Basically, I'm looking for something similar to LacZ/X-gal type of enzyme/substrate activity that produce insoluble (precipitate) product with color change but not with β-galactosidase.
I want to insert the gene (that code for enzyme, but not β-galactosidase) in e.coli and see with naked eye the result (color change) of its activity on its target substrate.
One I found is chitinase/4-Nitrophenyl N,N’-diacetyl-b-D-chitobioside activity but that is all I got.
Do you know anything else?
I think you should first determine which enzyme you want to insert before you look for substrates for an enzyme assay.
When you know which enzyme you want to insert you have to know the substrates and structure of the enzyme (binding site...) with such knowledge you could predict what your enzyme could take as artificial substrate too.
When you know your specific enzyme you could probably find activity assays already existing.
If you just want to screen if your enzyme is there, then I would tag it with for example GFP but that can of course change its activity.
Hope it helps...
horse radish peroxidase has a substrate that makes an insoluble product, but I don't know if it can be expressed in ecoli because of its heme group. Any oxidase would work, or alkaline phosphatase maybe. I don't know if AP makes an insoluble product, but I guess it would since it is also used for IHC staining.
Hi. I think that a good system for you could be a belatactamase (bla) with the chromogenic substrate nitrocefin. Even more, there is a kit (http://www.biovision.com/manuals/K803.pdf). Introducing a betalactamase in the chromosome is very easy. But you can do it in a plasmid too (any cloning vector). Regarding to solubility, depend on the conditions of the assay, can be both, soluble or insoluble. The substrate, once that is hydrolysed turn from yellow to red and is quantitative. If you want, with a maximum absorption around 490 nm. Is an enzyme which natural microenvironment are bacteria, so nothing to worry about expression. If you need more details, just let me know, but there is a lot of literature about it. Hope this help.
Thank you for lots of suggestions. Gave me new ideas and surly more papers to read :)
I have to clarify my original question. I want to create genetic circuits with the reporter gene at the end so if my circuit works as I designed, I see the product of the reporter gene. So, I want to find the reporter gene that is non-endogenous (non-constitutively expressed gene) in e.coli and that its activity produces visible (with naked eye) color change (no fluorescence microscope or blue light).
Right now I'm reading about HRP/AP and beta-lactamase to see if I can put them in my gene circuit.
If you have more suggestions or if my question is unclear, please let me know!
Ok, if this is the idea, the b-lactamase should work. Tem1 and derivative are non-endogenous alleles for normal strains that work very well in E. coli. You can use also use chloramphenicol acetyl transferase and one kit to detect it but here there is compilation of comertial enzymes and substrates (https://www.lifetechnologies.com/de/de/home/references/molecular-probes-the-handbook/enzyme-substrates/introduction-to-enzyme-substrates-and-their-reference-standards.html). If I were you I would use a lacZ minus strain to integrate the circuit and use the B-galactosidase/x-gal combination (the simpler, the better). Good luck.
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