Contents 1 Linear DNA clock: Richard 1.1 Initial idea 12 February 2010 1.2 Ongoing references 1.3 Development 1.3.1 MSc project meeting -- 30 March 2010 1.3.2 Linear DNA in crisis -- 02 April 2010 1.3.3 Another problem with linear DNA -- 05 April 2010 1.3.4 Renewed hope for linear DNA -- 06 April 2010

Linear DNA clock: Richard

Initial idea 12 February 2010

Builds on idea about protecting against mutations. Rather than wait for a mutation to 
happen, could guarantee that genetically-engineered BS cell will not survive after a certain 
time. Idea is to linearise prokaryotic dna and insert two genes: one a repressor protein at 
a variable short distance from the cleaved site, and 2) a gene that codes for a cell-
destroying protein of some sort which the repressor protein usually inhibits. 

Remember telomeres? Idea is that every time the BS divides, its dna will become progressively 
shorter from either end. Thus the gene for the repressor protein will get eaten away. After 
time x (can be set by experimenter) the repressor gene will be rendered non-functional and 
no repressor protein is produced. This means that the second gene we inserted, which codes 
for the cell-destroying protein is free to kill the cell. No mutation, no residual problem 
of removing BS after treatment by idea 3.

At some point in evolution in the move from prokaryotic to eukaryotic genomes, the dna became 
linearised, so shouldn’t be such a hard problem to solve if we want the dna to stay this way. 
Could insert a piece of linear dna containing linear transcription machinery on it as well.

Ongoing references

Baker et al. 2007 A Novel Linear Plasmid Mediates Flagellar Variation in Salmonella Typhi.Took linear plasmid from S.Typhi and transformed into E.coli [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876496/].

Baker et al. 2007 A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi. More info on this linear plasmid [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652032/].

Cui et al 2007 Escherichia coli with a linear genome. Linearises genome of E.coli [http://www.nature.com/embor/journal/v8/n2/abs/7400880.html].

Development

Literature research and plan [1]

Presentation delivered at informal meeting on 26 Feb [2]

MSc project meeting -- 30 March 2010

Start modelling using deterministic algorithms (no random exponential variables for cell division event). Assume bacteria in chemostat -- no need to model stages of bacterial growth yet. Can model gene-regulatory network, cell densities, log/lag.

Unlikely to produce working part for iGEM -- but lots of cudos for idea/working model.

List requirements/specification for component.

Essential parts: many genes on linear plasmid from S.Typi currently inessential; some needed for maintaining linearity/DNA replication: look on GenBank to see predicted functions of 33 coding regions.

Update wiki page with links.

Teach yourself Kapezi, Cell designer, SMBL shorthand.

Linear DNA in crisis -- 02 April 2010

Please help! I have attached an attempt to model the linear dna system [3]. If my assumptions are correct, then the clock can no longer be a guaranteed kill switch.

The problem is that the clock isn't synchronous. Basically, template stands are immutable once created, will persist, and will not shorten. I have attached a doc which explains. For example in the excel printscreen, follow the green or yellow dna molecule through the replications and see that they don't shorten!

The results are interesting for telomere biology but either we find a way to create blunt ends, accept a one-cell signal, use single-stranded dna, or go back to the drawing board.

I do hope I'm wrong,

Another problem with linear DNA -- 05 April 2010

Even though the previous problem could be solved by having a synchronous population clock with weak extracellular inhibitor signals and strong intracelllar inhibitors, there is still, much to my dissapointment, a greater problem.

The idea is that the linear dna molecule becomes shorter with each round of cell division. This is flawed because after 20 cell divisions, the bacterial population wil be over 1 millon in number afterwhich it cannot divide any further. If one cell division takes 20 mins, and the loss in bp is about 100, and a gene is about 1000, then it takes ten cell divisions to eat through a single gene. Secondly, if it takes 10 x 20 minutes to eat through a single gene, then each timer is really only limited to controlling one gene at a time over a long time period.

The only solution seems to be to have the linear dna plasmids copy themselves without cell division (as Anil suggested was possible). But would this create a useful component or a cell swamped by a hundred thousand linear plasmids?

It is extremely hard now to see how this could ever be applied to repairing cracks in concrete.

There was a mechanism in eukaryotic cells that could be exploited to speed up the shortening but it requires endonucleases and dna modifications.

Probably a good idea to draft alternative ideas now unless this can be salvaged

Renewed hope for linear DNA -- 06 April 2010

I have come up with a potential solution to both problems with the linear DNA idea that I highlighted over the Easter break. (I also apologise for the 3' and 5's being the wrong way round in my diagrams and code).

Please see first slide of attached PowerPoint [4]. The idea requires four things which I could research into but thought you would know about straight away. It uses the assumptions 1) that bacteria protect their dna from nuclease activity through various mechanisms (i.e. but viral dna entering the cell is not). 2) If the dna is not protected it is eaten away by endo/exo nucleases. 3) that these protective mechanisms can be disrupted using the telomere-shortening mechanism during cell division 4) genes are not protected.

It overcomes the immortal strand hypothesis problem because exonucleases would eat into these. It overcomes the time/cap of divisions problem because whole genes are removed at each step and a unit time delay is encoded by a single protective structure. (the length of the protective structure tails would be set to align with the number of bases removed from the telomeres at each step).