Post-translational synthetic circuits with split-inteins

We have designed and tested two systems to build post-translational synthetic circuits with split-inteins. Our systems offer a modular way to design protein-based pathways and can act as an extension to traditional genetic circuits or operate independently.

Using inteins as a computational basis is exciting for several reasons:

  • Inteins are self-catalyzing and therefore do not require any intracellular machinery to operate.
  • While inteins are found in all domains of life, they are relatively rare in any specific genome and the likelihood of host interactions is low. [Shah 2014]
  • Inteins can operate in a wide range of temperatures and environments allowing them to be used in almost any chassis (and even in-vitro).
  • Inteins are fast. Recently, four orthogonal split-inteins have been identified with reaction half-lives of less than 20 seconds. [Carvajal-Vallejos 2012]


Inteins are a pretty central part of our project so here is a quick overview of what they are and how they work:

Inteins are a type of protein that perform a self-splicing reaction to remove themselves from a host protein. This splicing reaction connects the two halves of the host protein with a peptide bond while removing the intein sequence. The host protein is usually referred to as the "extein." Since the splicing reaction changes the sequence of the extein, it can also change the structure and subsequently the function.

Figure 1 A cis-intein performs a splicing reaction to ligate two extein halves while removing itself from the protein. After splicing, the extein's enzymatic function is restored.

In nature, inteins are usually found embedded in proteins in a way that disrupts the original structure and prevents the native function of the extein. However, once the intein splices itself out, the extein regains functionality. In this way, inteins can act as a "protein switch" to activate a protein post-translationally. [Shah 2014]

Inteins come in two flavors: cis-inteins and trans-inteins (which are also called split-inteins). Cis-inteins are translated as a single piece inside of an extein while trans-inteins are translated in two separate pieces (Figure 2).

Figure 2 Two parts of a trans-intein associate through electrostatic interactions to form a bound complex. This bound complex then collapses and acts like a cis-intein to splice the extein halves together. [Shah 2013]

The beginning...

Our project development started in late May. We spent several weeks brainstorming ideas until we eventually discovered inteins, realized how cool they are, and settled on the idea of using split inteins to implement intracellular logic gates. Several previous iGEM teams inspired us including Heidelburg 2014 who used split inteins to create heat-resistant, circular proteins and Queens Canada 2018 who developed small-molecule biosensors with inteins.

Initially we viewed inteins as magical, robust, protein-splicing machines. However, as we dug into the literature we found that this is unfortunately not the case. While inteins work very well in their native environments, they are generally not very tolerable to new environments and can be difficult to work with in the lab. Early on we discovered some very important things to consider when working with inteins:

Figure 3 When splitting a protein-of-interest with an intein, there must be C,S or T in the +1 position for the splicing reaction to proceed. [Shah 2014]

Figure 4 Inteins do not tolerate changes to their native flanking sequence very well; it is important to conserve 3-5 amino acids on either side of the intein that will also be present in the spliced product. [Lockless 2009] [Amitai 2009] [Appleby-Tagoe 2011]

In order to obtain scar-less splicing, you need to find a site in the extein that closely resembles the native flanking sequence of the intein.

Engineering with inteins

Despite their switch-like behavior, there is little experimental evidence that inteins are used as regulatory proteins in nature [Shah 2014]. In the lab, however, there is nothing stopping enthusiastic synthetic biologists from engineering controllable inteins. Here we survey some recent achievements in designing controllable inteins and broadly classify them into three categories:

Class 1: Regulated cis-inteins

These are cis-inteins that cannot splice until some input is applied to change the conformation.

  • Photochemically activated splicing [Cook 1995] - A serine at the splice junction is replaced by a photocaged variant, blocking splicing until photolysis.
  • Estrogen induced splicing [Buskirk 2004] - An estrogen binding domain is introduced directly into a cis-intein sequence and specific splicing activity is restored through directed evolution.
  • Thyroid hormone induced splicing [Skretas 2005] - A thyroid hormone binding domain is introduced into the RecA mini intein and splicing activity is restored through directed evolution.
  • Temperature controlled splicing [Zeidler 2004] - Development of a temperature controlled intein.

Class 2: Proximity induced split-inteins

These are split-inteins that have a natural low affinity and must be brought close together by some type of chaperone before they can associate and splice.

  • Rapamycin-induced protein heterodimerization [Mootz 2002] - Rapamycin heterodimerizing domains are fused to split Sce VMA and induce association in the presense of rapamycin.
  • Light-induced heterodimerization [Tyszkiewicz 2008] - Two light induced heterozimerizing domains are fused to split Sce VMA to induce association.
  • Coiled-coil scaffold activation [Selgrade 2013] - Two coiled-coil domains are fused to split Sce VMA and association is induced in the presens of a coild-coil scaffold.

Class 3: High affinity controllable split-inteins

These are split inteins where the affinity is controlled rather than the ability to splice. So far, we have only found one example of this class.

  • Intein zymogens [Gramespacher 2017] - Split inteins are fused to their cognate partner with a protease recognition site that can be cleaved to "free" the intein.

Extending intein switches

All of the examples above are ways to transduce some input signal (small molecule, light, temperature, etc...) into a splicing event. Often times, a protein of interest can be split in such a way that this splicing event restores functionality and activates the protein. As a result, these constructs give bioengineers a powerful way to control protein activity post-translationally.

Figure 5 A single input induces intein splicing.

However, there are limits to the systems you can design with a single input and output. Ideally, we would be able to replicate (and exceed) the complex, post-translational pathways observed in nature. This is one of the main goals of synthetic biology after all.

Figure 6 A three-input synthetic pathway allows more complex control of protein activation.

We approached this challenge by designing an intein-based logic gate framework that can be used to implement fully post-translational synthetic circuits.

Digital logic

Digital logic is the basis of our design. In digital logic, values can exist in two states: on/off, 1/0, true/false... In electrical circuits, values are stored as high or low voltages. In our intein-based circuits, values are represented by the presence (or absense) of intermediate reactants.

Logic gates are a way to represent operations on these values. We focused primarily on the two basic two-input logic gates AND and OR:

Figure 7 An AND gate is active only when both inputs are active. In this diagram, both inputs need to be present to induce splicing of the target protein.
Figure 8 An OR gate is active when either input is active (or both). In this diagram, either input is enough to induce splicing of the target protein.

Pitteins Part 1 - "Nested inteins"

In our initial design, we were interested in exploring multi-input AND gates.

A single split intein can trivially be thought of as a two-input AND gate:

Figure 9 A single split-intein acts as an AND gate: if either half is missing, splicing cannot occur.

To add another AND gate to this circuit, we asked the question: "can you split a split-intein?" Our goal for this design was to take a second split-intein (in blue below) and fragment one of the original halves such that we can reconstruct it after splicing.

Figure 10 A second split intein is used to reconstruct one of the original inputs. Int-C is split into two pieces (Int-C 1/2 and Int-C 2/2). These fragments are spliced together by the second split intein (in blue) to form Int-C full. At this point, Int-C full can react with Int-N in a standard split-intein splicing reaction.

Now if we incorporate these constructs in the original circuit, we obtain the following:

Figure 11 The two blue intein fragments splice to reconstruct the gray Int-C fragment. This fragment then associates and splices with the Int-N fragment to splice the final target protein. This circuit is equivalent to a three-input AND gate where all inputs must be present to activate the gate.

By symmetry, we could also imagine splitting the Int-N half with a third split-intein (in orange) as follows:

Figure 12 A two-layer circuit with three AND gates (eqiuvalent to a four-input AND gate). Blue inteins splice to form the original Int-C construct. Orange inteins splice to form the original Int-N construct. If both the gray Int-C and the Int-N constructs are present, the target protein is spliced.

Pitteins Part 2 - "Split-linker" system

During the design and devlopment of our first system, we realized that it is relatively difficult to identify good location to split an extein. Essentially, there is a necessary comprimise between maintaining the extein sequence and maintaning the intein's flanking sequence. In order to circumvent this problem, we designed a second type of system where native flanking sequences can be preserved.

Our work is largely inspired by literature on the "proximity induced" Sce VMA split intein (see class 2 inteins above). Here are two papers highlighting the applications of this intein:

Figure 13 The Sce VMA split intein is fused to two rapamycin heterodimerizing domains that bind in the presence of rapamycin and induce association of VMA. [Mootz 2002]

Figure 14 The Sce VMA split intein is fused to two coiled-coil domains that bind to a coiled-coil "scaffold" and induce association of VMA. [Selgrade 2013]

We propose a similar system where the Sce VMA intein is fused to split inteins that splice in series to form a glycine-serine linker:

Figure 15 Three orthogonal split-inteins (yellow, green, blue) are configured to splice a linker in series. The dark line between intein fragments represents a flexible, glycine-serine linker that contains the native flanking sequences. The two gray inteins on either side are the Sce VMA split-intein. After all of the intermediate inteins splice, these inteins will be covalently connected with a glycine-serine linker and the barrier to associate and splice is much lower.

After the internal split-inteins react (yellow, green and blue in figure 15), the Sce VMA split-intein halves are covalently connected, in essense forming a pseudo-cis-intein:

Figure 16 After splicing, the Sce VMA split inteins are covalently connected with a flexible glycine-serine linker, forming a pseudo-cis-intein.

This cis-intein then automatically splices to activate the protein of interest. In this way, we preserve all native flanking sequences and are able to introduce more inteins while only splitting the target protein at one spot.

As a circuit, the split linker system implements multi-input AND logic:

Figure 17 The split-linker system implements multi-input AND logic. In this example, all four parts are required to form the pseudo-cis intermediate and spliced product.


[Amitai 2009]

Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G., & Belfort, M. (2009). Modulation of intein activity by its neighboring extein substrates. Proceedings of the National Academy of Sciences, 106(27), 11005-11010.

[Appleby-Tagoe 2011]

Appleby-Tagoe, J. H., Thiel, I. V., Wang, Y., Wang, Y., Mootz, H. D., & Liu, X. Q. (2011). Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. Journal of Biological Chemistry, 286(39), 34440-34447.

[Brophy 2014]

Brophy, J. A. N., & Voigt, C. A. (2014). Principles of genetic circuit design. Nature Methods, 11(5), 508-520.

[Buskirk 2004]

Buskirk, A. R., Ong, Y.-C., Gartner, Z. J., & Liu, D. R. (2004). Directed evolution of ligand dependence: Small-molecule-activated protein splicing. Proceedings of the National Academy of Sciences, 101(29), 10505-10510.

[Carvajal-Vallejos 2012]

Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D., & Schmidt, S. R. (2012). Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. Journal of Biological Chemistry, 287(34), 28686-28696.

[Cook 1995]

Cook, S.N., Jack, W.E., Xiong, X.F., Danley, L.E., Ellman, J.A., Schultz, P.G., and Noren, C.J. (1995). Photochemically initiated protein splicing. Angew. Chem. Int. Ed. 34, 1629-1630

[Gramespacher 2017]

Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. Journal of the American Chemical Society, 139(24), 8074-8077.

[Lockless 2009]

Lockless, S. W., & Muir, T. W. (2009). Traceless protein splicing utilizing evolved split inteins. Proceedings of the National Academy of Sciences of the United States of America, 106(27), 10999-11004.

[Mootz 2002]

Mootz, H. D., & Muir, T. W. (2002). Protein splicing triggered by a small molecule. Journal of the American Chemical Society, 124(31), 9044-5.

[Selgrade 2012]

Selgrade, D. F., Lohmueller, J. J., Lienert, F., & Silver, P. A. (2013). Protein scaffold-activated protein trans-splicing in mammalian cells. Journal of the American Chemical Society, 135(20), 7713-7719.

[Shah 2012]

Shah, N. H., Dann, G. P., Vila-Perelló, M., Liu, Z., & Muir, T. W. (2012). Ultrafast protein splicing is common among cyanobacterial split inteins: Implications for protein engineering. Journal of the American Chemical Society, 134(28), 11338-11341.

[Shah 2013]

Shah, N. H., Eryilmaz, E., Cowburn, D., & Muir, T. W. (2013). Naturally split inteins assemble through a "capture and collapse" mechanism. Journal of the American Chemical Society, 135(49), 18673-18681.

[Shah 2014]

Shah, N. H., & Muir, T. W. (2014). Inteins: Nature's gift to protein chemists. Chemical Science, 5(2), 446-461.

[Skretas 2005]

Skretas, G. and Wood, D.W. (2005). Regulation of protein activity with small-molecule-controlled inteins. Protein Sci. 14, 523-532.

[Tyszkiewicz 2008]

Tyszkiewicz, A. B., & Muir, T. W. (2008). Activation of protein splicing with light in yeast. Nature Methods, 5(4), 303-305.

[Zeidler 2004]

Zeidler, M. P., Tan, C., Bellaiche, Y., Cherry, S., Häder, S., Gayko, U., & Perrimon, N. (2004). Temperature-sensitive control of protein activity by conditionally splicing inteins. Nature Biotechnology, 22(7), 871-876.