Recombination pains in E.coli - (Jun/20/2006 )

HI all,

We are experiencing great pains with the sub-cloning and amplification of plasmid in E. coli for expression purposes. We are currently using the Pichia pastoris system for expression along with the pPICZ alpha vector system for soluble secreation of protein.

We have tried to amplify recombinant plasmid in DH5 alpha, TOP10F' (asrecommended) and JM109 cell lines. All of these cell lines have various recombination deficiencies. We obtain positive clones with screening of colonies from solid media. Unfortunately when we grow clones in liqiud culture to isolate plasmid, we obtain multiple bands with PCR screening. Sequence data reveals that all bands represents our insert with varying degrees of plasmid duplications inserted into the sequence. Furthermore we have found with some of the cell lines (TOP10F' and JM109) that though colonies grow on selective media and are positive for our insert after liquid culturing few or none contain insert. We have initially cultured cells at 37'C, but have decreased that temperature to 27'C to little effect.

Any suggestions will be greatly appreciated.

Thank you

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Some strains are more recombination deficient than the mentioned E. coli strains.
Invitrogen hast the stbl-strains and stratagene has the SURE strains. Maybe they can be of any help?

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Thank you for replying,

We are currently exploring the Stbl3 cell line from Invitrogen, which as yet has not produced satisfactory results. We are doing control reactions to asses if it is a problem with the cell batch.

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It sounds like your cloning construct is poisoning your cells and forcing a rearrangment. Some expression vectors will be much more highly expressed in E.coli when trying to shuttle the vector. The strains will then force rearrangments that are not very stable, even in differing media conditions with the same selection.

I've seen similar problems that were solved, by using a shuttle strain that contained the correct repressors (tet, lac, etc.). I don't know if there is one available for the pPICZ expression system. AOX1 promoter, correct?

You can also try a different lab strain (staph, bacillus, etc.) to shuttle. Or cut out the middle man altogether and tranform ligations directly into Pichia on repressing media.

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Thank you for the feedback,

In terms of the promoter utilized for the pPICZ expression system it is Alcohol oxidase gene activation and that induces expression in P.pastoris. I am not entirly sure if I will be able to transform my ligations directly into the yeast. Pastoris expression involves the incorporation of your gene of interest into its genome using AOX sites for the recombination. I will need between 5-10ug of recombinant plasmid wich must be linearized prior to transformation. This will likley require a large amount of starting material prior to ligation and effecient dephosphorylation of restricted plasmid to minimize false positive colonies. Thank you for the suggestion, this may yet be an avenue to explore.

The bacterial strain that we used initially (TOP10 F') is the strain that has been supplied with the expression system and they suggest other recA and andA mutant strains (like JM109 and DH5alpha F'). We have used these cells with little/no success and are looking at the Stbl 3 cell line from invitrogen.

Can you mabe suggest another that we might consider?

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Someone above mentioned the SURE strains, which are worth a try. We have also found that reducing the copy number of the plasmid can help. We use p15, SC101, or F plasmid origins to get lower copy numbers. Of special mention is the pSCANS plasmid backbone from BNL which has a constitutive F origin and an inducible ColE1 ori, so that the plasmid can be maintained at low copy number and then induced to high copy number when a plasmid prep is done.

You might also look at some plasmids that surround the cloning site with transcriptional terminators, such as the Lucigen plasmids. They also have some new low copy number plasmids which might be worth looking at. Sometimes transcription into or out of the cloning site can have a dramatic effect on the stability of the construct.

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What type of integration method are you using to insert the construct into the genome, sounds like double recombination if the construct is linear?

You can try a 3 part pcr. I've used this strategy to create linear KO constructs and expression constructs for filamentous fungal species and bacteria. If this is what you need, I'll try to dig up a protocol and post. Its a little complicated to write here. You can also find a protocol on the web.

basically you amplify your insert with flanking region for a 5' arm and flanking region for a 3' arm. The oligos for this amplification are long 40-45bp. Amplify your each arm seperately. purify each fragment and ligate with a PCR rxn. The arms act as primers for the entire construct and the flanking regions of the middle (insert) tie each part together. Then you can amplify the entire construct with outside primers. This will be clearer with a protocol.

I'll see what I can find.

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Thank you for your responses so far,

In answer to integration into Pichia, yes as far as I understand homologous recombination is utilized to insert the gene of interest into the genome at the AOX gene and place it under the control of the AOX promoter. Integration also allows the incorporation of not only a selectable marker (Antibiotic resistance) but also tags which are useful as reporters and for purification.

Are you suggesting I amplify the entire construct (including the sites for yeast recombination and my insert), perhaps I should have a look at the protocol so I may gain a better understanding of what you are proposing. I would like a copy of the said protocol if I can trouble you for it. Please mail me at s99073732@student.up.ac.za so I can have a look to see if I can use this approach. Thank you kindly.

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Hi Stutz,
i am having the same problem with pPICZA, and would like to ask you, how did u solve this problem.

thanks

Miraki

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