Hello my dear friend. I have an experiment like you

Prophage make up 12% of the enterohemorrhagic Escherichia coli genome, and

they play a prominent role in the evolution and virulence of this foodborne pathogen.

Acquisition, loss and rearrangements within prophage region are the primary causes of

differences in pulsed‐field gel electrophoresis (PFGE) patterns among strains of E. coli

O157:H7. Sp11 and Sp12 are two tandem integrated and putatively defective prophage

carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions

that occur within the Sp11‐Sp12 region at a frequency of ca. 7.74 × 10‐4. One deletion

involved a precise excision of Sp11 and the other two spanned the junction of Sp11 and

Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE.

Interestingly, we identified DNA sequencing reads that align with the Sp11‐Sp12 region

from the inducible prophage pool of E. coli O157:H7 clinical isolate PA10, suggesting

that the deleted regions may be packaged into mature phage particles. Unexpectedly,

deletion containing pchB and psrC, which are Sp11‐encoded regulators of type III

secretion, did not affect the secretion levels of the EspA or EspB effectors. Alignment of

the Sp11‐Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7

and O55:H7 strains suggested that homologous recombination rather than integrase‐

mediated excision is the mechanism behind these deletions. Therefore, this study

explains, in part, a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.

I hope this information is helpful to you. If the information helped you, tell me to send all of the protocol for you.

But want to add that upstream and downstream fragment of ~450bp have been used with success.

Thanks for all of the responses. I appologize, I was confusing in how I stated that the first time. I meant that the section of DNA that I want to replace with the cassette is only 65bp, specifically that there are 65 bps between the end of a gene and the terminator and I want to insert the 800bp cassette in there. Is there any president for this either working or not working?

Hi all,

I have a similar question. The genomic DNA region I want to replace is about 12.6 kb in size, while the antibiotic resistance cassette I want to insert is only ~2 kb. I use about 2 kb of homologous DNA flanking either side of my antibiotic resistance cassette, so the effective size of the genomic DNA region to replace is 8.6 kb. Will that work? (i.e. 2 kb cassette to replace 8.6 kb genomic region). Or do I need to target a smaller region to replace?

Thanks in advance.