iGEM 2020 — Team CSU_CHINA

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSU CHINA/Notebook

Proof of Concept

![](https://static.igem.org/mediawiki/2020/3/3a/T--CSU_CHINA--Logo.png)

CSU\_CHINA

[HOME](https://2020.igem.org/Team%3ACSU_CHINA)

PROJECT

[Description](https://2020.igem.org/Team%3ACSU_CHINA/Description)
[Design](https://2020.igem.org/Team%3ACSU_CHINA/Design)
[Results](https://2020.igem.org/Team%3ACSU_CHINA/Results)
[Proposed Implementation](https://2020.igem.org/Team%3ACSU_CHINA/Implementation)
[Proof of Concept](https://2020.igem.org/Team%3ACSU_CHINA/Proof_Of_Concept)

TEAM

[Team Members](https://2020.igem.org/Team%3ACSU_CHINA/Team)
[Attributions](https://2020.igem.org/Team%3ACSU_CHINA/Attributions)
[Collaborations](https://2020.igem.org/Team%3ACSU_CHINA/Collaborations)
[Partnership](https://2020.igem.org/Team%3ACSU_CHINA/Partnership)

LAB

[Experiments](https://2020.igem.org/Team%3ACSU_CHINA/Experiments)
[Notebook](https://2020.igem.org/Team%3ACSU_CHINA/Notebook)

PARTS

[Parts](https://2020.igem.org/Team%3ACSU_CHINA/Parts)
[Contribution](https://2020.igem.org/Team%3ACSU_CHINA/Contribution)
[Engineering Success](https://2020.igem.org/Team%3ACSU_CHINA/Engineering)

[MODEL](https://2020.igem.org/Team%3ACSU_CHINA/Model)

HUMAN PRACTICES

[Integrated Human Practices](https://2020.igem.org/Team%3ACSU_CHINA/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3ACSU_CHINA/Education)

[SAFETY](https://2020.igem.org/Team%3ACSU_CHINA/Safety)
[JUDGING FORM](https://igem.org/2020_Judging_Form?id=3452)

iGEM 2020

# Notebook

- Model -

## Week 1: (16-22 August)

After having identified the components that needed to be transferred to the engineered algae, our first task was to build them into plasmids that could be transferred to the aggregates. We used the enzyme digestion method to attach the component fragments purchased from GenScript to the PVZ322 plasmid.

## Week 2: (23-29 August)

To obtain a large amount of alga, 1ml of alga (OD=1) was pipetted into 40ml BG11 liquid medium in a 50ml centrifuge tube for propagation.

To determine whether the plasmid PVZ322 can be expressed in our synechocystis, we attempted to use natural transformation to transform the empty pVZ322 plasmid.

| Synechocystis | OD=0.8, 200μL |
| --- | --- |
| PVZ322 | 100ngμL, 50μL |
| Transformation Time | 6h |
| Medium | BG11, 1.5%Agar, sugar-free |

The new algal fluid turned green after 12 days, and the result was: no algal blooms on the resistant BG11 plate, indicating that the algal transformation method or operation was wrong.

## Week 3: (6-12 September)

In order to shorten the culture cycle of synechocystis, we pipetted algae seeds in the sugar-free BG11 liquid medium and 0.9% glucose BG11 liquid medium at a scale of 1:10.In order to eliminate the problem of conversion operation, we added parallel group, negative control and positive control.

The algal in sugar-free BG11 liquid medium turned green after 7 days, and the algal fluid in 0.9% glucose BG11 liquid medium turned green after 3 days, but one tube was infected with bacteria. The results showed that the sugar medium could shorten the culture period obviously, but increased the risk of bacterial infections.

## Week 4: (13-19 September)

To successfully transform the plasmid PVZ322 into the synechocystis, we improved the transformation method and used the solid medium BG11 with sugar to culture and screen the transformed algae.

| Synechocystis | OD=0.8, 200μL |
| --- | --- |
| PVZ322 | 100ng/μL, 50μL |
| Transformation Time | 6h |
| Medium | BG11, 1.5%Agar, 0.9% sugar |

To know exactly the growth period of wild-type synechocystis, we took samples every 1 day to detect the absorbance at 730nm.

| TIME(H) | OD730 |
| --- | --- |
| 0 | 0.754 |
| 29.5 | 1.052 |
| 48.5 | 1.298 |
| 71 | 1.4 |
| 130.5 | 2.09 |
| 185.5 | 2.45 |
| 209.5 | 2.47 |
| 233.2 | 2.51 |

![](https://static.igem.org/mediawiki/2020/7/75/T--CSU_CHINA--fig1.png)

The results showed that in our culture conditions, when the initial OD was 0.8, the synechocystis could be used for transformation for culturing about 30h.

## Week 5: (20-26 September)

We continued to measure OD of several bottles of new synechocystis to obtain more accurate growth curve.

Since the last transformation was not successful, the method still needs to be improved.

| Synechocystis | OD=0.8, 400μL |
| --- | --- |
| PVZ322 | 100ng/μL, 100μL |
| Transformation Time | 6h |
| Medium | BG11, 1.5%Agar, 0.9% sugar |

## Week 6: (27 September-3 October)

We went on to determine the OD value of synechocystis in the previous week.

Since the last transformation was not successful, the method still needs to be improved:

| Synechocystis | OD=0.8, 400μL | OD=0.8, 400μL |
| --- | --- | --- |
| Mixed Cellulose Ester | With Mixed Cellulose Ester | Without Mixed Cellulose Ester |
| PVZ322 | 100ng/μL, 100μL | 100ng/μL, 100μL |
| Transformation Time | 6h | 6h |
| Medium | BG11, 1.5%Agar, 0.9% sugar | BG11, 1.5%Agar, 0.9% sugar |

## Week 7: (4-10 October)

We attempted to test the cadmium tolerance curve of wild-type synechocystis.

Surprisingly, we stumbled upon the fact that the PVZ322 plasmid could not be transformed naturally, so trisophily conjugation was used.

## Week 8: (11-17 October)

10.12.

1. Test the availability of cadmium determination reagent kit

The sample order：A(1-6) standard sample in kit B(1-4) standard sample by me

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| A | 0 | 0.4 | 1.2 | 4.7 | 10.4 | 32.8 |
| B | 1 | 1 | 1 | 1 | \ | \ |

2. Result of availability (availability test1):

![](https://static.igem.org/mediawiki/2020/7/77/T--CSU_CHINA--fig3.png)

Theoretical concentration:0.976179401

Excuse me? The curve is absolutely upside down, but I got the right concentration?

Do it again the other day! With a new kit! (this one must be polluted by my dear friends)

10.13

1. different densities of Synechocystis deal with cadmium

50mL / OD=0.17, 0.46, 0.5, 0.61, 0.78 / deal with cadmium of 0.1mg/L (density gradient test1)

50mL medium with 5μL of 1g/L Cd2+ stock

Take sample after 0.5h,1h,1.5h,2h,4h,12h

2. prepare new generation of Synechocystis for futher use (We are short of Synechocystis !)

3. prepare SA and CaCl2 stock----200mL

4. Immobilize Synechocystis experiment(MBs test1)

50 Ca-Alg-MBs, 50 Ca-Alg-MBs with Synechocystis

Take sample after 0.5h,1h,1.5h,2h,4h,12h>

5. Result of availability (availability test2)

![](https://static.igem.org/mediawiki/2020/2/23/T--CSU_CHINA--fig4.png)

Oh, my holy \*\*\*\*, why the curve is always, always upside down!

I need a new kit!

10.14

Neaten all the Synechocystis we have:

OD=0.9----100mL

OD=0.5----50mL

OD=1.2----50mL

OD=1.0----50mL

1. different densities of Synechocystis deal with cadmium

30mL / OD=0.42, 0.62, 0.89, 1.16, 1.34, 1.45 / deal with cadmium of 0.1mg/L (density gradient test2)

2. Synechocystis deal with different concentration of cadmium

30mL / Initial OD=0.7 / 0.01,0.1,0.5,1,2 mg/L Cd (Cd gradient test1)

3. gain sample of medium in (density gradient test1) and (MBs test1)

4.finally! a new kit!

![](https://static.igem.org/mediawiki/2020/f/f1/T--CSU_CHINA--fig5.png)

What a beautiful curve!

10.15

Determination of the samples before

![](https://static.igem.org/mediawiki/2020/9/98/T--CSU_CHINA--fig2.png)
![](https://static.igem.org/mediawiki/2020/d/d4/T--CSU_CHINA--Nbfig6.png)
![](https://static.igem.org/mediawiki/2020/e/ef/T--CSU_CHINA--Nbfig7.png)
![](https://static.igem.org/mediawiki/2020/b/b4/T--CSU_CHINA--Nbfig8.png)
![](https://static.igem.org/mediawiki/2020/d/de/T--CSU_CHINA--Nbfig9.png)

Emmm, why we get so awful results out of so beautiful standard curve

I guess the protein in the solution will affect the kit, the experiment operation is also important. Discard the data, try it again

10.16

Try to determine abnormal data gained in yesterday

![](https://static.igem.org/mediawiki/2020/c/c8/T--CSU_CHINA--Nbfig10.png)

Emmm, even worse.

Don’t want to do more experiment today.

10.17

Do every experiment once again

density gradient test3: OD=0.42, 0.62, 0.89, 1.16, 1.34 (0.1mg/L, 2mg/L)

Cd gradient test2: 0.01, 0.1, 0.5, 1, 2 (OD=0.7)

MBs test2: 30mL/ 50 MBs with PCC6803+50 MBs+ PCC6803 / 0.1mg/L+ 2mg/L Cd2+

## Week 9: (18-25 October)

10.18

Gain the results seems to make sense(density gradient test3)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSU CHINA/Engineering

Engineering

![](https://static.igem.org/mediawiki/2020/3/3a/T--CSU_CHINA--Logo.png)

CSU\_CHINA

[HOME](https://2020.igem.org/Team%3ACSU_CHINA)

PROJECT

[Description](https://2020.igem.org/Team%3ACSU_CHINA/Description)
[Design](https://2020.igem.org/Team%3ACSU_CHINA/Design)
[Results](https://2020.igem.org/Team%3ACSU_CHINA/Results)
[Proposed Implementation](https://2020.igem.org/Team%3ACSU_CHINA/Implementation)
[Proof of Concept](https://2020.igem.org/Team%3ACSU_CHINA/Proof_Of_Concept)

TEAM

[Team Members](https://2020.igem.org/Team%3ACSU_CHINA/Team)
[Attributions](https://2020.igem.org/Team%3ACSU_CHINA/Attributions)
[Collaborations](https://2020.igem.org/Team%3ACSU_CHINA/Collaborations)
[Partnership](https://2020.igem.org/Team%3ACSU_CHINA/Partnership)

LAB

[Experiments](https://2020.igem.org/Team%3ACSU_CHINA/Experiments)
[Notebook](https://2020.igem.org/Team%3ACSU_CHINA/Notebook)

PARTS

[Parts](https://2020.igem.org/Team%3ACSU_CHINA/Parts)
[Contribution](https://2020.igem.org/Team%3ACSU_CHINA/Contribution)
[Engineering Success](https://2020.igem.org/Team%3ACSU_CHINA/Engineering)

[MODEL](https://2020.igem.org/Team%3ACSU_CHINA/Model)

HUMAN PRACTICES

[Integrated Human Practices](https://2020.igem.org/Team%3ACSU_CHINA/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3ACSU_CHINA/Education)

[SAFETY](https://2020.igem.org/Team%3ACSU_CHINA/Safety)
[JUDGING FORM](https://igem.org/2020_Judging_Form?id=3452)

iGEM 2020

# Engineering

- Model -

## Overiew

## 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](#1.4) for more details)

| NAME | DESCRIPTION | CATEGORY | FUNCTION |
| --- | --- | --- | --- |
| [BBA\_K3452000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3452000) | hMT-1A | chemoattractant protein | Chelating Cadmium to improve tolerance |
| [BBA\_K3452020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3452020) | APX2 | antioxidant protein | Reducing reactive oxygen species (ROS) to avoid apoptosis |
| [BBA\_K3452602](http://parts.igem.org/wiki/index.php?title=Part: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:

![](https://static.igem.org/mediawiki/2020/d/de/T--CSU_CHINA--Figure1-enginerring.png)

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:

1. The protein has better cadmium specificity, rather than being able to widely absorb all heavy metal ions
2. The protein has the literature to prove that it can be expressed in prokaryotes, and the expression effect is more satisfactory, preferably in cyanobacteria
3. 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)

![](https://static.igem.org/mediawiki/2020/2/24/T--CSU_CHINA--Figure2.png)

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).

![](https://static.igem.org/mediawiki/2020/a/ae/T--CSU_CHINA--figure-engineering_success.png)

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

![](https://static.igem.org/mediawiki/2020/5/56/T--CSU_CHINA--Figure3.1-enginerring.png)
![](https://static.igem.org/mediawiki/2020/4/44/T--CSU_CHINA--Figure3-enginerring.png)

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:

1. The symbiotic relationship between bacteria and algae is uncertain
2. In engineered algae, where cadmium removal is the core purpose, the symbiotic system is overly cumbersome and introduces organisms other than engineered algae
3. The loop is too complex to be expressed in the prokaryote needs to be confirmed
4. 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

![](https://static.igem.org/mediawiki/2020/c/c3/T--CSU_CHINA--Figure4-enginerring.png)

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

![](https://static.igem.org/mediawiki/2020/2/2a/T--CSU_CHINA--Figure5-enginerring.png)

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.

## References:

1. Ochoa-Fernandez, R., et al., Optogenetic control of gene expression in plants in the presence of ambient white light. Nat Methods, 2020. 17(7): p. 717-725.
2. Harms, A., et al., Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Molecular Cell, 2018. 70(5): p. 768-784.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSU CHINA/Experiments

PROTOCOLS AND EXPERIMENTS

![](https://static.igem.org/mediawiki/2020/3/3a/T--CSU_CHINA--Logo.png)

CSU\_CHINA

[HOME](https://2020.igem.org/Team%3ACSU_CHINA)

PROJECT

[Description](https://2020.igem.org/Team%3ACSU_CHINA/Description)
[Design](https://2020.igem.org/Team%3ACSU_CHINA/Design)
[Results](https://2020.igem.org/Team%3ACSU_CHINA/Results)
[Proposed Implementation](https://2020.igem.org/Team%3ACSU_CHINA/Implementation)
[Proof of Concept](https://2020.igem.org/Team%3ACSU_CHINA/Proof_Of_Concept)

TEAM

[Team Members](https://2020.igem.org/Team%3ACSU_CHINA/Team)
[Attributions](https://2020.igem.org/Team%3ACSU_CHINA/Attributions)
[Collaborations](https://2020.igem.org/Team%3ACSU_CHINA/Collaborations)
[Partnership](https://2020.igem.org/Team%3ACSU_CHINA/Partnership)

LAB

[Experiments](https://2020.igem.org/Team%3ACSU_CHINA/Experiments)
[Notebook](https://2020.igem.org/Team%3ACSU_CHINA/Notebook)

PARTS

[Parts](https://2020.igem.org/Team%3ACSU_CHINA/Parts)
[Contribution](https://2020.igem.org/Team%3ACSU_CHINA/Contribution)
[Engineering Success](https://2020.igem.org/Team%3ACSU_CHINA/Engineering)

[MODEL](https://2020.igem.org/Team%3ACSU_CHINA/Model)

HUMAN PRACTICES

[Integrated Human Practices](https://2020.igem.org/Team%3ACSU_CHINA/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3ACSU_CHINA/Education)

[SAFETY](https://2020.igem.org/Team%3ACSU_CHINA/Safety)
[JUDGING FORM](https://igem.org/2020_Judging_Form?id=3452)

iGEM 2020

# Experiments

- Model -

## Synechocystis PCC6803 Protocols

1. prepare a stock solution

|  |  |
| --- | --- |
| Chemical | Amount (g) |
| \(100\ \times\ BG11 (per L)\) | | | |
| \({NaNO}\_{3}\) | 149.6 |
| \({MgSO}\_{4}\) ·7 \({H}\_{2}O\) | 7.49 |
| \({CaCl}\_{2}\) ·2 \({H}\_{2}O\) | 3.6 |
| Citric acid | 0.6 |
| Add \(1.12\ ml\ 0.25\ M\ {Na}\_{2}EDTA\), \(pH\ 8.0\ 0.25\ M\) | | | |
| \({Na}\_{2}EDTA\), \(pH8.0\ (per\ 100 ml)\) | | | |
| \({Na}\_{2}EDTA\) | 9.3 |
| Trace elements \((per\ 100 ml)\) | | | |
| \({H}\_{3}{BO}\_{3}\) | 0.286 |
| \({MnCl}\_{2} ·4\) \({H}\_{2}O\) | 0.181 |
| \({ZnSO}\_{4}\ ·7\) \({H}\_{2}O\) | 0.022 |
| \({Na}\_{2}{MoO}\_{4}\ ·2\) \({H}\_{2}O\) | 0.039 |
| \({CuSO}\_{4}\ ·5{H}\_{2}O\) | 0.008 |
| \({Co(NO3)}\_{2}\ ·6\ {H}\_{2}O\) | 0.005 |
| Iron stock \((per\ 100ml)\) | | | |
| Ferric ammonium citrate | 1.11 |
| Phosphate stock \((per\ 100ml)\) | | | |
| \({K}\_{2}{HPO}\_{4}\) | 3.05 |
| \({Na}\_{2}{CO}\_3\) stock \((per\ 100ml)\) | | | |
| \({Na}\_{2}{CO}\_3\) | 2 |
| TES buffer, \(pH 8.2 (per\ 100ml)\) | | | |
| TES | 22.9 |
| \({NaHCO}\_{3}\) stock \((per\ 100 ml)\) | | | |
| \({NaHCO}\_{3}\) | 8.4 |
| HEPES, \(pH 8.2 (per\ 500 ml)\) | | | |
| HEPES | 119.15 |
| Vitamin B12 \((Per 50 ml)\) | | | |
| Cyanocobalamin | 0.02 |

2. Prepare BG11 medium

* 2.1. Prepare stock solutions of \(100\ \times\ BG11\), trace elements and iron stock in protocol1.
* 2.2. Prepare separate solutions of phosphate stock, \({Na}\_{2}{CO}\_3\) stock, N-[Tris (hydroxymethyl) methyl]-2-aminoethanesulfonic acid (TES) buffer and \({NaHCO}\_{3}\) in protocol1.
* 2.3. Autoclave the phosphate and \({Na}\_{2}{CO}\_3\) stocks—Filter-sterilize TES buffer and \({NaHCO}\_{3}\) with \(0.2 µm\) filters.
* 2.4. Prepare BG11 by combining 976 ml of water, \(10 ml\) of \(100\ \times\ BG11\), \(1 ml\) of trace elements and \(1 ml\) of iron stock and autoclave the solution. After this solution has cooled to room temperature, add 1 ml of phosphate stock, 1 ml of \({Na}\_{2}{CO}\_3\) stock and 10 ml of \({NaHCO}\_{3}\) .
* 2.5. For BG11 solid medium, add 15 g of agar and \(700 ml\) of water to one flask. To the second flask, add \(3 g\) of \({Na}\_{2}{S}\_{2}{O}\_{3}\), \(226 ml\) of water, \(10 ml\) of \(100\ \times\ BG11\), 1 ml of trace elements and 1 ml of iron stock. Autoclave both solutions. After these solutions have cooled to room temperature, combine them and add 1 ml of phosphate stock, 1 ml of \({Na}\_{2}{CO}\_3\) stock, 10 ml of TES buffer, and 10 ml of \({NaHCO}\_{3}\) . Note: Solutions are prepared separately to avoid precipitation of certain salts.

3. Culture Synechocystis PCC6803 and refresh medium

* 3.1. Culture: the Synechocystis was placed under the condition of \(30℃\), \(40 E/m2/s\) light, with fresh BG11 culture medium \({OD}\_{730}\)>0.3, to avoid the occurrence of photoinhibition), refresh medium every two days.
* 3.2. refresh medium: 30-50mL of Synechocystis was taken and centrifuged at 2300g for 5 min. The cell precipitation was washed twice with a fresh BG11 medium. The final suspension was either in the original volume of fresh BG11 culture medium (for preserving the original growth state) or in a larger volume of BG11 culture medium (for propagation).

4. Determine the growth of Synechocystis PCC6803

* 4.1. 1mL Synechocystis was taken from the culture medium and placed into a cuvette.
* 4.2. Turn on the spectrophotometer (UNICOSH, UV2355) and preheat it for 20 mins.
* 4.3. Choose the wavelength as 730nm.
* 4.4. Cuvette filled with distilled water must be scanned as a blank to provide a spectral background.
* 4.5. The growth condition was determined by \({OD}\_{730}\)

5. Cyanobacteria (Synechocystis) conjugation and transfer

* 5.1. 2 mL culture medium of cyanobacteria in the logarithmic phase \({OD}\_{730}\) is close to 0.7) was taken and centrifuged at 6000 rpm for 5 min. The cell precipitation was washed twice with a fresh BG11 medium. Finally, resuspend it in 200 L of BG11 medium.
* 5.2. E. coli strain E. coli HB101 (transferred with pRL443+pRL623+ plasmid) cultured overnight was transferred to a fresh LB medium for the logarithmic period (\({OD}\_{600}\) approximately 1.0).
* 5.3. 2 mL E. coli in the logarithmic phase was taken and centrifuged at 6000 rpm for 5 min. The cell precipitation was washed twice with fresh LB medium. Finally, they were resuspended in 200 L of LB medium.
* 5.4. Cyanobacteria suspension and Escherichia coli cell suspension were mixed. The Cyanobacteria -E. coli suspension was placed at 30℃, 30~50 E/m2/s and stood for 4 h.
* 5.5. The Cyanobacteria -E. coli suspension (100~200 L per plate) was coated on the BG11 plate covered with cellulose nitrate film (NC film). 20 hr was required under 30℃, 30~50 E/m2/s light conditions.
* 5.6. Then, the NC membrane was transferred to a solid BG11 plate containing antibiotics (concentration) and continued to be cultured under 30℃, 30~50 E/m2/s light conditions.
* 5.7. After a few days (4-7 days), there are single colonies of Cyanobacteria growing up; The single colonies were selected from solid plates containing antibiotics and then separated for cell passage at 30℃, 30~50 E/m2/s.

## Experiment protocols

1. prepare a stock solution

|  |  |
| --- | --- |
| Chemical | Amount (g) |
| \(1g/L\) \({Cd}\_{2+}\) stock \((per\ 50mL)\) | | | |
| \(3{CdSO}\_{3}•8\ {H}\_{2}O\) | 0.1141 |
| 6% sodium alginate \((per\ 100mL)\) | | | |
| sodium alginate | 6 |
| 4% calcium chloride \((per\ 100mL)\) | | | |
| calcium chloride | 4 |

2. Cadmium ion determination (Shanghai Youlong Biotech Co .Ltd, BE0241)

* 2.1. Take \(100μL\) of medium supernatant and add 300μL of sample extract, mix well. That is the sample solution to be tested.
* 2.2. Add \(50µL\) of cadmium ion standards (0, 0.4, 1.2, 3.6, 10.8, 32.4ppb) to each standard well, and add 50µL of sample solution to the sample well.
* 2.3. Add \(50μL\) of antibody solution and \(50ul\) of enzyme label solution to both standard wells and sample wells, shake gently to mix, and then incubate at 25°C for 30min.
* 2.4. After the incubation, shake off the liquid in the microwells, add about \(250μL\) of diluted washing solution to each well, leave it for 1 minute, and discard the liquid. Repeat the washing 4 times. Pour out the liquid and pat dry the plate on absorbent paper or dry towel.
* 2.5. Add \(100 µL\) of the color developer to each well, shake gently to mix, and develop color in a constant temperature incubator at 25°C for 15 minutes.
* 2.6. After the color reaction is over, add \(100μL\) stop solution to each well. Measure at 450nm with a microplate reader. It must be completed within 10 minutes after adding the stop solution.

3. Cadmium tolerance and absorption capacity test of wild type Synechocystis PCC6803 under different cadmium concentrations

The Synechocystis (\({OD}\_{730}\)=0.7) were treated with 0.1,0.5,1,2,5,10 (mg/L) cadmium concentration gradients.

* 3.1. Centrifuge and the collected Synechocystis was resuspended in 30mL fresh BG11 culture medium until \({OD}\_{730}\)=0.5. Then the Synechocystis grew to 0D730=0.7 after 36h culture.
* 3.2. After absorbance was determined, 3, 15, 30, 60, 150, and 300μL of 1g/L \({Cd}\_{2+}\) stocks were added to corresponded groups.
* 3.3. Samples were taken at 0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h to determine the concentration of cadmium ions in the culture medium (Shanghai Youlong Biotech Co .Ltd, BE0241) and the growth of Synechocystis (\({OD}\_{730}\)).

4. Determination of cadmium absorption ability of different densities of Synechocystis PCC6803 under high and low cadmium conditions

Five groups of Synechocystis (\({OD}\_{730}\)=0.3,0.6,0.8,1.1,1.4) were treated with 0.1mg/L (low cadmium group) and 2mg/L (high cadmium group), respectively.

* 4.1. 30mL of fresh BG11 culture medium was used to resuspense Synechocystis to \({OD}\_{730}\)=0.2, 0.4, 0.6, 0.8, 1.2. Then the Synechocystis grew to target \({OD}\_{730}\) after 36h culture.
* 4.2. After \({OD}\_{730}\) was determined, 3 μL and 60 μL of 1mg/L \({Cd}\_{2+}\) mother liquor were added to the corresponded group.
* 4.3. Samples were taken at 0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h to determine the concentration of cadmium ions in the culture medium (Shanghai, youlong biological company, BE0241) and the growth of algae (\({OD}\_{730}\)).

5. Embedding and fixation of Synechocystis PCC 6803

* 5.1. The logarithmic growth stage was taken, centrifuged at 2300g for 5min, and washed twice with fresh BG11 medium. Finally, it was resuspended in a fresh BG11 culture medium until \({OD}\_{730}\)=0.7.
* 5.2. The above Synechocystis was mixed with 6% sodium alginate 1:1. The mixed liquid was then dropped into 4% calcium chloride with a dripping device to produce 3.5mm calcium alginate microbean. Curing at 4℃ for 3h.
* 5.3. The 10 microbeans were dissolved in 10 mL sodium citrate solution with a mass fraction of 2%. The absorbance of the dissolved solution at 730 nm was determined as the biomass density of Synechocystis. Ten microbeans of calcium alginate were dissolved in the sodium citrate solution as the colorimetric control solution.

6. Influence of calcium alginate microbeans on cadmium absorption

The cadmium removal ability of the three calcium alginate microbean groups containing Synechocystis was compared by 0.1mg/L (low cadmium group) and 2mg/L (high cadmium group).

* 6.1. Experimental group 1: The Synechocystis (\({OD}\_{730}\)=0.7) mixed with 6% sodium alginate 1:1 was blown well. The mixture was dropped into 4% calcium chloride with a self-made device to produce 3.5mm calcium alginate microbeans. Curing at 4℃ for 3h. Put 50 microbeans in a 30mL volume medium.
* 6.2. Experimental group 2: BG11 medium and 6% sodium alginate 1:1 were mixed and blown well. The mixed liquid was dropped into 4% calcium chloride with a device to produce 5mm glueballs. Curing at 4℃ for 3h. Put 50 microbeans in a 30mL volume medium.
* 6.3. Experimental group 3: Put 5mL Synechocystis (\({OD}\_{730}\)=0.7)(equal to 50 microbeans) in 30mL volume.
* 6.4. Samples were taken at 0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h to determine the concentration of cadmium ions in the culture medium (Shanghai youlong biological company, BE0241) and the growth of Synechocystis (\({OD}\_{730}\)).

7. express parts in E.Coli and their reaction to cadmium

* 7.1. The E. coli cells, harboring pVZ322-parts, were grown overnight in LB medium containing corresponded antibiotics. Different flasks containing 20 ml LB were inoculated with this overnight culture. The cells were incubated at 37℃ in a shaker till \({OD}\_{600}\) of 0.7.
* 7.2. After an hour, 0.1mg/L of cadmium was added to cultures. Control cultures containing E. coli cells transformed with only pVZ322 vector (without insert) were also treated in the same way as to test cultures.
* 7.3. The \({OD}\_{600}\) was measured in 400μL bacterial medium taken every 30min, by which the growth curve was drawn.
* 7.4. Another 400 L bacterial medium was taken every 30min too and centrifuged at 8000rpm for 2min to determine cadmium concentration.

## General Molecular Protocols

1.PCR Reaction

|  |  |
| --- | --- |
| Reagent | Volume(μl) |
| 5X Buffer | 10 |
| DMSO | 4 |
| dNTP | 4 |
| Forward Primer | 2 |
| Reverse Primer | 2 |
| Targeted DNA fragment | 1 |
| Phusion | 1 |
| dd\({H}\_{2}O\) | 26 |
| In Total | 50 |

Program:

\(98℃(5min)\)

\(\left.
\begin{array}{l}
\text{98℃}&\left(30s\right)\\
\text{58℃}&\left(30s\right)\\
\text{72℃}&\left(30s/kb\right)
\end{array}
\right\}
\*40\)

\(72℃\ (10min)\)

\(4℃\)

2. Gel recycling（Omega）

* 2.1. Agarose EB gel was prepared, and electrophoresis was used to separate DNA fragments.
* 2.2. After enough time has passed, the required DNA fragments are carefully cut off under ultraviolet light. And try to get rid of the excess gel.
* 2.3. The weight of the empty centrifuge tube was weighed, and the gel with the target segment was cut off and placed in the 1.5mL centrifuge tube and weighed. The weight of the gel block was calculated, and its volume was approximately determined. A binding Buffer of the same gel volume was added, and the mixture was placed in a water bath of 55℃~65℃ for 7min to melt completely, during which the gel was mixed every 2-3 minutes.
* 2.4. Transfer 700 μl of DNA-agarose solution to a HiBindTM DNA column and place the column in a clean 2ml collection tube. Centrifuge at 10,000×g for 1min at room temperature.
* 2.5. Put the column back into the collection tube and add 300 μL Binding Buffer; Centrifuge at 10,000×g for 1 min at room temperature, then discard the filtered liquid.
* 2.6. Put the column back into the collection tube and add 700 μL SPW Wash buffer. Centrifuge at room temperature 10,000×g for 1 minute, then discard the filtered solution.
* 2.7. Put the column back into the collection tube and repeatedly add 700μ L SPW Wash buffer. Centrifuge it at 10,000×g for 1 minute at room temperature to discard the filtered solution.
* 2.8. Discard the liquid and reinsert the empty column back into the collection tube. Centrifuge it at 10,000×g for 1min to dump the remaining liquid.
* 2.9. The column was placed on a clean 1.5 mL centrifuge tube and added with a 50 μL sterilized water column film. The column was centrifuged at 10,000×g for 1 minute.

3. Assembly Reaction ( abm, Pro Ligation-Free Cloning Kit)

* 3.1. Quantify the insert and vector DNA by a UV spectrophotometer or by comparison of the target band against a known molecular weight marker run on the same gel.
* 3.2. Set up the following reaction on ice:

|  |  |  |
| --- | --- | --- |
| Reagent Cloning | Reaction | Example Reaction |
| 2X Pro Ligation-Free MasterMix | 10 µl | 10 µl |
| Linearized Vector | 3:1 insert-vector molar ratio | 60 ng (1 kb insert) |
| Nuclease Free \({H}\_{2}O\) | Up to 20 µl | Up to 20 µl |

To calculate amount of insert to add:

insert (ng)= 3\*insert (bp)/ vector (bp)\* vector (ng)

* 3.3. For 1 insert, incubate at 50°C for 15 minutes. For multiple inserts or complicated assemblies, incubate at 50°C for 1 hour. 4
* 3.4. Transform immediately or store samples at -20°C until transformation.

4. transfection of competent cells (DH5a CELL and HB101 CELL)

* 4.1.Put the cells on ice and let stand for 20min, then precool the EP tube, transfer 20μL of cells to the EP tube;
* 4.2.The 2 μL of plasmid was added to the cells and placed on the ice for 30min;
* 4.3.Heat shock 42℃, the 90s, followed by 5min on ice;
* 4.4.Add 500 mL of antibiotic-free liquid medium and incubate in a shaking table at 37℃ for 60min
* 4.5.coat plates, select monoclonal colony or add to 6mL LB medium added with 6μL antibiotics (\(1000×\))

5. protein extraction

* 5.1. Solutions and reagents: lysis buffers

+ NP-40 buffer

- 150 mM NaCl
- 1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
- 50 mM Tris-HCl, pH 8.0
- Protease inhibitors

+ RIPA buffer (radioimmunoprecipitation assay buffer)

- 150 mM NaCl
- 1% IGEPAL CA-630
- 0.5% sodium deoxycholate
- 0.1% SDS (sodium dodecyl sulphate)
- 50 mM Tris-HCl, pH 8.0
- Protease inhibitors

+ Tris-HCl

- 20 mM Tris-HCl
- Protease inhibitors

* 5.2. Centrifuge centrifugal tube containing E.Coli or Synechocystis in a centrifuge at 4°C.
* 5.3. Washed with PBS and then resuspend cells in lysis buffer (equal to the volume of precipitate) in a microcentrifuge tube.
* 5.4. Maintain constant agitation for 30 min at 4°C.
* 5.5. Centrifuge in a microcentrifuge at 4°C. 12000rpm, 10min.
* 5.6. Gently remove the tubes from the centrifuge, place it on the ice, aspirate the supernatant, put it in a fresh tube kept on ice, and discard the pellet.

6. Western blot

* 6.1. Remove a small volume of lysate to perform a protein quantification assay. Determine the protein concentration for each cell lysate.
* 6.2. Determine how much protein to load and add a 5×SDS sample buffer.
* 6.3. To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 10 min.
* 6.4. Run the gel for 1–2 h at 100 V.
* 6.5. The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack. The time and voltage of transfer may require some optimization. We recommend following the manufacturer’s instructions. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.
* 6.6. Block the membrane for 1 h at room temperature or overnight at 4°C using a blocking buffer.
* 6.7. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer.
* 6.8. Wash the membrane in three washes of TBST, 5 min each.
* 6.9. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.
* 6.10. Wash the membrane in three washes of TBST, 5 min each.
* 6.11. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap. Acquire images using darkroom development techniques for chemiluminescence or standard image scanning methods for colorimetric detection.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSU CHINA/Results

Result

![](https://static.igem.org/mediawiki/2020/3/3a/T--CSU_CHINA--Logo.png)

CSU\_CHINA

[HOME](https://2020.igem.org/Team%3ACSU_CHINA)

PROJECT

[Description](https://2020.igem.org/Team%3ACSU_CHINA/Description)
[Design](https://2020.igem.org/Team%3ACSU_CHINA/Design)
[Results](https://2020.igem.org/Team%3ACSU_CHINA/Results)
[Proposed Implementation](https://2020.igem.org/Team%3ACSU_CHINA/Implementation)
[Proof of Concept](https://2020.igem.org/Team%3ACSU_CHINA/Proof_Of_Concept)

TEAM

[Team Members](https://2020.igem.org/Team%3ACSU_CHINA/Team)
[Attributions](https://2020.igem.org/Team%3ACSU_CHINA/Attributions)
[Collaborations](https://2020.igem.org/Team%3ACSU_CHINA/Collaborations)
[Partnership](https://2020.igem.org/Team%3ACSU_CHINA/Partnership)

LAB

[Experiments](https://2020.igem.org/Team%3ACSU_CHINA/Experiments)
[Notebook](https://2020.igem.org/Team%3ACSU_CHINA/Notebook)

PARTS

[Parts](https://2020.igem.org/Team%3ACSU_CHINA/Parts)
[Contribution](https://2020.igem.org/Team%3ACSU_CHINA/Contribution)
[Engineering Success](https://2020.igem.org/Team%3ACSU_CHINA/Engineering)

[MODEL](https://2020.igem.org/Team%3ACSU_CHINA/Model)

HUMAN PRACTICES

[Integrated Human Practices](https://2020.igem.org/Team%3ACSU_CHINA/Human_Practices)
[Science Communication](https://2020.igem.org/Team%3ACSU_CHINA/Education)

[SAFETY](https://2020.igem.org/Team%3ACSU_CHINA/Safety)
[JUDGING FORM](https://igem.org/2020_Judging_Form?id=3452)

iGEM 2020

# Results

- Model -

As described in Design, our loop consists of a core “transport-chelation-redox reaction” system and an auxiliary “toxin-antitoxin” suicide system. Our project designed experiments to verify the “transport-chelation-redox reaction” system and verify the “suicide system” through modeling and other methods. The results are as follows:

## 1. Experiments in Synechocystis sp.PCC6803

The ultimate goal of our project is to use the modified Synechocystis sp.PCC6803 for the treatment of cadmium pollution in water bodies, therefore we carried out numerous experiments in Synechocystis sp.PCC6803 and obtained the following results:

1.1 Exploring the efficiency of cadmium uptake at different Synechocystis sp.PCC6803 concentrations

To preliminary investigate the cadmium uptake by wild Synechocystis sp.PCC6803, we designed different algae concentrations with OD values of 0.3, 0.6, 0.8, 1.1 and 1.4, respectively. We incubated the algae in low cadmium (0.1mg/L) and high cadmium (2mg/L). We measured the efficiency of cadmium absorption and growth. The results showed that (1) in the low cadmium group, the algae grew well, but in the high cadmium group, except for the algae with an OD of 1.4, the rest were inhibited, and the lower the concentration of algae, the more obvious the inhibition (Figure 1); (2) in the low cadmium group, the algae had a better effect of cadmium removal, while in the high cadmium group, except for the Synechocystis sp.PCC6803, with an OD value of 1.4, the effect of cadmium removal was not significant in other groups. What’s more, under high cadmium conditions, the cadmium uptake curve of Synechocystis sp.PCC6803 increases first and then decreases, indicating that cadmium is released after Synechocystis sp.PCC6803 death. (Figure 2). The above results show that wild Synechocystis sp. PCC6803 has a certain capacity to absorb cadmium, which is enhanced by the increase of initial algal concentration but is easily inhibited by high cadmium.

![](https://static.igem.org/mediawiki/2020/1/1f/T--CSU_CHINA--Figure9.jpeg)

Figure 1: Growth at different algal concentrations (0.1mg/L, left) and (2mg/L, right).

![](https://static.igem.org/mediawiki/2020/7/78/T--CSU_CHINA--Figure10.jpeg)

Figure 2: Removal of cadmium at different algal concentrations (0.1mg/L, left) and (2mg/L, right).

1.2 Further investigation of cadmium resistance and uptake by Synechocystis sp.PCC6803

To further investigate the ability of wild Synechocystis sp.PCC6803 to remove and tolerate cadmium, we designed a cadmium concentration gradient of 0.1 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L, 5 mg/L and 10 mg/L. The absorbance at 730 nm was measured at eight time points (0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h, respectively) to monitor the growth of Synechocystis sp.PCC6803. (figure3), it was observed that as the concentration of cadmium increased, the growth of Synechocystis sp.PCC6803 was inhibited more and more obvious. The OD value of algae in concentrations greater than or equal to 2 was lower than 0.7, which indicated that the growth of Synechocystis sp.PCC6803 was greatly inhibited. Afterward, the cadmium absorption efficiency of Synechocystis sp.PCC6803 under different cadmium concentrations were measured. The results showed that algae absorbed most of the cadmium in 0.5h and then entered the platform phase. And the cadmium absorption efficiency was lower under high cadmium condition than low cadmium condition, with the highest absorption efficiency at 0.1mg/L (figure4). The above results show that wild Synechocystis sp. PCC6803 can remove and absorb cadmium, but it needs further improvement.

![](https://static.igem.org/mediawiki/2020/9/9e/T--CSU_CHINA--Figure7.jpeg)

Figure3: Growth of Synechocystis sp.PCC6803 at different cadmium concentrations

The growth of algae is inhibited when the concentration is greater than 2 mg/L, and algae’s growth is better in the case of low cadmium.

![](https://static.igem.org/mediawiki/2020/a/a6/T--CSU_CHINA--Figure8.jpeg)

Figure 4: Cadmium uptake efficiency of Synechocystis sp.PCC6803 at different cadmium concentrations.

## 2. Embedding of Calcium-Alginate Microbeads (Ca-Alg MBs）

In order to improve the cadmium removal capacity and cadmium tolerance of Synechocystis sp.PCC6803, we proposed the Ca-Alg MBs embedding scheme, and the following are the experimental results we obtained:

2.1 Production of Ca-Alg MBs

Cell immobilization can effectively limit cell movement, reduce the escape of engineered algae, and prevent genetically engineered algae from harming the natural environment. In our project, we used sodium Ca-Alg MBs to embed the algae. We determined that the optimum concentration of sodium alginate is 3% by setting up a concentration gradient. The optimum concentration of calcium chloride is 4%. To ensure Ca-Alg MBs are square, we use pasteurization pipe and dropper to make our own rubber ball manufacturing pipe (Figure5). To facilitate modeling, ImageJ was used to calculate the diameter of Ca-Alg MBs (Table 1).

![](https://static.igem.org/mediawiki/2020/d/db/T--CSU_CHINA--Figure11.jpeg)
![](https://static.igem.org/mediawiki/2020/1/1b/T--CSU_CHINA--Figure11%282%29.jpeg)

Figure 5: Cadmium uptake efficiency of Synechocystis sp.PCC6803 at different cadmium concentrations.

2.2 Exploring the role of the Ca-Alg MBs

At the same time, in order to verify the effect of Ca-Alg MBs embedding on the cadmium absorption efficiency of algae, we designed a comparative experiment between embedded algae, sodium Ca-Alg MBs and unembedded algae, finding that Ca-Alg MBs can improve the cadmium absorption efficiency of algae and the Ca-Alg MBs has a natural ability to absorb cadmium (Figure6A/B). At the same time, we found that the Ca-Alg MBs have a certain protective effect on algae in a high cadmium environment (Figure7). We speculate that the reasons are as follows: Ca-Alg MBs have three procedures called liquid phase transfer, Solid phase transfer, metathesis to absorb Cd and perform an excellent cadmium removal ability. So, it can immobilize our biological chassis and improve the cadmium elimination in high Cd by acting as a ‘buffer zone’.

![](https://static.igem.org/mediawiki/2020/3/33/T--CSU_CHINA--Figure12.jpeg)

Figure6: Effect of Ca-Alg MBs on the ability of algae to absorb cadmium.

We determined the cadmium uptake efficiency of Ca-AIg MBs, PCC6803, Ca-AIg MBs + PCC6803 under high and low cadmium conditions, respectively. The results showed that Ca-AIg MBs have some cadmium removal capacity and can protect PCC6803 under high cadmium conditions and improve its cadmium removal capacity (0.1mg/L, left) and (2mg/L, right).

![](https://static.igem.org/mediawiki/2020/4/4a/T--CSU_CHINA--Figure13.jpeg)

Figure7: Protective effect of Ca-AIg MBs on algal growth in high cadmium environments

(0.1mg/L, left) and (2mg/L, right).

3.1.3 Life cycle of Ca-Alg MBs

![](https://static.igem.org/mediawiki/2020/6/6b/T--CSU_CHINA--Figure14.jpeg)

Figure 8: During 36 hours, these MBs will bubbl with time or even burst due to the oxygen, suggesting that we need to recycle these MB in time to avoid the leak of PCC6803.

## 3. Experiments in E. coli

To further improve the cadmium uptake and resistance of Synechocystis sp.PCC6803, we designed the core “transport-chelation-redox reaction” loop. However, due to the difficulty of conducting an experiment in Synechocystis sp.PCC6803 compared with E. coli, we first verify our loop in E. coli

3.1 Successful construction of plasmids

![](https://static.igem.org/mediawiki/2020/c/ce/T--CSU_CHINA--Figure1.png)

Figure9: the backbone of our plasmid and the OptimumGeneTM algorithm we used to accomplish codon optimization

![](https://static.igem.org/mediawiki/2020/2/24/T--CSU_CHINA--Figure2.png)

Figure10: our plasmids with different resistance

In our project, we selected nine proteins including three translocators: OsNRAMPS/MntH/IRT1, three chelating proteins: SmtA/hMT-1A/TMCd1 and three redox proteins: APX2/LOX1/Cu-SOD, and we successfully constructed the nine plasmids we needed to screen by using the vector pVZ322 (figure9), and we also added different resistances to our plasmids for next experiments (figure10).

3.2 Successful expression of our components in E. coli

We transfected E. coli using our constructed plasmids. We examined protein expression by western blot, and the results are as follows (figure11):

![](https://static.igem.org/mediawiki/2020/c/c6/T--CSU_CHINA--Figure3.png)

Figure11: the Western blotting results indicating the successful expression of our plasmids in E.Coli

3.3 Characteristics of the Engineering Synechocystis sp.PCC6803

To investigate the effect of successfully transferred plasmids on Synechocystis sp.PCC6803, we determined the growth curves of engineered and wild algae at certain cadmium concentrations, with the following results (figure12/13):

![](https://static.igem.org/mediawiki/2020/d/d3/T--CSU_CHINA--Figure4.png)
![](https://static.igem.org/mediawiki/2020/f/f4/T--CSU_CHINA--Figure4%282%29.png)
![](https://static.igem.org/mediawiki/2020/a/a1/T--CSU_CHINA--Figure4%283%29.png)

Figure12: Growth curves of chelated protein-expressed or redox protein-expressed or transport-chelation-redox protein-expressed E. coli in 0.1 mg/L of cadmium solution

We incubated E. coli expressing the protein TMCD1, hMT-1A, SmtA or APX2 and the E. coli expresses transport-chelation-redox protein in a cadmium solution concentration of 0.1 mg/L and determine the OD value in response to algal concentration. The results showed that these proteins could improve algae cadmium tolerance and protect their growth in a high cadmium environment.

To further verify our proteins' role, we incubated E. coli expressing hMT-1A, MntH, MntH&TMCd1&APX2 in different cadmium concentrations. The results showed that E. coli expressing transport-chelating-antioxidant and chelating proteins had higher cadmium tolerance, while E. coli expressing only transport proteins had poorer cadmium tolerance due to more cadmium transfer (figure5).

![](https://static.igem.org/mediawiki/2020/a/a1/T--CSU_CHINA--Figure5.png)

Figure 13: Growth curves of modified E. coli at different cadmium concentrations.

3.4 Efficiency of cadmium uptake in modified E. coli

To verify the change in cadmium uptake efficiency of E. coli after successful genetic modification, We cultured E. coli successfully expressing hMT-1A, MntH, MntH&TMCd1&APX2 in a medium with a cadmium concentration of 0.1 mg/L, determined the cadmium concentration at different times and plotted the cadmium uptake curve (figure14). The results showed that the expression of the transport protein MntH increases the efficiency of cadmium uptake by E. coli but substantially decreases its cadmium tolerance. And the E. coli was successfully expressing MntH&TMCd1&APX2 absorbed more cadmium than those expressing MntH alone and empty plasmid.

![](https://static.igem.org/mediawiki/2020/c/c5/T--CSU_CHINA--Figure6.png)

Figure 14: Cadmium uptake curve of engineered E. coli.

## 4. Enhancing safety

To further reduce the environmental damage caused by genetically modified algae, we designed a toxin-antitoxin suicide system. Blue light inhibition element was used, which can down-regulate the expression of the downstream protein. Due to the covid-19, the verification experiment of this system could not be carried out in time. Still, we have proved the system from the perspective of mathematical modeling. The results showed that the system can inhibit the toxin expression under blue light, and the algae can grow naturally. However, When the blue light disappears, the toxin is expressed in large quantities, leading to the algae’s death.

## 4. Outlook

To further improve our project, the following supplement and efforts will be carried out in nearly future:

4.1 Further verify the optical control system in the laboratory

In our experimental part, although we have confirmed the effect of the visual control system with the modeling method, its real impact remains to be further verified by experiments. In the future, we will synthesize optical control elements and demonstrate them through the transformation, selection and amplification and other molecular biology standard processes.

4.2 Explore the growth curve of the modified algae

Although the modified alga can remove cadmium effectively, its growth curve under different cadmium concentrations is unknown. In the following experiments, we will design different cadmium concentration gradients to observe the growth of algae to provide data support for the use cycle of subsequent engineering algae used in the environment

4.3 Verify safety and practicality in the real environment

Although our engineered algae’s cadmium removal efficiency has been verified in natural water bodies, its actual performance in large-scale water bodies is unknown. Next, we will select the bodies of water polluted by cadmium to ascertain the practicality of our algae

4.4 Promotion

After completing the above experiment supplement, we will promote our algae in various ways and apply it in the real environment

4.5 Molecular\_evolution\_of\_our\_proteins

In subsequent experiments, we will use a molecular evolutionary approach to screen our pre-selected proteins. Express the cadmium ion probe in Synechocystis sp.PCC6803, point mutates the domains related to cadmium ion recognition and express them in the algae. And then, screen the proteins with high fluorescence intensity of the probe by flow cytometry, isolate and sequence them to determine and select the more efficient proteins.

## 5. Considerations for replicating our experiments

The experiments we conducted are all routine experiments in molecular biology with standard operating procedures. However, the following two points should be noted during the replication of our experiments:

5.1 The protection of toxic substances

In our experiments, a large number of poisonous substances are used, including the toxin RelB…. Please pay attention to the protection

5.2 The treatment of engineering algae

After the modification, the engineered Synechocystis sp.PCC6803 has a robust antioxidant ability, and its survival ability has been enhanced, too. Therefore, when dealing with it, be careful not to let them escape and cause environmental damage.

## Demonstration

The core of our project is to improve the cadmium removal efficiency of Synechocystis sp.PCC6803 through synthetic biology methods under the premise of ensuring biosafety. Based on the above experimental results, we can draw the following conclusions:

1. Synechocystis sp. PCC6803 has a particular ability to remove cadmium. The efficiency of cadmium removal increases with an increasing initial concentration of Synechocystis sp. PCC6803.

2. Synechocystis sp. PCC6803 grows well under low cadmium conditions but is inhibited at cadmium concentrations above 2 mg/L.

3. Ca-AIg MBs have a particular ability to remove cadmium and can improve the ability of Synechocystis sp. PCC6803 to resist and remove cadmium.

4. The components we designed could be expressed in prokaryotes and can significantly improve cadmium resistance and removal.

5. Mathematical modeling demonstrates that our light-controlled suicide system could ensure the biosafety of our modified organisms

The above experimental results show that our design could help prokaryote effectively remove cadmium from the water with good biological safety