4 replies to this topic

[Luria Bertani]

Hi there

I have some genes that have been synthesised in pET-9a however the expression in these has not worked out well at all and they seem quite toxic (low or no expression on SDS-PAGE gel!). Even making plasmids from these things seems to be troublesome with low yield!

So I'm thinking of changing the vector to something else.
The other proteins we have are in a different vector pET 47b (+) and seem to be well behaved.

I've seen the pET manual and there seems to be heaps and heaps of different vectors which do similar things. We don't need anything fancy like tags or whatnot. Is there a relationship between vector and expression?


I am looking around at different synthetic gene suppliers; We usually use GeneArt but I have looked also at "DNA2.0"
Their website has some claims that I'd like to ask around about:

"Inserts are flanked by transcriptional terminators to allow cloning of inserts encoding proteins that are toxic to E. coli.  DNA2.0 can even guarantee that your gene will express in E. coli."

Exactly how does this work?

"Inserts are cloned nondirectionally to minimize potential toxicity of inserts."

What does this "directionality" mean?

"We also have vectors with low-copy origins for cloning genes that are problematic despite the transcriptional terminators."

I strongly suspect that my proteins are toxic. What is a low copy origin and why does it matter?

Finally some of their vectors have T5 promoters. I know T7 promoters are IPTG inducible, but T5? What's the difference there and can it be used interchangeably with other cell lines such as BL21 (DE3)?

Sorry if this might sound basic to some!!

Thanks in advance for your help!

Edited by Luria Bertani, 19 May 2011 - 01:41 AM.

[pDNA]

first of all you should switch to a vector that allows tight repression of your gene of interest like pET30a ...important thing is that downstream of your T7 promoter there is a lac operator binding site and a lacI on your plasmid backbone ...this will keep your promoter silent when no IPTG is present! Also use 1% glucose in the medium to lower basal expression! Detailed information can be found here! If your protein is really toxic this is of utmost importance!!!

"Inserts are flanked by transcriptional terminators to allow cloning of inserts encoding proteins that are toxic to E. coli.  DNA2.0 can even guarantee that your gene will express in E. coli."
--> this is to prevent read-through transcription of other promoters from your plasmid backbone e.g. antibiotic resistance gene, origin of replication ...both having promoters on its own ...transcriptional termination is never 100% no matter what terminator you have

"Inserts are cloned nondirectionally to minimize potential toxicity of inserts."
--> sometimes inserts are toxic in a certain directionality ...non-directional cloning selects for the direct that is less toxic!

"We also have vectors with low-copy origins for cloning genes that are problematic despite the transcriptional terminators."
--> copy number defines your gene-dosage ...some genes are only toxic at a certain dosage ...using lower copy numbers can prevent toxicity ...if you have the feeling that your gene is toxic go for a low-copy plasmid!

"Finally some of their vectors have T5 promoters. I know T7 promoters are IPTG inducible, but T5? What's the difference there and can it be used interchangeably with other cell lines such as BL21 (DE3)?"
--> T5 promoter is not recognized by the T7 RNA polymerase present in DE3-strains ...T5 can be used for in-vitro transcription of your gene.

Hope this helps!

Regards,
p

Luria Bertani, on 19 May 2011 - 01:40 AM, said:

Hi there

I have some genes that have been synthesised in pET-9a however the expression in these has not worked out well at all and they seem quite toxic (low or no expression on SDS-PAGE gel!). Even making plasmids from these things seems to be troublesome with low yield!

So I'm thinking of changing the vector to something else.
The other proteins we have are in a different vector pET 47b (+) and seem to be well behaved.

I've seen the pET manual and there seems to be heaps and heaps of different vectors which do similar things. We don't need anything fancy like tags or whatnot. Is there a relationship between vector and expression?


I am looking around at different synthetic gene suppliers; We usually use GeneArt but I have looked also at "DNA2.0"
Their website has some claims that I'd like to ask around about:

"Inserts are flanked by transcriptional terminators to allow cloning of inserts encoding proteins that are toxic to E. coli.  DNA2.0 can even guarantee that your gene will express in E. coli."

Exactly how does this work?

"Inserts are cloned nondirectionally to minimize potential toxicity of inserts."

What does this "directionality" mean?

"We also have vectors with low-copy origins for cloning genes that are problematic despite the transcriptional terminators."

I strongly suspect that my proteins are toxic. What is a low copy origin and why does it matter?

Finally some of their vectors have T5 promoters. I know T7 promoters are IPTG inducible, but T5? What's the difference there and can it be used interchangeably with other cell lines such as BL21 (DE3)?

Sorry if this might sound basic to some!!

Thanks in advance for your help!

[Luria Bertani]

Thank you pDNA!

Low copy plasmids-- does that imply low protein expression too? Or just low numbers of plasmid in the cells?

Will this be a problem making minipreps from this?

Edited by Luria Bertani, 19 May 2011 - 04:58 PM.

[pDNA]

in the case of the T7-system low copy most of the time means increased solubility of the protein ...since the copy number can be compensated by the high activity of the T7 polymerase the expression level is not that much affected.

With low copy number plasmids mini-preps can be tricky ...but most of the time expanding the volume of culture and processing more cells helps and gives you good yields.

Regards,
p

Luria Bertani, on 19 May 2011 - 04:55 PM, said:

Thank you pDNA!

Low copy plasmids-- does that imply low protein expression too? Or just low numbers of plasmid in the cells?

Will this be a problem making minipreps from this?

[Luria Bertani]

Much appreciated! Thank you