Team:Hainan China/Proof Of Concept

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Overview

The concept for our research is that enriched nutrition enables corals and their symbionts to resist bleaching caused by global climate change. It is proven by a well designed research plan and its implementation. We first developed a nutritional enhancement strategy to strengthen coral/Zooxanthellae against marine environmental stress. Several nutrients were screening and vitamin B12 was selected as the essential nutrient. P. denitrificans was used as cellular chassis and vgb gene from marine microalga Vitreoscilla sp. as biological part to minimize oxygen demand for effective VB₁₂ production within the framework of synthetic biology. Coral polyps, Zooxanthellae and probiotic P. denitrificans constructs were grown on the microfluidic system for our proof of concept.

More specifically, our proof of concept can be divided into three parts: 1. cultivation of Zooxanthellae in vitro for nutritional optimization, 2. synthetic biology of P. denitrificans for effective VB₁₂ production and 3. microfluidic chip for coral symbiosis cultures at single coral polyps level.

Cultivation of Zooxanthellae in vitro for optimal nutrition:

1. Ordinary growth of Zooxanthella and Pseudomonas denitrificans

We chose three clades of zooxanthella, clade B1, clade C92 and clade E1, in our study f/2 medium is used for ordinary growth of Zooxanthellae. It is found that the growth rate of zooxanthella clade E1 was higher than clade B1 and clade C92. The growth curve for clade E1 is shown in Figure 1.

Figure 1. Growth curves of Zooxanthellae clade E1 at 28°C in f/2 medium

Additionally, we have grown the bacterium Pseudomonas denitrificans (see Figure 2 or its growth curve). It is known that Pseudomonas denitrificans is capable of producing vitamin B₁₂ (V B₁₂) in bioindustry. Together with nitrogen fixation and phosphorus dissolving property. Therefore, this strain was selected for our project.

Figure2. Growth curves of Pseudomonas denitrificans in LB medium.

2. Determination of nutrients effective for health of zooxanthella under heat shock

After literature survey, we chose four nutritional elements, N, F_e, P and V B₁₂ that are critical to healthy growth of zooxanthellae for our experiments. N, F_e, P and VB₁₂ with concentrations of 1, 10 and 100 times that of the control group were added to the zooxanthella clade E1 cell cultures and then the Fv/Fm value was measured to find the optimal concentration. After measurement, zooxanthella will be cultured in a new medium.

Table 1 The Fv/Fm values of zooxanthella containing different concentrations of N, F_e, P and VB₁₂

Figure3. The Fv/Fm values of zooxanthella containing different concentrations of N, F_e, P and VB₁₂

After the experiment, we drew primary solution of the best concentration of each element for resisting coral bleaching. According the collected data, the approximate best concentration of P was between 1x and 10x; the best concentrations of Fe was between 10x and 100x and that of VB₁₂ was between 1x and 10x. However, we had not discovered the approximate best concentration of N from the data.

It was also found that Fe was relatively less effective. N, P and VB₁₂ were selected for further investigation.

3. Determination of optimal nutrient concentrations for zooxanthella against heat shock

In the further experiment, we still took the two groups of zooxanthella B1 and C92 and added N, P and VB₁₂ with concentrations of 1, 10 and 100 times respectively. In one of the C92 corals, we particularly added VB₁₂ with concentrations of 20, 40, 60 and 80 beside the concentrations of 1, 10 and 100. Then in the first three days of the experiment, the samples were taken out of the light for 5 minutes in every measurement and their Fv/Fm values were measured. On the third day, zooxanthella was bleached, and the Fv/Fm values of the samples were measured every two days after the bleaching

Table 2 The Fv/Fm values of zooxanthella B1 with different P concentrations

Table 3 The Fv/Fm values of zooxanthella C92 with different P concentration

Table 4 The Fv/Fm values of zooxanthella B1 with different VB12 concentration

Table 5 The Fv/Fm values of zooxanthella C92 with different VB12 concentration

Table 6 The Fv/Fm values of zooxanthella B1 with different N and P concentration

Table 7 The Fv/Fm values of zooxanthella C92 with different N and P concentration

Synthetic biology of P. denitrificans for effective VB₁₂ production

Pseudomonas denitrificans is used in our project as the cellular chassis for the production of vitamin B₁₂, It is safe-to-use with fast growth rate. P. denitrificans can dissolve phosphorus, release potassium, fix nitrogen. Most importantly, it is capable of production of vitamin B₁₂ in industrial scale. However, P. denitrificans would exhaust a high level of oxygen when synthesizing vitamin B₁₂ which may retard its growth and metabolism. Traditional, vitamin B₁₂ production will be enhanced mainly with the increase of oxygen supply using higher oxygen pumping speed. This measure leads to higher energy consumption. The secret of our success is that we have introduced the vgb gene from marine microalga as a biological part into P. denitrificans to minimize oxygen demands for VB₁₂ production. vgb gene has been proven to transfer oxygen through the respiratory chain for cellular oxygen supply.

The plasmid pOJ260 is used to construct a transformation vector with vgb gene, a constitutive promoter, and the homologous arm of the P. denitrificans . Below is the engineered plasmid pOJ260⁺:

Figure 4.

After conjugation, the biological part with vgb gene was loaded onto the cellular chassis for an efficient VB₁₂ synthesis. It could become a probiotic bacterium to provide nutrition for the coral symbiotic system.

Figure 5.

The plasmid pOJ260 is transferred into E. coli S17, knocking down the target genes in the S17. The E. coliS17 is then transferred to P. denitrificans through conjugal transfer, and then a homologous single exchange will occur in the P. denitrificans, so the information in the edited pOJ260 will remain inside the bacteria.

1. Construction of Recombinant Plasmid and Engineering Bacteria

The full-length of homologous arm-Provgb:: vgb is 1555 bp which was obtained from the total gene synthesis (Figure 6. A). The whole sequence and pOJ260 were digested with the XbaI and EcoRI restriction enzyme (Figure 6. B) then linked together with the ligase. The plasmid was inserted into E. coli for replication and storage. Colony PCR was implemented to confirm successful transformation of the whole module into the host E. coli S17 cells. Electrophoresis image demonstrates the presence of a gene of length 1555 bp in the E. coli constructs (Figure 6. C, D) . After sequencing to verify the gene orientation, the correct recombinant plasmid was transferred into the Pseudomonas denitrificans> through the conjugal transfer. The results (Figure 6. E, F) showed that we have successfully obtained the Modified Engineering Bacteria.

Figure 6. Construction of Recombinant Plasmid. (A) PCR result of the insert gene module. (B) double enzyme digested gene module (1) and linearized pOJ260 (2). (C) transformation result of the E.coli S17. (D) PCR verification of the single colony. (E) conjugal transformation result of the Pseudomonas denitrificans. (F) PCR verification of the single colony. M DS5000 marker.

2. VB₁₂ production from engineered Pseudomonas denitrificans

Both wild type and engineered Pseudomonas denitrificans was cultivated for VB 12 production. Fig. 7 indicates that Pseudomonas denitrificans construct with vgb gene insertion produced higher VB₁₂ than that of wild type.

Figure 7. The yield of VB₁₂ by wild type and engineered Pseudomonas denitrificans

Microfluidic chip:

In order to have a long term observation on the growing conditions of zooxanthellae in response to the variations in nutrient concentrations, we designed a microfluidic chip, and call it “coral-in-a-chip, to do the long-term cultivation of coral polyps with symbionts. The microfluidic chip can be directly observed under the microscope.
Figure 8 is a microfluidic system consisting of a pump with injection poles, micro fluidic chips, and waste collection beakers. The medium is added into the chip by the pump. The the medium fluid will be distributed by the channels and arrive at the chambers, where coral polyps and zooxanthellae are cultivated.

Figure 8. Coral-in-a-chip

In Figure 9, we can see the microfluidic device on the left for the precise determination of nutrient dilution rate. Medium of different nutrient concentrations is added by pumps into channel A and B, then the fluid will be diluted the four parallel lines of chambers where coral tissue is cultivated.

Figure 9. Schematic diagram of the coral-in-a-chip for coral symbiosis

On the upper right in Figure 9, we can see the microfluidic system. This is microfluidic system for experimentation. The pump used to add fluid, water to control temperature, LED light to control the light intensity, and beaker to collect metabolic wastes.
On the lower part, single coral tissue with zooxanthellae are cultivated in the round chamber and the the medium and nutrients can be added through the fluid flow.

Figure 10. Fluorescence microscopic images

Figure 10. upper: control (28 °C, healthy growth）, lower left: Zooxanthellae without enriched nutrients after 2 hour heat shock (38 °C, it was bleached) and lower right: Zooxanthellae with enriched nutrients after 2 hour heat shock (38 °C, it was not bleached)
It can be concluded that the nutritional enhancement strategy performs well in prevention of Zooxanthellae from bleaching. So the proof of concept indicates that idea of probiotics to enhance coral symbiosis works well in coral bleaching prevention!