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pET14b vector with insert transformed in BL21(DE3) RIPL cells


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#1 Fluffy

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Posted 05 November 2012 - 07:00 AM

Hello Everyone,

Hope you are all doing fine.

I will be starting protein expression work soon. I am currently setting up an experimental design to get started. I came across an obstacle and would love to hear your inputs about it.
I will be first transforming my pET14b plasmid (has my target insert) in BL21(DE3) RIPL cells. Now, my vector has an Ampicillin resistance marker. The cells also have their own vector that possesses an antibiotic resistance marker called Chloramphenicol. When plating should my LB-agar plates contain Ampicillin or Chloramphenicol? I personally thought it should be Ampicillin (or carbenicillin-can be used instead of Amp) so that way only the cells with my pET vector survive.

Is that correct?

Thank you :)

#2 John Forsberg

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Posted 05 November 2012 - 02:17 PM

If your cells have a plasmid with a Chloramphenicol resistance marker in it, you'll want to put your transformations on plates with both Ampicillin (I definitely prefer Carbenicillin, as I get fewer satellite colonies) and Chloramphenicol. The RIPL plasmid in your BL21 cells is there to help with expressing proteins with codons that are rare in E. coli, so if you're expressing a eukaryotic protein, you'll definitely want to select for cells that keep that plasmid too.

We usually use 30 µg/ml Chloramphenicol and 100 µg/ml Carbenicillin for LB plates.

#3 ascacioc

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Posted 05 November 2012 - 02:43 PM

In addition: also the preculture should contain both antibiotics. However for the main expression I would use only ampicillin because 2 antibiotics at once during expression make to much stress on the cells.

Andreea

#4 Fluffy

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Posted 06 November 2012 - 07:33 AM

John: Thanks for the reply. I will use Carbenicillin instead of Ampicillin. Thanks for providing the concentrations you have tried in your lab.

Ascacioc: hmm..just to be clear that I understand what you said..you prefer that I only use Ampicillin (or carbenicillin) for both my LB medium and LB-Agar plates during my protein expression work. You don't think it is a good idea to use both antibiotics as in Amp and Chloramphenicol?
Correct?
And thanks to you dear :)

#5 ascacioc

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Posted 06 November 2012 - 03:54 PM

No-no: I said: put both on agar plate and the overnight preculture, but when you make your big culture, put only ampicilin: aka the 1-6 L of LB which you induce with IPTG should contain only one antibiotic because 2 of them will be too stressful.

Andreea

Edited by ascacioc, 06 November 2012 - 06:24 PM.


#6 Fluffy

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Posted 06 November 2012 - 06:11 PM

Thanks for your replies.

I am sorry Andreea for my misunderstanding :)
I want you all to be patient with me. I am hesitant to start this. Since I am a fresh starter, I will need your feedback on this.
I read websites and papers (including the papers you sent me Andreea-they were really helpful :)) and they all have slightly different ways of starting their work.
I understand that I have to first transform my pET14b vector in BL21-(DE3)RIPL cells (Andreea when plating I will put both antibiotics correct?), then I will take a single or two colonies and inoculate with LB medium (Andreea this will have both antibiotics as well correct?) at 37C overnight. Now I am not sure what is the best volume of LB medium to use at this point? I saw different volumes being used by different websites and papers..After that I add that culture to LB medium (Andreea this time I only put Amp antibiotic correct?)..but samething here I am not sure for a starter like me what would be the best volume of LB medium to add the culture to?...
Note that I will be dividing it into an induced and uninduced control

Next would be growing it to midlog phase (OD600 0.5-1.0)-what is the best way to measure it? I have a microplate reader and is it best to measure it at different time intervals throughout the 2-3hours of growth..and should I dilute my aliquots to get accurate readings?..

For now these are my concerns..Sorry for my really long inquiries ..

Thanks all :):)

#7 ascacioc

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Posted 06 November 2012 - 06:23 PM

You got it all right with the culture, as I explained.

Do not worry about how different people do the same thing in many ways. Choose whatever works for you, and stick to it.
I do 50-100 mL of preculture. Depends on what flask I have available :) I usually make 1/10 of the volume of the flask i.e. for 500 mL flask I do 50 mL preculture.
Usually, I measure OD600 in a cuvette not with the microplate reader (too complicated and takes too long); I just take 1 mL culture, put in a cuvette and stick it in the spectrophotomer. Moreover, measure the culture every 1 hour or even 30 min. Keep in mind that the duplication time of BL21 derived bacteria is 20 min (around, depends on what protein you are expressing) so if you are measuring every 2 hours you get 0.1 and next point is >6.

I usually do 6 L of main induced culture but you need the flasks and shaker capacity for this kind of expression and BTW, I do X-ray crystallography so I need tons of my protein :P Another important aspect: I use 1/5 of the volume of the flask for expression i.e. in a 5 L flask, I put 1 L of media. This is needed for good aeration.

And another thing: before I waste my time with 6 L of culture, I make an expression test in 100-200 mL of culture from which I take several time points 50 mL and harvest the cells, resuspend in 5 mL, brake them with a sonicator, and run a gel with pellet, soluble protein (supernatant of the disrupted cells) and I also incubate for 1-2 hours on rotation 2 mL of supernatant = soluble protein solution with 20 uL of the affinity beads i.e. Ni-sepharose for His-tagged proteins to get a sample of enriched protein (for the SDS-PAGE, I wash these beads 3 times at low centrifugation speed and then boil them in loading buffer)

Andreea

#8 Fluffy

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Posted 08 November 2012 - 08:49 AM

Hello again :)..

Awesome Andreea..I appreciate your time with this:)
I sat down and made a protocol of what I will do next week willingly. If anyone thinks wants to add or make changes to my protocol please let me know, all your comments would help.

1. Plate my transformations on LB-Agar plates containing Carbenicillin and Chloramphenicol antibiotics.
2. Inoculate a single colony with 3mL of LB media containing Carbenicillin and Chloramphenicol (it will also have 0.5-1% glucose to reduce basal expression levels, pH will be around 8.2 to overcome low pH problems) in a culture tube.
3. Incubate the culture prepared in step 2 at 37C at 250rpm overnight.
4. Next day, add the 3ml culture to a 100ml of LB media containing carbenicillin antibiotic only.
5. Shake the culture prepared at step 4 at 37C for 2-3hours until OD600 is 0.5-1.0. Then cool the culture to 18C.
6. Before induction, divide the culture from step 5 into two 50ml cultures. One of them will serve as the uninduced control. The other 50ml will be induced by adding IPTG to a final concentration of 0.4mM. Both cultures will then be incubated with vigorous shaking at 18C for 16-20hr.

I will also keep in mind to use proper culture tubes and flasks and try to keep volume 10%-20% of tube or flask volume for better aeration.

What do you think :)

Thanks and have a nice day!

#9 ascacioc

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Posted 08 November 2012 - 06:55 PM

Step 4: add only 1 mL in 100 mL: usually it is 1 in 100 dilution. The rest is beyond perfect. I know that you said that you read the materials from me, but now you even proved it :P

Andreea

#10 Fluffy

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Posted 09 November 2012 - 04:44 PM

Oh great thanks for pointing that out! I did not know about that :). I am glad that it is a good protocol, I cross my fingers that it would actually work in the lab. I will let you know how things turn out.
You have been great help, thanks alot Andreea! Best of luck to you in everything :)
Cheers :)

#11 Fluffy

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Posted 10 November 2012 - 08:02 PM

Hi again,

A question came to my mind and thought I should ask about it to know the answer ahead of time :)

After step 6. of my protocol, the culture I get (that is the one I incubated with vigorous shaking at 18C for 16-20hr) can it be stored for a couple of days? I want to use it to get total cell protein fraction, the soluble and insoluble cytoplasmic forms and run on SDS-gel, but I do not want to do it the next day after the 16-20hr incubation. Is there a way to store that culture for a couple of days and continue my work later with it?

Thanks everyone :)

#12 ascacioc

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Posted 11 November 2012 - 04:03 AM

I usually spin it down and discard the media and then freeze the pellets at -20 until I have time to analyze them.

Andreea

#13 Fluffy

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Posted 12 November 2012 - 05:06 PM

Perfect idea Andreea. Thanks :)

For analysis, I will be doing SDS-PAGE with coomassie blue staining first, then Western Blotting. I will focus on SDS-PAGE now. I noticed different protocols online. I do not know which is best. I think it also depends on the protein size. My protein sizes are 16.52KDA and 21.38KDA. Now, what do you normally prepare in terms of the sample loading buffer concentration, running buffer, stacking gel ,and running gel? Oh and do you normally prepare ahead of time and store at a certain temperature, and use them when needed.

Sorry all this is new to me, so please bear with me.

#14 ascacioc

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Posted 12 November 2012 - 05:26 PM

In your case, I would use the 15% SDS-PAGE gels as described in Sambrook and Maniatis molecular cloning: http://www.amazon.de...l/dp/0879695773

Loading buffer I prepare and aliquot in 1 mL Eppis at -20; the rest I keep at RT. I never prepare gels ahead of time and then store them. People do that and keep cast gels in the fridge but over time the H+ gradient between the stacking and the resolving (which are usually at different pHs) will diffuse and the resolution of the gels will be sub-optimal.

Andreea

#15 Fluffy

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Posted 12 November 2012 - 05:43 PM

Ok hmm I can't really access the manual..hehe..do I have to buy it inorder for me to see its contents? Is there another way possibly.




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