Problem on growing up bugs - (Apr/23/2011 )

Hey guys.

I was able to grow good transformants in my LB plate, then I picked one colony, grow it in 5ml LB media, works perfectly fine. I made a glycerol stock from that.

Then I made up an overnight culture using the glycerol stock. The overnight culture grows well. I inoculated this to 1L LB media. Unfortunately, it didn't grow. Waited for 5 hours, but the media stuck at OD600 = ~0.1.

This happened to me three times already. I know it works before, as I managed to do a grow-up with this construct. I don't know what went wrong.

I recently managed to grow-up bugs with a different construct very well. So, I don't really get what's wrong right now.

Thanks for any inputs!

Sounds like a bad batch of medium or the wrong antibiotic.

How much of your overnight culture did you add to your 1L culture?

phage434 on Sun Apr 24 00:18:40 2011 said:

Don't think so. It happened to me on this specific construct (different transformants) three times. Antibiotic should be right as I use the same strain of E.coli as the successful grow-ups.

leelee on Sun Apr 24 03:22:55 2011 said:

About 5ml of overnight culture. The grow-up I've done before this is 10ml, result still the same.


I started at 12:30PM yesterday and check the OD at 4:30pm and 5:30pm. Not much difference. I went to lab this morning at 4:30am... checked and the bugs grew to 1.0. I induced it anyway and see if it will work.

Anyone seen this happening before? Sounds to me the bugs are struggling to grow (but doesn't make sense if it grows so fast on small cultures).

if it's not the media (e.g. the pH is adjusted to 7? ...have you checked that?) than it must be an issue of plasmid stability.

What cells are using? It looks like your cells have lost the plasmid during overnight culture and you are just transfering plasmid-free cells to your new culture resulting in slow/no growth!

What antibiotic do you use?

Regards,
p

pDNA on Sun Apr 24 07:55:09 2011 said:

I usually don't check the pH of my LB media... maybe I should?

I'm using BL21 pLysS. And I use ampicillin as antibiotic.

Any recommendation what should I do? How to stabilised plasmid over the course of overnight culture? It looks like my protein expression is leaky, my friend asked me to add glucose... could it be that my protein is responsible for slow growth?

I got a construct which have two plasmids that will produce this protein and another protein. I'll try to work on that if it is construct specific, protein specific or merely... batch (again?!).

adding glucose to your medium is for sure a good idea for the overnight culture ...to prevent a strong selection towards plasmid-free cells!

You can find information on supplementing your LB medium with 1% of glucose here ...see this link!

What pET vector do you use for expression?

Ampicilling is also a bit of problem since it gets secreted in the culture medium and therefore makes it possible that plasmid free cells are able to survive since the Ampicillin is totally degraded with time. So it is also an option to use carbicillin since it gets degraded slower than ampicillin.

Good luck!

Regards,
p

pDNA on Mon Apr 25 08:03:56 2011 said:

The construct is using pET14b vector.

Thanks for the help. Will do the glucose route and see how it goes!

uhhh ...pET14b is a pain when leaky expression of your gene is really detrimental to the cell because 14b tends to high levels of leaky expression!
There is no lacO repressor downstream of the T7 promoter therefore control is only based on availability of the T7 RNAP. Vectors like pET30a would be a much better choice since it has an lacI on the backbone and an additional lacO binding site downstream of the T7 promoter.

Maybe you can improve the situation a bit by adding glucose.

If you are really interested in plasmid stability you can try to determine the fraction of plasmid carrying cells by plating dilutions of your broth on LB and LB-Amp plates and calculate the fraction.

Good luck!

Regards,
p

Xerophytes on Mon Apr 25 10:07:13 2011 said: