E. coli with pVIB not glowing, why?

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MC

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Feb 25, 2017, 2:01:04 AM2/25/17
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My failed pVIB E. coli experiment has been bugging me and I'm wondering if anyone has any ideas as to why. I got the pre-transformed kit from The Odin which was simple enough. My initial kit did not glow out of the box so Josiah was kind enough to send another one which I verified was glowing. I made additional LB plates and streaked them using the original glowing plate.

Easy enough right? Except none of my plates glowed. Zero. I tried different temps, giving them more time to grow, etc. There was also no visible contamination and the cultures appeared to be growing fine. What could be the cause of the lack of glow?

Nathan McCorkle

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Feb 25, 2017, 2:12:01 AM2/25/17
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Did you grow with ampicillin?
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MC

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Feb 25, 2017, 2:24:03 AM2/25/17
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Did you grow with ampicillin?

Yup, it came with amp media. Couldn't have been easier. 

Nathan McCorkle

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Feb 25, 2017, 2:40:05 AM2/25/17
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When I was reading about the strain, a lot of folks complained about
recombination being possible (at least for long term maintenance of
the plasmid, or for any kind of sequence-sensitive work, like storing
a DNA library prior to sequencing)... I was actually thinking of
getting that strain and trying to select for reversions back to the
parent plasmid, using tetracycline...
https://en.wikipedia.org/wiki/PBR322

If you still have some culture around, you might try picking up some
tetracycline from a pet store and making amp+tet plates and seeing if
anything will grow.

Hard to say what happened, as I'm not too familiar with the
bioluminescent pathway and conditions for optimal light production...
but that is what comes to mind from my recent reading on pVIB and
BL21... waiting on an order myself :)

On Fri, Feb 24, 2017 at 11:24 PM, MC <marky...@gmail.com> wrote:
>> Did you grow with ampicillin?
>
>
> Yup, it came with amp media. Couldn't have been easier.
>
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MC

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Feb 25, 2017, 12:56:19 PM2/25/17
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That's really interesting, I'm going to have to do some reading on recombination. Both my cultures were DH10B. I have some BL21 I'm checking if are still alive from the fridge. It'll be fun to transform BL21 and see if it's any different. Thanks for the insight!

Mega [Andreas Stuermer]

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Feb 26, 2017, 6:03:31 AM2/26/17
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I transformed it at university 4-5 years ago. Replated it on LB without Amp lots of times and it got less bright (plasmid loss), but even after 6 subcultures it was still possible to see with the naked eye. Then I left the Amp culture in the Uni fridge over the summer holidays, and after like 3 month put it on fresh LB Amp - glowed again.

So from my experience pVIB worked fine. I don't know which exact coli strain we had at Uni, they were commercial ones

Nathan McCorkle

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Feb 26, 2017, 12:35:16 PM2/26/17
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On Feb 25, 2017 9:56 AM, "MC" <marky...@gmail.com> wrote:
>
> That's really interesting, I'm going to have to do some reading on recombination. Both my cultures were DH10B.

Oh, ok, I guess the-odin changed how the sell pVIB... their product page showed this week that they ship pVIB in BL21.

Hard to say what could've happened from my end, then.

Sebastian

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Jul 30, 2017, 8:48:07 PM7/30/17
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Did you grow it at 37C or 30C? 37 deactivates the enzyme

MC

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Aug 1, 2017, 12:12:12 PM8/1/17
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Closer to 30C, room temperature while the weather was mild.

Otto Heringer

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Aug 3, 2017, 11:29:38 AM8/3/17
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Just giving a shot: any chance of antibiotic degradation for a longer time than you think? (like before even you get the plate)
Did you tested something without resistance growing in the plate?

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a a

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Sep 11, 2017, 8:49:59 AM9/11/17
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Just to be clear, pVIB requires a luciferin substrate, right?
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