Hello, what difference your bacterial cell transformed or not, you can use just classical method for DNA extraction from bacteria.

Dear Karen,

Thanks a lot for your response.Actually I want to know the protocol for replacing Bacterial genome with transfected plasmid in a viable cells.

Dear Manirujjaman,

I would like to know what is the reason for you to remove the bacteria's (I assume it is the expression host) genome content?! Is it for a specific reason?! From your question, I understand that you are transforming your bacteria (expression host) with a plasmid construct. Plasmids are extra-chromosomal units, which contain your gene of interest. Moreover, if you have chosen a commercially available plasmid (such as pRSET, pET etc.,), you would have transformed them into an expression host (such as BL 21 - DE 3 etc.,). Such expression hosts would have the RNA polymerase genes homologously integrated into their genome. This RNA polymerase, when expressed during induction, would then initiate the transcription process for the gene (ligated) in your plasmid. SO practically, if you have done what I have mentioned above, there is no possibility for you to remove the genome content of the bacteria. Additionally, the essential proteins necessary for the metabolism of your bacteria would be coded by its self genome. SO if you remove the whole genome, your bacteria is no longer viable at all.

Furthermore, I would like to know what exactly are you attempting to do?!

Dear Navnit

Actually I have read some articles about synthetic genome.The researchers have replaced the bacterial own genome with the synthetic one.So I am interested to know the protocol of this experiment.I could not find the detail protocol.I have also intention to do such type of research but for different purposes.

Thanks

Dear manirujjaman,

I assume you are asking about the whole genome replacement of the host. It cannot be done using a plasmid transformation into the host. Neither can you extract the whole genome of the host. I assume you have read the article in May 2019 about the whole synethetic genome replacement. This could have done by homologous recombination of segments of your genome, step-by-step; this would finally result in complete genome replacement by a newly synthesized sequence, as intended by you. Articles published as early as 1990 deal with the whole genome replacement of e.coli K 12 using lambda red homologous recombination. With respct to the exact protocol for homologous recombination using various techniques, i am providing you with a few links. There are commercially available kits, which would help you with homologous recombination.

https://www.biocat.com/recombineering

https://www.thermofisher.com/order/catalog/product/K601001

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044638

https://www.google.co.in/amp/s/phys.org/news/2019-05-genes-coli-bacteria-genome.amp

Dear Navnit

Thanks lot for providing the links.

Dear Navnit

If you have time please check the following article. I am feeling difficulty about the fig. no-3 of this article.

"Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast"

Dear Manirujjaman,

I will read the paper, attempt understanding it, and then get back to you.

Thanks a lot

Dear Manirujjaman,

I went through the paper. I am elaborating "my understanding" of the work and the figure, you have referred to.

1. the authors wanted to completely transfer (transplant) the genome of M. mycoides to M. capricolum.

2. they initially synthesized a fragment of the genome (M.mycoides) chemically, and homologously recombined it with the genome of E.coli. Since larger sequence assembly is impractical in E.coli, they used a vector to integrate the gene sequence with yeast's genome. Later, within yeast, the whole genome of M.mycoides was rengineered (re-done).

3. After this process, the yeast gDNA containing the "synthesized genome" was transplanted into the host M.capricolum using a PEG mediated method. Selection markers and genome profiling have helped them assess their transplantation process.

4 As for fig 3, I assume (may be right) that there are a few errors. It should have been, insertion of bacterial genome (containing the synthetic fragment) into yeast vector; Yeast vector with the genome (synthetic fragment); from step 3 it is clear.

5. For the protocol for transplantation, please refer to the title of an article by the same author. It has been given in detail; I hope it helps.

"Genome Transplantation in Bacteria:Changing One Species to Another"

regards,

Navnit KR

DearNavnith,Thanks for your kind effort.Already I have read the article you referred.I have not found the meaning of ‘’Resolution’’ in figure no.3.Initially there were two plasmids.One transfected and another one may be bacterial genome.Aftet resolution there is only one genome.I am confused about this one.Best Regards,Manirujjaman

Dear Maniujjaman,

I tried my level best to understand what the authors meant by "Resolution". But, I still have not been able to figure it out. I modestly assume it (resolution) could be the homologous recombination of the synthetic genome architecture with the host genome; they have shown the synthetic genome in blue and the host genome in green. It could be that after "transplantation", your host genome is entirely synthetic; the next image in the cycle shows the bacteria with a transplanted synthetic genome (blue). My perception may or may not be accurate.

Dear Navnith,Thanks a lot.I am also thinking about this point.My assumption is same as your perceprion.May be the authors wanted to meanhomologous recombination by Resolution .I am also trying to contact with the authors.Thanks again.Sincerely,Manirujjaman