I have a fluorescent protein in a pBAD vector and I want to express and purify it.
At first, I cultured a colony in 100 ml LB medium at 37 °C for over night. Then, I inoculated 25 ml from over night culture to 1.5 L 2YT medium. After 3 hours, the OD600 of the culture was around 0.6 at 37 °C. Later, for the expression of protein, I added 0.02% arabinose and incubated at 18 °C for over night.
So, after incubation, I could not express my protein.
Dose any one know how can i express my gene in pBAD vector?
Naghmeh -
There are several issues here that should be addressed.
Good luck!
Naghmeh -
There are several issues here that should be addressed.
Good luck!
Naghmeh,
Dante has covered all obvious issues. I am guessing that you are probably not using D-arabinose for induction. Usually GFP expression is quite robust so I am guessing that you may be using a plasmid that was designed to test putative ribosomal binding sites. In other words, the plasmid may lack a Shine-Dalgarno sequence. Check the sequence of the plasmid. If missing, either add one or simply acquire the correct version of plasmid.
For expression of certain genes that tend to express poorly, I have on occasions increased the concentration of Arabinose to 0.05-0.1%. If all else checks out, try increasing concentration of L-Arabinose.
Best...
Dear Dante and Tausif,
Thanks for your comments.
I got the plasmid from addgene (mRuby2-pBAD, Plasmid #54771) from Michaeal Davidson Lab (http://www.addgene.org/54771/).
I am not sure about the presence of Shine-Dalgarno sequence but I used L-Arabinose.
So, I will try different concentration of L-Arabinose.
Thanks,
Naghmeh
Hi there,
I've checked plasmid sequence from the site : there is a Shine Dalgarno sequence (AGGAG) 8 nucleotides upstream translation initiator ATG.
Hi All,
I expressed my protein with higher concentration of L-Arabinose.
Best...
Hello Naghmeh,
May I know what concentration was using for the induction of protein, temp and time of incubation. And what type of media was using for growth of bacteria either minimal or LB media.
I have a related question, the araB gene is the second gene in the metabolism of arabinose into the central carbon metabolism. For expression with pBAD vectors, does it needs a modified E. coli without araB so that it cannot use arabinose as carbon source and instead of being consumed is used for expression? Or if it is not required why?
Hello Luis Peña,
It would largely depend on the purpose of and the media used in the experiment. If the media has a high glucose supplementation or is a rich media like LB, the cells would preferentially go for glucose as a carbon source. Hence under such conditions you really do not need to worry about arabinose utilization. But then again depending on the purpose of the experiment, the media you can use would chance since glucose is also an inhibitor of arabinose induction. If you only intend to express your gene and then purify the product, LB and similar media should be just fine (same principle as IPTG induction). But in case,@ for what ever reason your media lacks glucose, I would suggest you either search for another carbon source preferred over arabinose for your AraB+ cells or use AraB- cells with maybe glycerol as the carbon source.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression