where is the problem, in induction of cloned genes or in cloning?

In Induction of cloned gene-the sequence has been sequenced and found to be i correct orientation and with histidine tag too -I am not getting any difference between uninduced and induced pattern

Hello Gautam! I work with BL21DE3 as well and don´t have problems before induction, cause T7-dependent promotors doesn´t seemed to be that leaky. So your problem is the expression before adding IPTG or the absence of expression at all? In this case try autoinduction medium which contains glucose AND alternative sugars such as lactose and glycerol. Glucose should repress expression until its metabolized (and the culture has high OD) and expression should start during growth on lactose. This may favour production of toxic prodeins, but I have to admit,that I don´t have many experiences with that method. If the latter is the fact, you can also try other strains such as C41 or C43.I had some problems with membrane-proteins in BL, too, but changing the strain solved the problem. And yes, higher efficency of competent cells may help too. 

Anja,

Yes I can see no difference in profiles between uninduced and induced and its as if addition of IPTG did not make any difference at all.I agree that leaky expression in pET system should not be such a big issue but I ve seen this pattern throughout and tried out catabolite repression experiments with addition of glucose and now I am changing the media to Terrific and maybe M9 to ""reduce"" background.The 2 suggestions you have I will explore -Is autoinduction medium available from NEB?Are you suggesting use of lactose and glycerol after adding IPTG.Could you explain that a bit?

Thanks 

Hello Gautam, I´m not sure if you can buy AIM from NEB but you´ll find some protocolls here: http://www.formedium.com/uk/EscherichiaColi/EscherichiaColi_6.htm

The use of IPTG is not necessary, cause lactose will induce the expression. just add the carbon-sources and inoculate your culture. After glucose is used up the cells will start growing on lactose and induce theirself - that´s way its called auto induction

If you still have problems try diffenrent temperatures after you add IPTG. We´ve got good results with 4-5h at 28°C or 16° over night.

Have you checked your induction with westernBlot as well?

Ok will follow up -No ive not done the Western blot-I am waiting for the protein to show up so that i can confirm its identity by binding to nickel NTA column as it has a his tag

Hello Gautam, how did you know that their is no difference between the induced and uninduced? Guess through OD reading and SDS-PAGE? Your reply.

yes through SDS profiles-there is no difference.I also checked the ODS .At times there is a difference where the induced shows much less than uninduced but still there is no difference between the 2 samples on the gel .I assume there is leaky expression or loss of plasmid and hve been trying with addition of glucose to solve the issue -no success so far 

Firstly, your OD result is not out of place (its correct) but since you used the right antibiotics and there is growth at least indicate that the plasmid is there, you could verify with controls anyway.

Secondly, what media are you using? - LB? Confirm that your plasmid is intact and that the protein of your interest is expressed through the band weight because whether induction or no induction once you run SDS-PAGE, you will always see bands especially with E. coli species in general. Also, you could run soluble and insoluble fractions of your growth broth before and at intervals after induction. Maybe you are having insoluble expression. You could run a control with empty BL 21DE3 (ie without plasmid) and BL21DE3 containing your empty plasmid(ie plasmid without your gene of interest).

More technically, what type of plasmid and promoter are you using? Be sure that you are not using a constitutive promoter instead of an inducible one OR that your medium (if LB) does not contain an inducer to your promoter. Similarly confirm in the sequence that the right inducible promoter is in place if thats what you are using.

More so, confirm that your medium (fermentation broth) is intact, that is without "mysterious" contamination of an inducer before or during use. Alternatively, use BL21DE3 pLysS it is more stringent against leaky expression.

Thanks a ton- I too was contemplating these controls for my experiment-maybe was not aware of the blank BL21DE3 -will keep you posted and yes must try   sonicating and running soluble and insoluble for larger volumes -i do it for 1.5ml cultures maybe 20 ml is a  better idea(1.5 ml out of 20-which I spin down and sonicate-maybe better to do 20 ml -sonicate and run supe and pellet

Further to yr queries (Valentine's) I am using the pET21A system in BL21DE3  host so it is IPTG inducible promoter-as the basal expression is high I tried using glucose but could not suppress the same .

How do I check plasmids being intact?Would BL21DE3 give  a good  yield of DNA ?

I guess as regards mysterious contamination are you asking me to clean up the apparatus in use to ensure no cross contamination of inducer in this case IPTG??Are you also referring to yeast extract in LB medium having an inducer in the form of galactose which contributes to high basal expression.So I m trying to use other medium like TB ,TB + glucose which have known to suppress basal expression.This is the report which highlights M9 and TB PLUS glucose as suppressing basal expression of GFP in pET28 A 

http://www.biotechniques.com/multimedia/archive/00010/00296st03_10412a.pdf

As regards controls I am starting an experiment today with PET 21A transformed in BL21DE3 as a control -I wonder why we need BL21DE3 as it will not grow on ampicillin -so should I grow the same without ampicillin to check background expression??

Guatam Its pointing towards a clear case of leaky expression; use of a more stringent host and promoter is advisable. But explore the control measures to be sure before going into this. Like I said earlier have you confirmed that your protein is been expressed either as soluble or insoluble? or is not even expressed?

You can use miniprep plasmid isolation, restriction digest and agarose gel electrophoresis to confirm that your plasmid is intact. Not necessarily from yeast extract but any form of inducer contamination should be checked and corrected.

Vakentine-no I cannot yet say whether its expressed -except once at 25 ml scale and at OD1 -when repeated it did not express it-since then ive tried these

1.37 degress 3 hrs 0.6,0.8,1.0

2.30 degrees 6 hrs at 0.6,0.8 ,1.0

3.25 DEGREES 0/N at 0.6 and 1.0 OD

4.Later I frehsly trransformed the DNA and again didnt see any difference

5.Then I did addition of glucose to suppress basal expression but to no avail

In short this is the whole story -will keep you posted -thanks for yr  help-ive also seen other reports of leaky expression with PET plasmids and switched to another clone to check if it was a DNA specific problem-my guide is ordering for plySs  which should help out but i hope to master it before then

SOmeone has also suggested autoinduction and varying glucose amounts 

• Use BL21-pLysS bearing strains in T7-based systems
• Use strains that are better for the expression of toxic proteins (C41 or C43