Transcription inhibition

[canalon]

I am trying to develop a project where I can select bacteria for an insertion event (likely rare) in the genome. I know where the insertion will take place, but the ca. 200bp site cannot be modified and cannot be used for transcription (Stop codons in all 3 frames), but will be slightly modified after the insertion take place.
So what I am thinking is to put a resistance gene downstream of the insertion site and use a transcription inhibitor to prevent its transcription after the insertion event is supposed to have taken place. The idea being that the WT (no insertion) will therefore not express the resistance gene from the promoter upstream of the insertion site, while the modifications during the insertion will be enough to allow the transcription to run and allow the selection of the resistant bacteria where an insertion happened.

But I see plenty of miRNA, siRNA and morpholinos for eukaryotic system, but is there a reliable system for prokaryotes? Any suggestions are welcome.

Thanks

[jonmoulton]

Hi Canalon,

Since they do not rely on cellular systems for causing knockdowns, Morpholinos can be used in any cell type (animals, plants, protists, bacteria) — if they can be introduced into the cells. Delivery is the key. Bruce Geller’s lab at Oregon State University has been working on Morpholino delivery in bacteria. They are using a conjugate of a Morpholino with a cell-penetrating peptide, which is not a commercially available construct. I suspect that doing the conjugation yourself is more of a project than you were looking for (unless you are a closet o-chemist), but at least the Morpholinos are an option for bacterial knockdowns.

Here are some of their literature.

Greenberg DE, Marshall-Batty KR, Brinster LR, Zarember KA, Shaw PA, Mellbye BL, Iversen PL, Holland SM, Geller BL. Antisense Phosphorodiamidate Morpholino Oligomers Targeted to an Essential Gene Inhibit Burkholderia cepacia Complex. J Infect Dis. 2010 May 3. [Epub ahead of print]

Mellbye BL, Weller DD, Hassinger JN, Reeves MD, Lovejoy CE, Iversen PL, Geller BL. Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo. J Antimicrob Chemother. 2009 Nov 1. [Epub ahead of print]

Mitev GM, Mellbye BL, Iversen PL, Geller BL. Inhibition of Intracellular Growth of Salmonella enterica serovar typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer. Antimicrob Agents Chemother. 2009 Jul 6. [Epub ahead of print]

Shen N, Ko JH, Xiao G, Wesolowski D, Shan G, Geller B, Izadjoo M, Altman S. Inactivation of expression of several genes in a variety of bacterial species by EGS technology. Proc Natl Acad Sci U S A. 2009 Apr 30. [Epub ahead of print]

Mellbye BL, Puckett SE, Tilley LD, Iversen PL, Geller BL. Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency Against Escherichia coli in vitro and in vivo. Antimicrob Agents Chemother. 2009 Feb;53(2):525-30. Epub 2008 Nov 17.

Tilley LD, Mellbye BL, Puckett SE, Iversen PL, Geller BL. Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose-response in mice infected with Escherichia coli. J Antimicrob Chemother. 2007 Jan;59(1):66-73. Epub 2006 Oct 31.

Tilley LD, Hine OS, Kellogg JA, Hassinger JN, Weller DD, Iversen PL, Geller BL. Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture. Antimicrob Agents Chemother. 2006 Aug;50(8):2789-96.

Deere J, Iversen P, Geller BL. Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition in Escherichia coli. Antimicrob Agents Chemother. 2005 Jan;49(1):249-55.

Geller BL, Deere J, Tilley L, Iversen PL. Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitis. J Antimicrob Chemother. 2005 Jun;55(6):983-8. Epub 2005 May 4.

Geller BL. Antibacterial antisense. Curr Opin Mol Ther 2005 7(2):109-13.

Geller BL, Deere JD, Stein DA, Kroeker AD, Moulton HM, Iversen PL. Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers. Antimicrob Agents Chemother. 2003 Oct;47(10):3233-9.

Best wishes,

– Jon

[canalon]

Thanks,
Yeah Morpholinos appear interesting, but I am no closet-o-chemist at all, and since I am not working with E. coli nor B. cepacia and do not have the time, or the experience, to create a delivery mechanism for my favourite bug I will have to find some other idea. I guess a plasmid coding for siRNA…

But thanks for the suggestion.

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