Engineering

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1. The determination of the core loop

Just like Design page statement, many parts have been designed to implement the "transport-Chelation-Redox reaction" system and Blue light regulated suicide system. However, due to the CO0VID-19, we do not have enough time to verify all parts. we made full use of the limited time to construct all proteins of the "transport-chelation-Redox reaction" system and verified hMT-1A (BBA_K3452000), APX2(BBa_K3452020),and MntH(BBA_K3452602).(See 1.4 for more details)

NAME	DESCRIPTION	CATEGORY	FUNCTION
BBA_K3452000	hMT-1A	chemoattractant protein	Chelating Cadmium to improve tolerance
BBA_K3452020	APX2	antioxidant protein	Reducing reactive oxygen species (ROS) to avoid apoptosis
BBA_K3452602	MntH	transport protein	Transporting Cadmium into cells

Table 1: the summary of the new part

we achieved “engineering success” in three different aspects including core loop, auxiliary loop and experimental methods by following the engineering design cycle, which are described minutely as follows:

Figure1: The core “transport-chelation-redox reaction” system of our project

1.1 Identification of core ideas

The choice of engineering algae: our initial idea is to design an engineering algae or engineering bacteria, we found that the Synechocystis and Anabaena themselves have a certain degree of cadmium tolerance and adsorption, and for prokaryotic organisms, transformation is relatively easy, the laboratory also has its corresponding growth conditions, so it is expected to select from the two. After laboratory culture found Anabaena, the low growth of Synechocystis sp. PCC6803. was finally identified as a candidate for transformation.

1.2 Determining the functions of our engineered algae

In order to have a high spheresorption efficiency, we thought of a way to chelate Cd on the cell wall and then transport it efficiently into the algae cell. However, high toxicity can cause cells to die rapidly and release Cd due to intracellular redox imbalance, photosynthetic failure, and blocked protein synthesis. Our cytosolic algae must be both highly absorbable and highly tolerant. We have considered magnetosomes: magnetosomes are enzyme systems that generate magnetosomes from magnetostrophic bacteria and mineralize cadmium ions to preserve them in magnetosomes[6]. However, the enzyme system is very complicated. There are some difficulties in control. If we use magnetotactic bacteria as a cadmium container, the culture conditions are harsh. We also thought of nitrogen fixation enzymes. Some nitrogen fixation enzymes can reduce some metal ions, including cadmium ions. But the current difficulty is that there are no existing efficient enzymes for cadmium reduction nitrogen fixation. The reduction of metal ions is not very specific, and we believe that next year's team has the opportunity to carry out targeted screening. Finally, we thought of intracellular chelating and redox proteins, efficient and straightforward to improve the cadmium tolerance.(Figure1)

1.3 Protein selection and determination

Protein selection and identification: each function can be screened by the literature to many proteins. To narrow down the scope, we considered the following aspects:

The protein has better cadmium specificity, rather than being able to widely absorb all heavy metal ions
The protein has the literature to prove that it can be expressed in prokaryotes, and the expression effect is more satisfactory, preferably in cyanobacteria
The mechanism of the Already relatively straightforward and known functions perform efficiently

Taking all these considerations into account, we finally selected nine functional proteins(please see the "Design" part)(Figure 2)

Figure2 : our plasmids with different resistance

In addition to this, our proteins were expected to be cadmium initiated, so we also looked into the cadmium promoter. Then, we realized that High Cd starts to express the chelat protein and transport protein will result in a delay. It may not be absorbed when it meets cadmium, and as the concentration of the Cd is absorbed decreases. The cadmium initiated protein expression may also be affected, so we still ended up taking the regular promoter to initiate expression and simplify the system as much as possible.

1.4 Validation of protein function

To demonstrate the feasibility of our components, we have successfully designed and constructed transfer, chelating, and antioxidant components, and designed experiments to verify their function:

We successfully constructed plasmids containing transport proteins (MntH, NARMP5, IRT1), chemoattractant proteins (SmtA, hMT-1A, TMCd1), and antioxidant proteins (APX2, LOX1), and successfully expressed them in E. coli DH5α (figure3. a), and the analysis showed that the expression of SmtA, hMT-1A, TMCd1, APX2 increased the cadmium tolerance of E. coli (figure3. b, c). It is noteworthy that the expression of MntH alone increased the efficiency of cadmium uptake by DH5α, but decreased the cadmium tolerance of DH5α (figure3. d, e). In order to increase the cadmium uptake capacity and ensure the cadmium tolerance of E. coli, we co-expressed MntH, TMCd1 and APX2 in the same E. coli, and the results showed that the cadmium tolerance and removal efficiency were greatly enhanced (figure3. d, e).

Figure3: (a) WB proves the successful expression of our protein, (b) Growth curves of chelated protein (SmtA, hMT-1A, TMCd1)-expressed E. coli, (c) Growth curves of APX2-expressed E. coli, (d) Cadmium uptake curve of (MntH / hMT-1A / MntH & TMCd1 & APX2)-expressed E. coli, (e) Growth curves of (MntH / hMT-1A / MntH & TMCd1 & APX2)-expressed E. coli.

1.5 Security and fixation considerations

Since our algae spread around when placed directly in the water column, the prokaryotic organism's small size makes it impossible to control and capture effectively. We thought of installing it in a fixture with a filter membrane, which is easy to salvage. Still, the development of the fixture had to take into account the design of the filter holes, which is not accessible to mass production. We decided to use the most environmentally friendly and standard immobilization method: Calcium-Alginate Microbeads (Ca-Alg MBs). They are easy to use. Preparation, diameter and other data are also easy to obtain and modeling. Algae in Ca-Alg MBs also have better adaptability. Nutrients and cadmium can be absorbed by the algae through it. However, we later realized that the Ca-Alg MBs itself has a certain degree of separation and adsorption. In the experimental principle's rigor, we measure and analyze the cadmium ion absorption of these aspects: the Ca-Alg MBs, the algae, and the algae MBs.

Also, the possibility of the algae escaping from the specific Ca-Alg MBs environment has to be taken into account, which is why we have improved the supporting ideas (see the auxiliary loop).

1.6 Recycling of Ca-Alg MBs

Our original idea was to burn the salvaged globs to destroy the cell walls and globs and recover the cadmium, but given that a large part of the cadmium contamination is from atmospheric pollution, this method was ruled out due to the possibility of gas leakage. Thus, we will concentrate MBs in a confined device, add the right amount of sodium citrate hot solution to dissolve the Ca-Alg MBs. Finally, the closed device will be handed over to the cadmium treatment plant for proper recycling.

2. The determination of auxiliary loop

In order to achieve the purpose of biosafety, we designed and improved the auxiliary loop for many times, and finally determined the final toxin-antitoxin system under blue light regulation by means of modeling and experiment. The specific improvement measures are as follows:

2.1 First-generation system: nutrition-deficient symbiotic system

Figure4: First-generation system the Nutrition-deficient symbiotic system. The system consists of tryptophan-deficient Synechocystis sp.PCC6803 and a bacterium complementary to his nutrients, thus our system can only survive when both are present together, which ensures the biosafety.

In order to restrict our engineered algae to a specific environment and reduce environmental pollution, we designed our first-generation nutrient-deficient symbiosis system (Figure 4), which consists of a leucine-deficient tryptophan-producing bacterium that senses the concentration of cadmium in water and a leucine-producing tryptophan-deficient Synechocystis sp.PCC6803 which can efficiently removes cadmium from water body, which are embedded in sodium alginate together. Under normal circumstances, the two express mutually defective amino acids to complement each other and grow together normally; but when the engineered algae escape, they die due to the lack of tryptophan, thus achieving biosecurity. However, subsequent review of the literature and discussion revealed the following problems with this system:

The symbiotic relationship between bacteria and algae is uncertain
In engineered algae, where cadmium removal is the core purpose, the symbiotic system is overly cumbersome and introduces organisms other than engineered algae
The loop is too complex to be expressed in the prokaryote needs to be confirmed
The pore size of the cadmium-transport protein is designed to allow access to amino acids and it is uncertain whether it interferes with the pore size of the cadmium-transport protein

Based on the above 4 points, we have improved the auxiliary system and designed the following second-generation system.

2.2 Second-generation system: Red and blue light control system

Figure5: Second-generation system the Red and blue light control system. HTH-LOV-REP: the Blue light receptor protein, PhyB: the red light receptor protein

In order to avoid the complexity of regulation and interference from other organisms, we turned our thoughts to the regulation of a simple photoreceptor system. After reviewing the literature, we selected PhyB and LOV proteins from a large number of photoreceptor proteins as red and blue light-sensing proteins, respectively, and designed the following red-blue light control second-generation system (Figure5)[7]. In this system, the algae grow normally and are unaffected by blue light or red light + blue light; when and only when red light is present, the algae initiate an external excretion system to discharge the intracellular cadmium to outside; when no light or low light is present, the algae express the toxin RelE in large quantities and initiate a suicide system. The above system seems to be perfect, it can prevent the escape of the algae and discharge cadmium to reuse the algae, but it is obvious that it is more complicated than our first generation system, and whether through experiments, modeling or theoretical verification are more difficult, so we made the following simplification, and the final three generations of the system was determined

2.3 Third-generation system: Blue light regulated suicide system

Figure6: Third-generation system the Blue light regulated suicide system. The suicide system is regulated by blue light. In the presence of blue light, HTH-LOV-REP dimerizes and inhibits the expression of the toxin RelE, and when blue light is lost, RelE is abundantly expressed and kills the algae. HTH-LOV-REP: the Blue light receptor protein, RelE: toxin, RelB: antitoxin

Based on the complexity of red and blue light regulation and the uneconomical of reusing algae, we have simplified the above system by eliminating the Cadmium efflux system, which is under the control of red light, and establish our final auxiliary idea (Figure 6). The system is only regulated by blue light. When blue light is present, the expression of toxin RelE is inhibited, and Synechocystis sp.PCC6803 grow normally[8]. However, when it escapes from the environment that provides artificial blue light, the toxin is expressed in large quantities and the engineered algae will be killed quickly, thus reducing the harm to the environment. At the same time, the system was verified by mathematical modeling (Figure4a/b). Modeling shows that when blue light was irradiated, the toxin RelE content decreases rapidly over time. However, when they escape from the artificial light environment, the toxin content increases rapidly with the increase of distance, and finally kills the engineered algae to play a vital role in environmental protection.

3. The Improvement of experimental methods

In the previous experiments, due to the problem of plasmid carrier, we spent a lot of time in the construction of plasmid. Moreover, unlike E. coli, the culture, growth observation and transformation of Synechocystis sp.PCC6803 require constant exploration. Therefore, we made a lot of mistakes in this process, but we also made continuous improvements.

3.1 Transformation of Synechocystis sp.PCC6803

Due to the transformation of Synechocystis sp.PCC6803 is different from E. coil, in our early experiments we use the same way in E. coil to directly transform of Synechocystis sp.PCC6803, but has not gotten the result we want. Through a review of the literature and the guidance of our teachers, we switched to a triple-philic system for the transformation of the cytosolic algae by the carrier plasmid (pRL-443), auxiliary plasmid (PRL-623) and splice plasmid (Prl-25C-chyb) and finally achieved success.

3.2 The selection of chassis

Due to a lag in transformation, we were unable to quickly obtain the engineered algae used to test the efficiency of cadmium uptake, so we turned to E. coli, which also is a prokaryotic organism, to validate our components first in it. It was with this shift that we successfully demonstrated that our components can express and enhance cadmium uptake in prokaryotes.

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