Download Autonomous linear DNA clock

AUTONOMOUS LINEAR
DNA CLOCK
Richard J Crossland
Purpose
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Internal count-down timer to any cellular event
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Deployment of function at target site or at correct time
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Internal control mechanism to prevent GM organisms
evolving (does not require external signal)
Extra-chromosomal – can contain all GM genes
Mechanism – no telomerase
repression
repressor gene
dna replication
kill switch
dna replication
telomere shortening
cell death
no repression
Identified problems
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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
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Two processes:
 1.
linear plasmid replication
 2. patching gaps/ TIRS (terminal inverted repeats)
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Disarm step 2
280 nt
(Casjens, 1999)
Replication machinery -- given
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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
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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
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S.Typi pBSSB2: 27kpb linear plasmid, 33 coding sequences
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Contains z66 flagellin antigen (flijBz66 gene)
lambda red recombinase
pBSSB1 + kanamycin resistance cassette (1,432-bp)

pBSSB2.
electro-transformation
pBSSB2
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
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.
cont ...
Horizontal gene transfer
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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
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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