Team:BIT-China/Design
Basic Circuits
○ Resistance subsystem
In this circuits, three functional genes, NhaA, NhaB and glsA, are employed for resisting both acidic and alkaline environment. NhaA and NhaB can encode two kinds of Na+/H+ antiporter, ensuring the survival of bacteria under alkaline circumstances. Under acidic condition, bacteria can maintain homeostasis due to the function of glsA (details in project). Since all these proteins of functional genes can be regulated by pH (details in project), the strong constitutive promoter J23119 is applied to guarantee the steady transcription of three functional genes. Thus, our resistance subsystem can make E.coli survive in an expanded pH range.
○ Regulation subsystem
In this subsystem, two pH-responsive promoters, P-asr and P-atp2, are employed. P-asr induces transcription in acidic environment from pH 4.0 ~ 5.0, and shows low activity at neutral pH. P-atp2 can respond at pH 7.0 ~ 9.0, especially at alkaline pH. These two pH-responsive promoters are applied to open the synthesis of acid and alkali. ldhA encodes an lactate dehydrogenase (ldh) which converts pyruvate to lactate. gadA, one of a functional genes for producing alkali, is used in our circuit to convert glutamate to GABA(γ-amino butyric acid), a certain kind of alkaline substance. This subsystem is used to regulate the external environment pH.
Fine-regulation circuits
○ Cre-LoxP & Flp-FRT circuits
In fermentation process, the environment usually turn acidic, so we produce alkali (γ-aminobutyric acid, GABA) in the primary state. And alkali-induced promoter P-atp2 and acid-induced promoter P-asr are employed to produce the recombinase Cre and Flp respectively. When the pH of environment reaches the P-atp2’s responsive range, Cre would express and recognize the LoxP sites at both side of strong constitutive promoter J23119 and J23119 would be inverted. Therefore the bacteria stop producing GABA and start the product of lactate. Similarly, when the pH of environment reaches the P-asr’s responsive range, J23119 would be inverted again. The bacteria will produce GABA again. Through the combine of multiple LoxP sites and FTR sites, we can realize multi-regulation and fine-regulation. To prevent gene from wrong inverting, we designed two kinds of sRNA to inhibit Cre and Flp’s function. When one of recombinases expresses, the sRNA for the other recombinase will be produced at the same time. The sRNA would inhibit the translation of the other recombinase and avoid the wrong inversion. sRNA consists of two parts, the scaffold structure MicC and a target-binding sequence. Target-binding sequence, an anti-sequence about 15~20 bp, can inhibit the translation through binding with mRNA. These circuits are used when the functional acid and the alkali are both medium.
○ Bxb1 circuit
Bxb1 integrase is a DNA recombinase (details in project page). In the primary state, the bacteria also produce alkali. Since the alkali is strong, the pH of environment could reach the responsible range of P-atp2 easily. It can lead Bxb1 to work. The promoter J23119 could be inverted persistently until the inverted efficacy reaches half and half. In this process, the bacteria produce the acid and the alkali at the same time, it can create a wave-motion of pH. And the pH of environment will stable finally. The final pH is influenced by the promoter sensing pH and the strength of acid and alkali. This circuit is applied when the functional acid and alkali are both strong.
○ FimE circuit
FimE is different from other kind’s recombinase. Its recognized sites on the upstream (IRL) and downstream (IRR) are not same. It means that after the recombination mediated by FimE happens, the sign sites would be exchanged, so that the new sign sites couldn’t be recognized by FimE again. So the recombination mediated by FimE is irreversible. Granted that the bacteria produce the strong alkali in the primary, the environment would turn alkaline quickly. So P-atp2 can correspond the change of environment and start the expression of FimE. FimE recognizes IRL and IRR, then catalyze the inversion of J23119. The weak acid would be generated at that time. For the environment, due to the production of the weak acid, it would turn neutral gradually.
Basic circuits model
○ Overview of Basic model
Modeling of basic circuits is divided into two parts: resistance subsystem model and regulation subsystem model. The main processes analyzed in models include promoter induction, transcription, translation, catalytic process of enzymes and the effects of products on environmental. Differential equations are applied to simulate the concentration change of all substances. Factors considered in our model are reversible reaction and materials production and degradation.
○ Resistance Model
In resistance subsystem, the constitutive promoter J23119 is applied, and we focused on the efficiency of three functional genes (Fig.1). GlsA encodes glutaminase which can catalyze the transformation from glutamine to glutamate and ammonia. Ammonia will neutralize the intracellular H+. The alkali-resistance functional genes NhaA and NhaB encode two types of Na+/H+ antiporters. These antiporters catalyze the exchange of sodium ions for protons and therefore maintain the homeostasis of bacteria.
Fig.1 Basic resistance circuit of pH Controller.
In resistance subsystem model, three key processes considered are the transcription and translation of two functional genes and the degradation of their products.
Three resistance devices’ models were constructed separately. To prove the function of these devices, we set up initial intracellular pH to 3.0 in program to examine the function of GlsA, while the initial pH was set to 9.0 to examine the function of NhaA and NhaB antiporters.
Results show that resistance subsystem can regulate intracellular pH to a suitable range (Fig.2, 3, 4).
Fig.2 Intracellular pH is regulated by acid resistance device. To prove the function of this device, initial pH is set up to 3.0 in program. pH can effect activity of GlsA, while GlsA conducts gaseous ammonia production. With the enzyme activity changing, pH will be finally stabilized to a suitable level.
Fig.3 Intracellular pH change regulated by alkali resistance device led by NhaB. NhaB is not effected by pH, and can assist NhaA to regulate alkaline environment. Here we assumed that initial pH was 9.0 and NhaB had already been activated.
Fig.4 Intracellular pH change regulated by alkali resistance device led by NhaA. NhaA will be induced at pH 7.0 ~ 8.2, while NhaB will function when NhaA cannot function normally. Here we assumed that initial pH was 8.0 and NhaA had already been activated.
○ Regulation Model
As for regulation subsystem model, pH-responsive promoters are combined with functional genes. The acid regulation device consists of acid-responsive promoter P-asr and alkali-synthesis gene gadA (Fig.5). gadA catalyzes the proton-consuming conversion of glutamate to γ-aminobutyric acid (GABA), and GABA can be excreted outside and neutralize the micro-environment.
The alkali regulation device is made up of alkali-responsive promoter P-atp2 and functional gene ldhA (Fig.6). ldhA encodes a lactate dehydrogenase which converts pyruvate to lactate. Lactate will be secreted outside the cell and neutralize the OH-.
We assumed that the H+ or OH- was straightly contacted with the promoters and induced the transcription process. For functional genes, their translation, degradation and catalysis processes are mainly analyzed in this model because the products of these two genes are both enzymes.
The final simulation results are shown below (Fig.7). In acidic condition, the regulation subsystem will produce GABA and reduce the extracellular pH. When environmental pH becomes alkaline, regulation subsystem will make lactate to neutralize the outside pH. Eventually the environmental pH will be constrained between the pH range where two promoters are both repressed (but with fluctuation).
Fig.7 Extracellular pH regulated by basic regulation subsystem. In normal fermentation environment, pH will tend to decrease and enter acidic level. Basing on this phenomenon, we assumed that the initial pH was 4.0, and our device was functioning.
Fine-regulation circuits models
Overview of Fine-regulation Circuits Model
Three fine regulation circuits are designed so that we can apply different regulation circuits according to different fermentation situations. The external environment can be sensed by the pH-responsive promoters. We ligated different kinds of recombinase down pH-responsive promoters. The recombinase can invert downstream constitutive promoter. This function is applied for conversion between acid and alkali parts.
Main processes considered in this model are similar to basic circuit model, this model is special for its cyclic processes. To simulate the inversion function of recombinase, we need to switch different groups of equations by detecting some key values of pH.
○ Cre/Flp regulation circuit
Cre-LoxP and Flp-FRT circuits are shown in Figure.8. To describe the function of recombinases, we set cyclic processes in our model.
Fig.8 Gene circuits of Cre/Flp regulation system
Our results show that with the application of Cre/Flp circuit, pH Controller can achieve automatic switch in acid and alkali production and stabilize environmental pH level (Fig.9).
Fig.9 Extracellular pH regulated by Cre/Flp regulation circuit. The final pH level fluctuates between the range where both promoters are repressed. To prove the function of circuit, initial environmental pH was set up to 8.0, indicating that our circuit had already been induced.
○ Bxb1 Regulation Circuit
The second fine-regulation circuit is led by recombinase Bxb1 (Fig.10). Bxb1 can invert the constitutive promoter to both sides, thus, the eventual pH level is determined by strength of both acid and alkali.
Fig.10 Gene circuit of Bxb1
Here we assumed that Bxb1 had reached stable condition, where the inversion efficiency of Bxb1 to both sides were the same. Results show that with the Bxb1 regulation circuit, pH could be regulated to a suitable level (Fig.11).
Fig.11 pH level change led by Bxb1 regulation circuit. Bxb1 can invert the constitutive promoter to both sides, thus, the eventual pH level is determined by strength of both acid and alkali. To prove the function of circuit, initial environmental pH was set up to 8.0, indicating that our circuit had already been induced. Eventual pH is determined by functional genes, and can reach other value if functional genes are replaced.
○ FimE Regulation Circuit
The last fine-regulation circuit is led by recombinase FimE (Fig.12). This recombinase can only invert the promoter for one time.
Fig.12 Gene circuits of FimE.
Results prove that with the application of FimE regulation circuit, pH Controller can regulate environmental pH to the level determined by the strength of acid (Fig.13). ldhA could be replaced by other functional genes which could produce acid with different strength.
Fig.13 pH level change led by FimE regulation circuit. J23119 conducts the transcription of ldhA which catalyzes lactic acid production. To prove the function of circuit, initial environmental pH was set up to 8.0, indicating that our circuit had already been induced. Eventually, extracellular pH is determined by strength of acid.