Similar to Alexandra's message, I have been working to use Q5 polymerase to amplify very large pieces of DNA (23 kb) for cloning of biosynthetic gene clusters, such as in your case. Please see my two papers for the method. If you need any more help with this, please don't hesitate to contact me.

Article Direct Pathway Cloning (DiPaC) to Unlock Natural Product Bio...

Article Direct Pathway Cloning Combined with Sequence- and Ligation-...

Depending on the GC content of the cluster and the operon organisation will determine how difficult this cloning strategy is.

Also, selecting to use either the native promoters or inducible promoter is also very important. This will depend on the relatedness of the native and heterologous host.

I would also suggest to use pCC1FOS. We have observed the inability of pET based vectors to replicate once their size exceeds 45kb.

Another method I could suggest to use is Rec/ET recombineering.

https://www.nature.com/articles/nprot.2016.054

Which method is best to you all depends on your specific gene cluster.

Good luck!

You could probably use Phusion or Q5 polymerase to amplify 5, ~10 kbp products then use Gibson Assembly to make them them into a vector.

The original paper describing the method used it to make ~300 kbp BACs

https://www.nature.com/articles/nmeth.1318

In the article it's called "One Step Isothermal Assembly"

NEB sells a mix that's a derivative of Gibson Assembly which is relatively cheap:

https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix#Product%20Information

Since the efficiency might be a bit low, I'd suggest using highly electrocompetent E. coli and transforming the whole desalted reaction, then giving them an extended recovery time. Using a BAC as the backbone might also be a good idea to make sure the construct is stable.

As a disclaimer, I haven't assembled anything this big personally but I have used Q5 and Phusion to amplify ~13 kbp fragments and electroporated a ~200 kbp plasmid into Top 10 E. coli without issue.

Hi Bikash,

We had used pCC1FOS fosmid (Epicentre) to clone 40 Kb to construct metagenomic library and express the genes for protein screening.

https://www.lucigen.com/CopyControl-Fosmid-Library-Production-Kits/

The use of BAC or YAC may help you for bigger inserts.

Similar to Alexandra's message, I have been working to use Q5 polymerase to amplify very large pieces of DNA (23 kb) for cloning of biosynthetic gene clusters, such as in your case. Please see my two papers for the method. If you need any more help with this, please don't hesitate to contact me.

Article Direct Pathway Cloning (DiPaC) to Unlock Natural Product Bio...

Article Direct Pathway Cloning Combined with Sequence- and Ligation-...

Depending on the GC content of the cluster and the operon organisation will determine how difficult this cloning strategy is.

Also, selecting to use either the native promoters or inducible promoter is also very important. This will depend on the relatedness of the native and heterologous host.

I would also suggest to use pCC1FOS. We have observed the inability of pET based vectors to replicate once their size exceeds 45kb.

Another method I could suggest to use is Rec/ET recombineering.

https://www.nature.com/articles/nprot.2016.054

Which method is best to you all depends on your specific gene cluster.

Good luck!

thank you all.... Alexandra, Oualid and Paul.

Paul, I have been using the same nature's protocol that you have attached. However, I am still encountering some challenges to clone the whole biosynthetic cluster in P15A plasmid. Do you have an idea why is it so?? Thanks

How large is your vector? I do not have much experience with the P15A vector but if your cluster is >50kb, size it probably the issue, but you should be able to find this information somewhere. As suggested by Oualic, you should try moving to the pCC1FOS vector.

and what are the problems exactly (no colonies? all negative colonies)?

Few of the plates do have colonies, but seems to be the self-circularized vectors (after checking with endonuclease).... and few don't have it at all...

try to use the direct cloning of BGC using TAR cloning or LLHR / ExoCET. they are some examples

Hi,

Yes I agree with Xiaoying Bian. Its better to use TAR cloning to capture your BGC. You can also try CRISPR-Cas9 mediated TAR cloning, that may give you additional flexibility to capture your BGC.

Hello Paul, do I need to digest the vector before doing the PCR amplification to get the homology arms??? I have not done it before, and the colonies that I got were all negative. Can you please suggest on this one. I am using p15A vector. Thank you in advance

Thank you everyone...

Hi Bikash,

Just some questions firstly.

Are you amplifying both the vector backbone and the insert to get your two linear DNA fragments before DNA assembly?

If the answer is Yes, you need to digest the vector PCR sample after amplification using DpnI. DpnI digests all of the vector template while not digesting your vector PCR product because it is active on methylated DNA only and the PCR product is not methylated (but your miniprepped vector DNA template is methylated). The reason you need to remove template DNA is because it will have the same antibiotic resistance cassette as your newly constructed vector and the smaller size means it will be transformed preferentially, which is likely why you have all negative clones. Digesting with DpnI removes this background. I always clean the vector using gel extraction after DpnI digest.

If the answer is no, you must have linearised your vector using a restriction digest and in this case, dephosphorylation of the vector is required to stop the empty vector from re-annealing and becoming background in your transformation.

I hope this is helpful.... If not, perhaps you could message/email me a detailed protocol of what you have done and I will be able to provide you with better help :)

Cheers,

Paul

Thank you Paul. Could you please send me your e-mail address? I will send you an email soon.

Hi Paul, I am not amplifying the whole vector as a backbone, but the only part which has the selection marker and Ori for replication. I am going to linearlize the vector by an enzyme and dephosphorylate it at the same time. I guess amplifying (performing PCR) by this way can have the homology arms hanging which should be able to capture the target BGC after the electroporation. I hope to receive few negative clones this time. By the way, the protocol I am following is of this paper: https://www.nature.com/articles/nprot.2016.054

Thank you and looking for your comments on the same.

Regards,

Bikash

Hi Bikash,

Did you have any luck with the cloning?

Cheers,

Paul