Team:Amsterdam/shadow/project/overview

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Summary

A chosen sensor gets activated which then promotes transcription of its specific coupled phusion protein, consisting of a ZN-finger and a methyltransferase (ZN-MTase).

Newly translated ZN-MTase moves to and binds to its specific ZN-finger motif binding site and correlating MTase binding site on the introduced plasmid, allowing the MT to methylate its specific motif which is closely located to the Zn-finger binding site. One point of methylation corresponds with 1 bit of information stored on the plasmid.

Storage

Digest
A methylation dependent restriction enzyme (RE) with the same motif as the MTase in the ZN-MTase is subsequently introduced in the cell. Depending on whether its restriction site is methylated or not it will be able to digest the DNA.

Read out of the data will be done using PCR and gel electrophoresis and analysis of the band lengths observed showing whether digestion has occurred. This way a Boolean (true or false) for the presence of each signal in E. coli’s environment is stored.

Primer tails
Specifically designed primers can anneal on either sites and close to the methylated site both with a predetermined length of the tail. Then in the PCR primers can be used that anneal to the introduced primer tails.

Without methylation; the first pair of primers can be ligated forming a piece of DNA of predetermined length that can then be found on agarose gel after PCR with primer tail specific primers.

With methylation; ligation of the first primers isn’t possible. This would mean that in a PCR no bands can be formed. Different lengths of the tails provide different sizes of bands for different conditions.

Near-site primers
Specifically designed primers anneal on a predetermined distance from the methylation site.

Without methylation; the primers can anneal and be transcribed creating a band to be found in gel.

With methylation; the primers can still anneal but transcription cannot occur since the methylation stops the full polymerase.

Different distances from the methylation site gives bands of different sizes to distinguish conditions.