Intein-based controllers

A method for constant-level expression of an output, said method comprising the steps:

• a. providing a cell or a cell-free system, wherein the cell or the cell-free system is capable of expressing a gene encoding the output;
• b. inserting an expression system into said cell or cell-free system, wherein the expression system comprises:
• i. a gene encoding a controller, wherein the controller comprises:
• I. an optional first effector;
• II. a first split-intein;
• and
• III. an optional second effector;
• ii. a gene encoding an anti-controller, wherein the anti-controller comprises:
• I. an optional third effector;
• II. a second split-intein;
• and
• III. an optional fourth effector;
• wherein
• at least one of the optional effectors is present;
• one of the first split-intein and the second split-intein is an N-intein and the other one is a C-intein;
• the controller and the anti-controller are capable of undergoing a trans-splicing reaction via the steps:
• excising the first split-intein from the controller, and excising the second split-intein from the anti-controller, and
• a) if the first split-intein is an N-intein, and the second split-intein is a C-intein:
• rejoining
• the first effector if present with
• the fourth effector if present
• yielding a first splice-product;
• assembling a protein complex of
• the N-intein
• bound to the second effector if present
• with
• the C-intein
• bound to the third effector if present
• yielding a second splice-product;
• b) if the first split-intein is a C-intein, and the second split-intein is an N-intein:
• rejoining
• the second effector if present with
• the third effector if present yielding a first splice-product;
• assembling a protein complex of
• the N-intein
• bound to the fourth effector if present
• with
• the C-intein
• bound to the first effector if present
• yielding a second splice-product;
• wherein at least the second splice-product is yielded;
• the anti-controller is capable of downregulating the controller stoichiometrically via undergoing the trans-splicing reaction;
• the second splice-product and the optional first splice-product have a weaker, neutral, or opposite effect on the concentration of the output in comparison to the effect of the controller;
• constant-level expression of the output is achieved via a route selected from the group consisting of:
• (1)
• the controller upregulates the concentration of the output;
• the output upregulates the concentration of the anti-controller;
• (2)
• the controller downregulates the concentration of the output;
• the output downregulates the concentration of the anti-controller;
• (3)
• the controller upregulates the concentration of the output;
• the output downregulates the concentration of the controller;
• (4)
• the controller downregulates the concentration of the output;
• the output upregulates the concentration of the controller;
• c. exposing said cell or cell-free system to a condition, wherein the expression level of the output is perturbed from a target level to a perturbed level;
• d. equilibration of the expression level of the output back to the target level;
• wherein the first, second, third, and fourth effector are peptides or polypeptides, and the first and second intein, the controller, and the anti-controller are polypeptides, and the output is a peptide, a polypeptide, or an mRNA.