Difference: ProtocolsWorkingWithVibrioNatriegens (14 vs. 15)

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Working with Vibrio natriegens (Vmax)

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The Vibrio species in unable to detoxify reactive oxygen species at colder temperatures...
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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. Colder temperatures reduce the activity and expression of the catalase gene. 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.


Natural DNase Production
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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