ProtocolsWorkingWithVibrioNatriegens < Lab

-- Main.JaredEller - 14 Dec 2017

Vibrio natriegens has the fastest doubling time of any known organism and has the potential to shorten experimental timelines and increase bioproduction levels with realtively easy to use protocols. We received several V. nat. cultures from the Dalia Lab at Indiana University. These cells have been stored in glycerol stocks and categorized as follows:

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)

Most of these methods were adapted from Dalia Lab or Gibson Lab methods:

http://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00116

http://www.nature.com/articles/nmeth.3970

Vibrio natriegens is meant to largely function using E. coli laboratory methods but there are a few factors that mark important differences between the two.

*Media Conditions:*

V. nat. cells can grow in LB or BHI for liquid cultures, but grow faster in liquid media that have been supplemented with v2 salts (v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2). I have had the most success growing V.nat strains in BHIv2, especially during reproliferation. V. nat will grow on LB agar plates with no special changes compared to E. coli. 

*Temperature Conditions:*

V.nat cells grow rapidly at 37C and grow well at 30C also. Plates or liquid cultures containing V.nat should not be stored at 4C, as this will prevent reproliferation from these sources. 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 V.nat can be stored in glycerol stocks (~15%-25%) at -80C.

*Antibiotic Resistance:*

V.nat has a low-level natural resistance to kanamycin but is more susceptible to other antibiotics. When selecting on kan plates, V.nat with KanR will simply grow _faster_ than V.nat without KanR. Larger colonies=KanR. Other antibiotics select against V.nat 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

<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.
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Topic revision: r11 - 2018-10-08 - GabrielSuarez