Frequently Asked Questions

Why not use a fluorescent protein?

Using a fluorescent protein has its advantages over our system but as does our system compared to a fluorescent protein(FP). The main advantage that our system might have over a FP is that there is no limit to expandability. When there are around 3 to more FP’s emitting a degree of interference starts to occur.

Wont endogenous methylation cause interference?

E. coli posses several own methyltransferases which are used for epigenetic purposes of E. coli’s own genome. Our scan of these endogenous methyltransferases do not indicate that any of them posses the ability to bind to our M.ScaI-recognition site and are thus not able to methylate the site.

Will the fusion-protein fold correctly or be processed by E. coli’s native systems?

At the moment there is no indication that this will be the case. Separately the parts of the protein have been translated and expressed in E. coli without complications.

Will the introduced methyl-tranferase cause problems inside the microorganism?

Right now all our experiments are conducted in the DH5alpha strain of E. coli which does not posses the M.ScaI methyltransferase we use. Actually the M.ScaI is a type 4 methyltransferase, of which none are or any recognition sites endogenously occur in E. coli. After BLAST searching the E. coli genome we also did not find any potential binding site for our M.ScaI.

Wont the memory plasmid be cut or processed by the endogenous restriction enzymes?

The chosen restriction sites in our plasmid are not recognized by E. coli’s endogenous restriction enzymes. The plasmid backbones and vectors that are used have all been previously used or expressed in E. coli without any complications.

If there is leaky expression of the protein, would this cause interference?

Replication creates a progeny without a logged signal, eventually you would lose the logged signals? This perspective differs in the application the system is used for. If something is being sensed that continues to activate the sensors linked to the system this isn’t a problem. If there sensed compound is not degradable there also is not a problem. If a time-indication based sensing is done the loss of logged signal to the progeny is actually desired. Also there is the factor of using a high- or a low-copy plasmid. In the case of using a high-copy plasmid there will be enough of the fusion protein divided during replication to keep the progeny methylated.

How much specificity is created by the Zinc-finger?

The Zinc-finger uses an unique 18 bp sequence which isn’t found anywhere else on the plasmid or in the genome of E. coli. Attached to the M.ScaI which has its own probability of binding (PoB) it multiplies the overall PoB to a much higher specificity.

Would constitutive active promoters cause random or unspecific methylation?

This is fine-tuned and/or dependent on the conditions that the experiment is being done in. Factors contributing in this are; the degradation time of the fusion protein, high or low copy plasmid and the amount of different sites present. If the degradation time is really fast then a constitutive active promoter is desirable. If slow then it is unwanted. When using a low copy plasmid it can be desirable, when using a high copy plasmid it could be unwanted.

When is choosing between a high or low copy plasmid the best approach?

If the intention is to store the occurrence of something for a longer period the best approach would be the high-copy plasmid.

In the event of a time-concentration storage a low-copy plasmid could be used since there is less chance of the progeny receiving any Mtase during replication.

How long before the fusion protein is degraded?

This depends on the three parts of which the fusion protein is composed of. First of all the degradation of the methyltransferase is the main part which will cause lose of function when degraded and is where the main focus for this question should lie on.

The Zn-finger would, when degraded, only lower the specificity of the fusion protein and enable the M.ScaI part to bind in random places.

The Myc-linker part would also create an unfortunate circumstance when degraded by releasing the Mtase into the cytoplasm for free binding at random sites.