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Bacterial knockout by double crossover

Designing the DNA

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4 replies to this topic

#1 rmbio

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Posted 06 September 2015 - 08:31 PM

Hi all

 

I wish to knock out certain genes in bacteria by double crossover. For transformation, I am planning to use linear DNA containing antibiotic reistance gene flanked by the region homologous to the gene to be knocked out. I have following doubts.
1) Is it neccessary that the antibiotic reisistance gene be cloned in-frame with the 5' fragment of the target gene? I think it's not.
2) As far as I know, I just need to include cds of the antibiotic resistance gene and it will be expressed using the target genes promoter. Please correct me if I am wrong in stating this.
3) Is it neccessary that the 5' flanking fragment of the target gene on the DNA to be trasformed should be as small as possible? So that the start codon of antibiotic resistance genes is closer to the target gene's promoter and is also easilty recognized during translation?

 

Thanks



#2 phage434

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Posted 23 September 2015 - 10:17 AM

1) You are correct.

2) You should add a good RBS upstream of the start codon. A promoter is not necessary, probably, although if you have a good resistance expressing cassette, I would include the promoter as well.

3) Irrelevant if you add a promoter, but perhaps important for inserting only an RBS + ORF.  The size of the overlap region strongly controls the recombination efficiency, so short ones may recombine poorly.



#3 rmbio

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Posted 29 February 2016 - 02:37 AM



1) You are correct.

2) You should add a good RBS upstream of the start codon. A promoter is not necessary, probably, although if you have a good resistance expressing cassette, I would include the promoter as well.

3) Irrelevant if you add a promoter, but perhaps important for inserting only an RBS + ORF.  The size of the overlap region strongly controls the recombination efficiency, so short ones may recombine poorly.

Thanks for your reply phage343! And sorry for delyaed response. I somehow missed to realize that some has replied.

I am now ready with the DNA to be transformed: both linear fragment and the one cloned in a plasmid.

Please see the image below. My original plan was to follow B. But since I now have the plasmid also (which is easy to produce than the linear fragment shown in B, I was also thinking of plan A but I am feared that it would result in 2, wherein the yellow gene that I wish to install has not been installed! I am sure 1 is not going to happen. 

Please let me know your suggestions and correct me if I am wrong.

 

Many thanks

 

tmhR4ny.jpg


Edited by rmbio, 29 February 2016 - 02:38 AM.


#4 rmbio

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Posted 29 February 2016 - 02:44 AM

 



1) You are correct.

2) You should add a good RBS upstream of the start codon. A promoter is not necessary, probably, although if you have a good resistance expressing cassette, I would include the promoter as well.

3) Irrelevant if you add a promoter, but perhaps important for inserting only an RBS + ORF.  The size of the overlap region strongly controls the recombination efficiency, so short ones may recombine poorly.

Thanks for your reply phage343! And sorry for delyaed response. I somehow missed to realize that some has replied.

I am now ready with the DNA to be transformed: both linear fragment and the one cloned in a plasmid.

Please see the image below. My original plan was to follow B. But since I now have the plasmid also (which is easy to produce than the linear fragment shown in B, I was also thinking of plan A but I am feared that it would result in 2, wherein the yellow gene that I wish to install has not been installed! I am sure 1 is not going to happen. 

Please let me know your suggestions and correct me if I am wrong.

 

Many thanks

 

tmhR4ny.jpg

 

 

....and would C (below) work? The black part flanking the fragment being electroporated is a part of vector not present in genomic DNA

 

HzlPxR4.jpg



#5 phage434

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Posted 29 February 2016 - 05:37 AM

2) cannot happen. The red and green regions in 2) are in the reverse orientation and won't recombine in that orientation (well, they won't recombine to yield the product you drew).






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