I have cloned my gene in pET28(a) vector and transformed the recombinant plasmid into E. coli BL21 (DE3) and also in E. coli JM109 (DE3). I have checked the putativeness of the clones to be positive by restriction digestion.
Now, I am trying to check the over-expession of the gene by induction with IPTG. My gene codes for a large protein of around 86 kDa.
For small scale protein synthesis, I induced the transformed E. coli BL21 (DE3) and E. coli JM109 (DE3) with 0.1mM IPTG and incubated at 18 degree C and 200 rpm for 18 hours. I took 1ml of the induced culture, pelletted the cells by spinning it at 10,000 rpm for 1 minute, aspirated the supernatant and resuspended the pellet in 100ul 5X gel loading dye followed by heating it at 100 degree C for 3 minutes, stored it at 4 degree C till loading on 10% SDS-PAGE gel. On running the PAGE, and after staining and destaining the gel, there were no over-expression in the induced cells.
The same steps were repeated by inducing the fresh 50 ml with 0.1mM IPTG at 37 degree C for 6 hours. Same result was observed.
What should be the possible reasons for no over-expression? Is it because of issues with in-frame cloning, and therefore no translation? How to check whether the cloning is in-frame or not? kindly suggest..
Just sequence your miniprep all the way through (from that ATG startsite to the stopcodon, and ideally ±50-100 bases up-downstream of that as well). Check whether your gene is in-frame or not. One should always sequence their clones before using them for experiments, it's the only way to know whether your gene is exactly as you think it is... It's really important to do (essential I should add, especially when using PCR to amplify the cDNA of interest), and it could save you loads of time in downstream experiments (or even more important, it could save you from publishing something wrong. What if you make a cool discovery, publish it, and have someone lateron find out it's actually a mutant that caused this phenotype? That's a big scientific no-no...).
Hence, just sequence it. You'll know within a few days for a couple of $$ only...
Cheers
Just sequence your miniprep all the way through (from that ATG startsite to the stopcodon, and ideally ±50-100 bases up-downstream of that as well). Check whether your gene is in-frame or not. One should always sequence their clones before using them for experiments, it's the only way to know whether your gene is exactly as you think it is... It's really important to do (essential I should add, especially when using PCR to amplify the cDNA of interest), and it could save you loads of time in downstream experiments (or even more important, it could save you from publishing something wrong. What if you make a cool discovery, publish it, and have someone lateron find out it's actually a mutant that caused this phenotype? That's a big scientific no-no...).
Hence, just sequence it. You'll know within a few days for a couple of $$ only...
Cheers
i think some things are wrong in your work!your IPTG concentration is very low!you should induce your culture at least with 1mMIPTG!after that you should have negative control for your expriment! empty vector that you induce is negative control,(empty pet28)-and another thing,after harvesting your culture you should suspend pellet with lysis buffer.it offer condition like inner cell for your bacteria.i offer induce culture with different concentrate of IPTG.good luck
As Robert Jan wrote, simply sequence it.
As Parastou wrote, your IPTG concentration is very low! Usually one uses between 1-10 mM.
Another thing, do not expect to see some huge band on Comassie-stained gel. Better would be Western or activity measurement.
Agree with a comments. Your bacteria may form inclusion bodies. An experienced eye can see these. Special detergents are required to lyse these.
I think you should check your pellet. The probability of getting inclusion bodies is very high. Check out the probability of forming inclusion bodies in silica:
http://mbs.cbrc.jp/ESPRESSO/Submission.php
Just a few remarks.
1. Robert is very right, you always sequence completely a PCR product. The frame could be one problem, but point mutations introduced by the Taq are another possibility, even using a proof-reading.
2. Check that in your cloning strategy the Shine-Dalgarno sequence was left in the right place, 8 nt before the ATG.
3. Parastou is also right, you can induce with 1 mM IPTG or higher. You will need controls, as he says, at least a non-transformed clone and a non-induced clone in parallel. Also, I believe it works better growing the culture at RT for 20 hrs without any induction, just to have the bacteria at density 0.6-0.8 and then induce at 37 °C for 2, 4, 6, 8 hrs and O/N (expression time course).
4. I don't think you are at the stage of checking inclusion bodies or pellets, your lysis condition in loading buffer is fine, and solubilizes everything, I don't think you have any pellet. You may improve it a bit by denaturing for 10 min, vortexing every 2 min (this will shear the DNA and protein bands on your gel will be more defined). Indeed you didn't obtain expression yet. The DE3 cells are delicate, they should always be grown at RT (also to prepare the competent ones) because they tend to mutagenize the T7 polymerase, since they don't need it (Vethanayagam & Flower, Microbial Cell factories, 2005, 4:3). Only for induction you can grow them at 37°C.
Good luck!
- Badges
- Science topic
- Similar topics
- Gels