Team:Potsdam/outlook
To empirically prove that metabolic channeling occurs as we have envisioned it, we would need to plan a lot more experiments:
1. Stoichiometry of bound and unbound complexes
We have to verify how effective our dCas9/IAA complex can bind the target cassettes to include that data in the isotope dilution experiments (mentioned below for proof of channeling).
2. Compare different Target cassettes
We need to measure the number of targets that our target cassette constructs offer and how the differences between the cassettes affect stoichiometry, so we would have to measure plasmid copy number and stoichiometric data (as mentioned above already). This is needed to normalize the data and precisely measure the effect of the different distances between the target sites and rule out other factors that may come into play.
3. Gather protein expression data
To be able to compare the final construct with the control (no deadCas9 and sgRNAs), we need to measure how much (ideally: functional) respective protein is produced with each construct.
4. Check for protein integrity
We have to verify if the fusion proteins we want to produce are even present as expected in vivo. This stems from the unpleasant experience Dr. Joachim Kopka from our group had, when a GFP fusion turned out to have been hydrolyzed and the GFP signal was just freely diffusing enzymes.
For both projects, we would need to make an isotope dilution experiment to confirm metabolic channeling in vivo.
The isotope dilution method uses the accessibility of the channeling complex to freely diffusing intermediate to determine the effectiveness of the channel. [1] The principle of this method can be explained with this Scheme:
Figure 1: Scheme to explain the isotope dilution method. Adapted after Spivey [1]
The precursor of the reaction S* (for us L-Tryptophan) is added and, after some time, a large amount of unlabeled intermediate I (Indolacetamide) is added, too. If the substrates reach the active centers of the proteins solely by free diffusion, we would expect the addition of I to “dilute” the radioactive signal of the product P* relative to S*. If the dilution is relatively low or non-existent (in an ideal situation), channeling may be concluded. The idea behind this method is this: A perfect channel has the intermediates directly transferred between the enzymes of the pathway, thus no intermediate from the bulk phase is used and no unlabeled product occurs, so the ratio of P*/S* is 1.
[1]Spivey, H. O. & Ova´di, J. Substrate channeling. Methods 19, 306–321 (1999).
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