Expression in E.coli
Posted 24 June 2010 - 02:11 PM
Posted 24 June 2010 - 06:06 PM
Posted 24 June 2010 - 09:10 PM
Ok @ram, dumb question, but do each of the non-expressing clones have a start codon? I presume you've put the sequences into your program of choice and asked it to show you the expressed sequence.
Posted 24 June 2010 - 10:12 PM
@ HomeBrew: Because I also had read somewhere that pLysS has reduced levels of uninduced expression, so is good for expressing toxic proteins. I don't know whether my protein is toxic to E coli or not; but having a thought that it would be better than just BL21(DE3), I use pLysS.
@ Swanny: Yes! There are start codons in all of the plasmids. And the molecular weights that I have mentioned are from in silico translation in MEGA.
Edited by ram, 24 June 2010 - 10:12 PM.
Posted 25 June 2010 - 03:17 AM
Yes, I knew that -- my questions (albeit crudely constructed) were actually on two unrelated topics. Let me try again...
What you have said about pLysS is true -- the reason I asked you about it is because its presence suggested to me that you suspected that your gene(s) were toxic to E. coli. So now my question is "do the cultures of the six clones that won't express grow normally (compared to the one clone that works) during your IPTG induction period, when the effects of pLysS are negated?" More directly, since you suspected at one point that one or more of your genes might be toxic to E. coli, how have you now concluded they aren't? Are BL21(DE3) cells without pLysS viable when transformed with your plasmids?
The second part of my original post was directed at seeing whether what was stopping your over-expression was an abundance of codons in your coding sequences that are rarely used in E. coli, to see whether one of the specialized E. coli strains containing pRARE (the Rosetta or Rosetta 2 strains, as swammy alludes to) might help overcome your expression problems.
Do the six clones that won't express contain a lot of codons that are rare in E. coli?
Posted 27 June 2010 - 03:48 AM
Plasmid getting expressed:
arg 5 (AGG:2, AGA:2, CGA:1)
Leu 3 (CTA)
Ile 5 (ATA)
Pro 1 (CCC)
NOT getting expressed
Arg 9 to 10 (AGG:4 to 5, AGA:4, CGA:0 to 1)
Leu 2 to 5 (CTA)
Ile 2 to 6 (ATA)
Pro 2 to 4 (CCC)
Is this difference significant?
Posted 27 June 2010 - 05:58 AM
Yes, but TOP10 cells don't have the T7 RNA polymerase necessary to express the gene in your pEXP5 CT TOPO clone, so the ability of these plasmids to be maintained in this strain gives no information about their toxicity. It is true that a toxic gene cloned in a BL21(DE3) pLysS background will be stably maintained because the T7 lysozyme encoded by pLysS suppresses the chromosomally-encoded T7 RNA polymerase, but as soon as you add IPTG this suppression effect is overcome, and the cloned gene is expressed. If this gene is toxic when expressed, the cells containing it will die under IPTG induction, and your ability to get over-expression of this protein from your IPTG-induced culture is nullified.
I suggest you try and determine definitively whether your cloned genes are toxic to E. coli when expressed by transforming them into a BL21(DE3) strain without pLysS.
I don't know -- I'm uncertain how to decipher the information you posted. What does this site have to say? My understanding is that it's not only the absolute number of rare E. coli codons used by the foreign gene, but their placement in the coding sequence as well. For example, you may be successful at expressing a gene containing four rare E. coli codons scattered throughout the gene, but be unsuccessful at expressing a gene with four rare E. coli codons in close proximity to one another.
Posted 27 June 2010 - 03:39 PM
Have you done a timecourse of the expression? That is, does induction lead to death of the cells, or does the OD plateau?
Posted 27 June 2010 - 05:20 PM
Posted 28 June 2010 - 12:35 AM
No, I did not see what happens to the growth after induction. May be I will observe it in the next experiment.
@ HomeBrew. I would have definitely liked to check the expression in BL21(DE3). But I don't have the cells right now I will see if I can get those from someone.
Posted 01 July 2010 - 09:28 PM
Posted 27 July 2010 - 07:13 AM
Edited by ram, 27 July 2010 - 07:19 AM.
Posted 11 September 2010 - 09:15 AM
Posted 11 September 2010 - 04:24 PM
I didn't get your question
Anyways...I did't do anything with the insoluble fraction of any of the 5 remaining clones. I was just trying get the protein in the soluble form. Are you trying to ask how can the protein be solubalized?
As mentioned in my previous post, out of several changes made to get the soluble protein, those done using LB media having sorbitol and betaine succeeded to some extent and right now I am proceeding with those since I get at least some protein out of it
Edited by ram, 11 September 2010 - 04:27 PM.
Posted 12 September 2010 - 04:01 AM