30°C</li>
</ul>

<hr>

*Antibiotic Resistance:*

Vmax has a low-level natural resistance to kanamycin but is more susceptible to other antibiotics. When selecting on Kan plates, Vmax with KanR will simply grow _faster_ than Vmax without KanR. Larger colonies=KanR. Other antibiotics select against Vmax but must have a dosage change compared to _E. coli_. (These dosages are from the literature. May need to do further controls.)

Liquid media Antibiotics : Solid Media Antibiotics:

Amp/Crb = 2-25 ug/mL : 2-50 ug/mL

Kan = 200 ug/mL : 200 ug/mL 

Tetracycline = 2.5 ug/mL : 2.5 ug/mL

Chloramphenicol = 12.5-25 ug/mL : 12.5-25 ug/mL

<hr>

<strong>Plasmid Transformation:</strong>


Creation of Vibrio natriegens Electrocompetent Cells:

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



 Transformation using Electroporation:

1. Add 2 uL of DNA to cells on ice.<br />2. Transfer cells to cold 0.1cm cuvette.<br />3. Electroporate at 700V<br />4. Immediately recover with 500 uL BHIv2<br />5. Recover for 1 hr at 37C in shaker incubator.<br />6. Plate 100 uL of reaction.<br />7. Grow overnight at 37C. 

<strong>Notes on Natural Transformation of Vibrio natriegens (see also -> [[http://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00116][MUGENT]]):</strong>
Gabo has successfully used [[https://pubs.acs.org/doi/suppl/10.1021/acssynbio.7b00116][Dalia's natural transformation protocol]] (supplement of MUGENT paper), *exactly* as described. For kanamycin selection, use 200ug/mL concentration, we find that lower concentrations (eg., the typical 50ug/mL) will result in high number of background/false positives due to V.natriegens low-level kanamycin resistance. In fact, we saw no escapes on noDNA control plates in LB-Kan (200ug/mL) - plated 50uL straight from transformation mix. In our hands, BHIv2 media didn't work even with Instant Ocean salt step, just LBv2 media worked (not important to add v2 salts to kan plates). Additionally, adding more DNA (e.g., 500ng/350uL transf.) may inhibit transformation given our yields were about 2-fold lower than using 50g tDNA in the 350uL transformation mix. Transformation frequency with 50ng GGpcr (Golden Gate assembled tdk-Kan with ~1kB homologies targeting dns gene that was further amplified by PCR) of Dalia’s SAD1306 tfox Vibrio (Vc), with ocean salts (4hr step) and 2hr outgrowth with added LBv2 was 1.77*10^-6 (average of 3 plates), so about 2 cells per million. We find that longer than 2hr overgrowth incubation (> 12hrs) can easily increase transformation frequencies ~10-fold, therefore this is highly recommended. We also find that increasing IPTG induction to 0.5mM (instead of 0.1mM) during the instant ocean salts step can also increase transformation 10-fold. 

The entry plasmid from the yeast toolkit (pYTK001) replicates inside V. natriegens, therefore, it is possible you can use this protocol for natural transformation to select for Golden Gate assembled plasmids and use this strain as platform to obtain high yields of a desired plasmid part. Yet, the ability of this plasmid to replicate inside Vibrio makes it problematic for [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAcinetobacterGoldenTransformation][Golden Transformation]] as used for ADP1 if genome modifications are desired; that is, if transformation doesn't generate a clear phenotype, you would have to PCR check multiple colonies to distinguish plasmid transformants from true genome transformants. Furthermore, our attempts to perform [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAcinetobacterGoldenTransformation][Golden Transformation]] on Vibrio failed to yield colonies with the targeted genome modifications, only kan-resistant pYTK001 plasmid escapes were observed.  While this indicated that the promoter in the kanR gene of the employed tdk-kan cassette works in Vibrio, we only recommend using PCR product amplified from a Golden Gate assembly (GGpcr) as described in the previous paragraph, linear DNA from a traditional overlap/extension PCR or purified plasmid.

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.

<br>
<h5 id="C4">ROS</h5>

At 4°C, Vibrio species reach a “viable but nonculturable” state because they are unable to detoxify the lethal reactive oxygen species that is present in the culture medium, especially in solid media. Many bacteria naturally produce highly reactive hydrogen peroxide as they oxidize flavoprotein with oxygen, and hydrogen peroxide damages cell parts. Some species can produce catalase to break down H2O2, and while Vmax does carry the catalase gene, colder temperatures reduce the gene's activity and expression. This can be prevented by supplementing the media with sodium pyruvate or catalase. Another way to circumvent this issue is by engineering Vmax with a plasmid that either (1) contains an E coli operon for the catalase gene, or (2), encodes for the E coli gene that produces catalase. 

<br>
<h5 id="C4">Natural DNase Production </h5>
Many vibrio species naturally produce DNase which is sent out to the periplasmic space, resulting in notably lower transformation efficiency. The dns and xds extracellular nucleases are responsible for the production of the extracellular DNase (Manning 1991). Of the two, dns seems to play a bigger role, and without it, transformation efficiency is significantly higher (Blokesch 2008) One may overcome this issue by transforming with highly concentrated samples (>550 ng of DNA). Another option is to engineer a dns gene knockout strain, as outlined in the Dalia 2017 paper. A third option is specific to when making competent cells: subject the cells to osmotic shock to remove the DNase from the extracellular environment, then wash in Mg2+ to stabilize the outer membrane, as described in the Kawagishi 1994 paper. 


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<hr>
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*References*
<ol>
 <li>https://www.biorxiv.org/content/biorxiv/early/2016/06/12/058487.full.pdf
</ol>
This topic: Lab > WebHome > ProtocolList > ProtocolsWorkingWithVibrioNatriegens
Topic revision: r17 - 2019-07-23 - NoorRadde