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)

Extra-chromosomal – can contain all GM genes

dna replication

dna replication

repressor gene

kill switch

cell death

repression

telomere shortening

Mechanism – no telomerase

no repression

Identified problems

Telomerase repair mechanism Replication machinery Repressor protein and cell death Getting linear DNA into cells other than

Streptomyces, Borellia. Horizontal gene transfer

Telomerase repair mechanism

Two processes: 1. linear plasmid replication 2. patching gaps/ TIRS (terminal inverted repeats)

Disarm step 2

(Casjens, 1999)

280 nt

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!

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

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

cont ...

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.

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