pJE202 pVIB in liquid culture

[Josiah Zayner]

Has anyone tried and been successful to see pJE202 or pVIB glow in liquid culture in E. coli?

Thanks,
Josiah

[Josiah Zayner]

To be more specific, when I grow it on a plate I see it fine. But when I grow it in liquid culture alongside the plate I don't see anything. Since the liquid culture is more dense I would expect to see it better? I know in the original paper they added a shit ton of stuff to the culture. Wondering if any of that is actually required? If there is a specific strain of E. coli that works better or worse? If quorum sensing can help or inhibit it(should help?)?

[Koeng]

I've never been able to see it in liquid culture. I am going to try the "luxbrick" igem project plasmid to see if I can get better expression. It appears that their's is successful in liquid culture since it is induced with arabinose

http://2010.igem.org/Team:Cambridge/Bioluminescence/G28

[Josiah Zayner]

Cool. Let me know how it goes.

[Mega [Andreas Stuermer]]

It works in liquid culture. I'll send you the fotos. After few minutes it gets dim due to oxygen depletion, then you shake it and it glows again

[Mega [Andreas Stuermer]]

I just used LB Amp to grow it in. No substances added. They constitutively produce the autoinducer anyway

[Mega [Andreas Stuermer]]

Ah damn it, I think the pictures were on the computer that died due to CryptoWall.

If I haven't backed them up somewhere they're gone...

However, I used 5 mL and 10 mL in a 50 mL tube. Put it on the lab desk horizontally (to maximize surface oxygen exposure)

After 2 days at room temperature see the glowing.

No 37°C, no shake incubator.
Remember >30°C denatures bacterial luciferase

[Josiah Zayner]

What strain of bacteria?

On Sun, Jul 20, 2014 at 12:05 PM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

I just used LB Amp to grow it in. No substances added. They constitutively produce the autoinducer anyway

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[Josiah Zayner]

Yes, I know not to grow at 37C that's not the issue.

Why not a shaking incubator?

[Mega [Andreas Stuermer]]

Some kind of self-made competent E Coli. Probably dh5 alpha.
http://diyspartanbiotech.wordpress.com/2013/12/03/found-old-unposted-pics-of-pvib/
Here are pictures of them on a petri dish

Aaaah I could still have them on my camera!! Gotta look that up immediately.

On Sunday, July 20, 2014 8:45:51 AM UTC+2, Josiah Zayner wrote:

[Mega [Andreas Stuermer]]

http://diyspartanbiotech.files.wordpress.com/2013/12/petri-disk-alight6.jpg

On Sunday, July 20, 2014 8:45:51 AM UTC+2, Josiah Zayner wrote:

[Mega [Andreas Stuermer]]

I wanted to do it diy style. It probably (very very likely) works if you use one though.
I just had it lying around, 5 mL in a 50 mL tube, it made a thin layer horizontally

On Sunday, July 20, 2014 8:45:51 AM UTC+2, Josiah Zayner wrote:

[Mega [Andreas Stuermer]]

Hallelujah.
How about that.

On Sunday, July 20, 2014 8:45:51 AM UTC+2, Josiah Zayner wrote:

[Mega [Andreas Stuermer]]

It's long ago I tried this. Much LB -> much glow. It was visible nicely, but the camera didn't like it. Plus you had to open the cover before taking a photograph due to oxygen depletion.

On Sunday, July 20, 2014 8:45:51 AM UTC+2, Josiah Zayner wrote:

[Josiah Zayner]

Cool thanks.

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[Josiah Zayner]

So I tested a few different medias. I think the problem was that I was using SOC. I thought using a more defined, nutrient rich media would help but instead it does the opposite. You can't really see any glow. I also found out that BL21 cells glow much better than DH10B. 

So using pJE202 pVIB with LB Amp and BL21 works best for me so far. 

Going to try some other stuff before cloning into a overexpression plasmid.

[Andreas Stuermer]

What do you intend to do? Inserting into a T7 promoter plasmid may either give loooooots of glow, or kill the bugs due to metabolic burden.

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http://diyspartanbiotech.files.wordpress.com/2014/03/genetic-engineering-and-synbio-for-beginners-v1.pdf