Can anyone share an experimental procedure for the direct transformation of a bacillus bacterium using PCR product or other non-circular DNA fragment?
I think it is possible to introduce DNA trough the Bacillus bacterium membrane as we perform with electroporation and heat shock for example. But, without a plasmid with replication origin and selective methods, the fragment won't be stable and inherited and it will be harder to detect the transformants, respectively. To do this, you probably need to insert this fragment in (genomic) circular DNA from Bacillus. Then, I do not know any method for inserting DNA fragments as a PCR product straightforward in the (genomic) circular DNA. You need supplementary mechanisms as homologous recombination. I am looking forward to see other answers and I will be surprised if there is (are) some method(s) to do this.
Please see this useful discussion at RG: Circular plasmid or linearized plasmid, which one is better for transformation?
PCR product is an linear product
https://www.researchgate.net/post/Circular_plasmid_or_linearized_plasmid_which_one_is_better_for_transformation
Electroporation is the simple and efficient method. Transformation of PCR fragments or linearized DNA is usually only used for gene transformation or transcient expression since the transformed DNA fragment is going to quickly degraded.
As long as the PCR product, (linear DNA) is not a part of cloning vehicle or does not get integrated into genomic DNA, it will not replicate. In fact, even if it makes entry inside the cell, it will be degraded by exonucleases.
Thank you for all your answer.
This question needed more precisions : Yes, the aim is to perform homologuous recombination at the end, using 2 homologuous genes of at least 500bp long. The tricky part is that the PCR product to transform Bacillus with will be around 40kb long (from assembly of long PCR products). I can't find any answer on the maximum DNA fragment size that can be integrated in B. subtilis for example using natural competence, chemical- or electro- transformation.
1. Since your 40-kb long PCR is all set up with two ends having homologous to two internal genes, you should just go ahead and try out
2. Once they (PCR product) get into the cells, do you have any system to promote the homologous recombination activity in the cells? For E. Coli, people have used Red system to promote the homologous recombination activity between the 'PCR' product and endogenous DNA fragments. See: https://www.researchgate.net/post/Do_you_have_experience_using_red_system_to_create_mutants_in_E_coli
PCR products can be transformed directly into bacillus cells as long as the PCR product is flanked with two 400 to 900 bp homologous sequences at both upstream and downstream of the chromosome. In term of the DNA size, million-base pair-long genomic DNA can be transformed into Bacillus.
http://www.sciencedirect.com/science/article/pii/S138917230670576X
Could I ask if there has been any update on whether this was successful or not. Attempting something very similar and would like to know the stability of linear PCR products for homologous recombination within the cell?
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