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Autonomous linear DNA clock

Autonomous linear DNA clock. Richard J Crossland. Purpose. Internal count-down timer to any cellular event Deployment of function at target site or at correct time Internal control mechanism to prevent GM organisms evolving (does not require external signal)

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Autonomous linear DNA clock

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  1. Autonomous linear DNA clock Richard J Crossland

  2. Purpose • Internal count-down timer to any cellular event • Deployment of function at target site or at correct time • Internal control mechanism to prevent GM organisms evolving (does not require external signal) • Extra-chromosomal – can contain all GM genes

  3. Mechanism – no telomerase repression repressor gene dna replication kill switch dna replication telomere shortening cell death no repression

  4. Identified problems • Telomerase repair mechanism • Replication machinery • Repressor protein and cell death • Getting linear DNA into cells other than Streptomyces, Borellia. • Horizontal gene transfer

  5. Telomerase repair mechanism • Two processes: • 1. linear plasmid replication • 2. patching gaps/ TIRS (terminal inverted repeats) • Disarm step 2 280 nt (Casjens, 1999)

  6. Replication machinery -- given • Bi-directional linear replication from central internal origin • Streptomyces, Borellia • Also in: Yersinia enterocolitica, E.coli, Klebsiella oxytoca, Salmonella Typhi. • Either already on linear plasmid – viral RNA polymerase, viral DNA polymerase • Or see case study – it works!

  7. Repressor protein and cell death • Tap into existing functionality, new sub-project • loads of possible mechanisms to choose from • nuclease • mazEF stress-induced toxin-antitoxin suicide module (E.coli) • skf and sdp operons in Bacillus Subtilis - nutrient limitation (Engleberg-Kulka et al. 2006) • B.S – go into sporulation and don’t germinate

  8. Case study: Baker et al. 2007 • S.Typi pBSSB2: 27kpb linear plasmid, 33 coding sequences • Contains z66 flagellin antigen (flijBz66 gene) pBSSB1 + kanamycin resistance cassette (1,432-bp)  pBSSB2. pBSSB2  E.coli SGB33 • Plasmid isolated (alkaline lysis method), sequenced, and shown to be capable of autonomous, existence and stability in E.coli. • E.coli expressed antibiotic resistance. • Although antigen could not be detected (interaction with flagella regulation machinery) when retransformed E.coli SGB33 plasmid into S.Typhi, it was stably maintained and z66 antigen was dominantly expressed. lambda red recombinase electro-transformation

  9. cont ...

  10. Horizontal gene transfer • All GM genes on the linear plasmid • Transferred to non-GM bacteria --> it becomes GM and dies (death plasmid) • Loss of GM linear DNA from GM bacteria --> it is no longer GM, so no problem.

  11. Conclusion • 1. Linear DNA exists, • 2. It is stable, is expressed and replicated when transformed into other bacteria ( E.coli) • 3. It is large enough to contain all GM genes • 4. What we need to achieve: • create a linear dna molecule with two genes (a repressor and a cell death protein) • knock-out the telomere patching mechanism • Transform into B.Subtilis

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