Dear Norelle

There are 2 main reason because generally we do not use the strain suitable for protein expression (as eg the BL21 which are derived from E.coli B):

1) For cloning is preferable use strains as the DH5alpha, DH10B, Mach1 which carryng the following mutations:

• recA1and DNA mutation to reduce DNA ricombination and improve DNA insert stability.

Generally those strains are derived from K12 E.coli and is it possible to obtain also chemically competent cells with good efficience (>106 cfu/ug)

On the contrary the strains used for protein expression,, to maximize their growth rate, are missing some proteases gene but not recA1 and endA. Moreover generally this are derived from E.coli B, and this strains are more difficult to be transformed (lower level of competency acheived with chemical treatments)

Therefore, is it possible in some cases clone directly your gene in the expression host, but it is not something feasible with many clones in parallel because togheter the lower efficiency of the competent cells and the lower stability of the DNA, may decrease success rate due to fail of the transfromation after ligases or due to uncorrect sequence obtained for unaxpected clone ricombination.

This do not means that it is not possible and that in some laboratories people did not have developed some specific expression strains able to overcome the cloning problems.

good luck

Manuele

Dear Norelle

There are 2 main reason because generally we do not use the strain suitable for protein expression (as eg the BL21 which are derived from E.coli B):

1) For cloning is preferable use strains as the DH5alpha, DH10B, Mach1 which carryng the following mutations:

• recA1and DNA mutation to reduce DNA ricombination and improve DNA insert stability.

Generally those strains are derived from K12 E.coli and is it possible to obtain also chemically competent cells with good efficience (>106 cfu/ug)

On the contrary the strains used for protein expression,, to maximize their growth rate, are missing some proteases gene but not recA1 and endA. Moreover generally this are derived from E.coli B, and this strains are more difficult to be transformed (lower level of competency acheived with chemical treatments)

Therefore, is it possible in some cases clone directly your gene in the expression host, but it is not something feasible with many clones in parallel because togheter the lower efficiency of the competent cells and the lower stability of the DNA, may decrease success rate due to fail of the transfromation after ligases or due to uncorrect sequence obtained for unaxpected clone ricombination.

This do not means that it is not possible and that in some laboratories people did not have developed some specific expression strains able to overcome the cloning problems.

good luck

Manuele

it has less transformation efficiency when compared with Ecoli dh5 alpha.

Hi,

I assume you in fine want to produce a recombinant protein of which you subcloned the ORF insert in a given vector.

For the reasons mentioned above you indeed prefer doing your molecular biology with DH5a and then transform your BL21 (or even Rosetta to increase yield if you have eucaroytic codons rarely used by bacteria) to produce your recombinant protein.

I had found this for a colleague facing cloning problems (answer by Benjamin S Glick September 6, 2012)

https://www.researchgate.net/post/Is_my_gene_toxic_to_the_bacteria_A_mysterious_cloning_problem

You can also try to grow your transformed DH5a after ligation at a lower temperature. I've seen people leaving their agar plates on the bench over weekend to avoid come get them out of the incubator. This strategy can be used to reduce toxicity too. You may have lesser yield but likely enough to isolate your cloning product with a low copy nb protocol and transform it into BL21 for your production. I've seen recombinant proteins productions kept as low as 16°C after IPTG induction to prevent toxicity of the produced protein so you could also grow them longer and slower at 30°C to reach the wanted DO while preventing toxicity of leaky transcription.

Hope this helps

Hi Manuele Martinelli

Thanks for your answer. We have been using the Alpha5 line from NEB and only after 5 days do we get colonies (25C or 37C, doesn't matter it's the same).

When we do colony PCR we have our inset in ~75% of the picked colonies. The key problem is getting liquid culture afterwards (not from PCR tubes, but from the other portion picked colony that was put in 10 uL LB for 1-2hrs*).

Anyway, we ordered some Mach1 and will hope this works.

*That is we pick the colony put it in 10 uL LB. 5 uL is used for colony PCR and the other 5 stays at 37C until we know it is positive. IF positive we add LB up to 3 mL for liquid culture.

Dear Norelle

Is you protein cloned in pet vector? Or in a vector carryng a different promoter?

Because i think that alpha5 line do not carryng T7 polimerase in its genome and theoretically the toxicity of your insert cannot be the problem of your slowth growth because with out polimesase cells cannot express the protein.

On the contrary of you use a different e.coli promoter as tac then it is possibile that you have basal expression also in alpha5 and this may delay the growth of your colonies.

However for the pcr screening you do not need to growth the coli in liqyis culture, you cam just pick the colony, duplicate it in an empty agar plate and aftereard instert the inoculation nedlrr in the pcr reaction. Dont worried about the possibility that all the cella remake attached to the plate, pcr is very sensitive and few cells are enough to show results in colony screening.

You can find a video regarding this procedure in my blog ProteoCool

https://proteocool.blogspot.com/

ProteoCool n°4 (PCR Colony screening)

Ciao

Manuele

Hi Manuele,

We are using the pSUMO1 vector from Addgene https://www.addgene.org/52258/

Our insert is 494 bp and we are using the HiFi Kit from NEB for PCR cloning.

We have also tried the 2 plate method you describe in your video, but the problem is the same - no colonies on the plate.

Best,

Norelle