We have several VNp plasmids available on addgene, and are adding more all the time.

If you are looking to clone your own cargo into our DNA plasmids, the best options are:

• pRSFDuet-1_VNp6-mNeongreen: VNp6 is the most versatile of the VNps, it produces optimal yield and is compatible with a wide range of cargoes. It functions at a wider range of temperatures than our other VNp sequences. This plasmid also contains an mNeonGreen tag, which allows you to see at a glance whether your protein of interest has been expressed and exported into the media, and a His-tag for purification. We recommend starting with this version.
• pRSFDuet-1_VNp15-mNeongreen: VNp15 is the shortest version of VNp. This plasmid also contains an mNeonGreen tag and a His-tag.
• pRSFDuet-1_VNp-His6: this plasmid contains VNp2, which is sometimes optimal for expressing more complex proteins (we recommend trying VNp6 before VNp2, as it produces higher yields for most of the proteins we have tested). This plasmid also contains a His-tag.
• pRSFDuet-1_VNp-mNeongreen: this plasmid is the same as pRSFDuet-1_VNp-His6, but also includes an mNeonGreen tag.

Yes, the protocol for culturing bacteria using the VNp system differs from normal bacterial cultures in several key ways. You can find our full protocol and video on JoVE.

The key factors to bear in mind are:

• Surface area:volume ratio. The VNp system requires a high level of oxygenation. For optimisation runs we advise 25ml of media in a 500ml flask. DO NOT use baffled flasks as this can disrupt the formation of external vesicles.
• Optical density. Induction of recombinant protein expression should be done during the late-log phase, when the optical density (OD₆₀₀) is between 0.8 and 1.0.
• Induction. The final IPTG concentration should be 20 µg/mL (84 µM) at MOST, as higher concentration can cause toxicity.
• Antibiotic. Most of our VNp plasmids use Kanamycin resistance.

The VNp system requires a high level of oxygenation. For optimisation runs we advise 25ml of media in a 500ml flask. DO NOT use baffled flasks as this can disrupt the formation of external vesicles.

Most of our plasmids use kanamycin or chloramphenicol resistance. When using our VNp system we recommend avoiding antibiotics which target the cell wall, such as ampicillin. 

For optimal yields, protein expression should be induced during the late-log phase, when the optical density (OD₆₀₀) is between 0.8 and 1.0. 

The final concentration of IPTG should be AT MOST 20µg/mL, more than this leads to cell lysis.

We recommend using a rich media such as TB.

The optimal temperature for all VNp sequences is 37℃. If lower temperatures are needed, we recommend using VNp6. 

Most proteins are exported out of the cell and into the media in vesicles. Some difficult to produce and larger proteins or protein complexes do not export and are instead incorporated into cytosolic membrane bound vesicles. 

All of our plasmids are available to academics and nonprofits on addgene. For commercial uses please get in touch using our contact form.

If you have another question please get in touch using our contact form.

Alternatively you can send an email to:

d.p.mulvihill@kent.ac.uk