Team:Hainan China/Engineering

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Engineering

Research and Imagine

Bleaching of coral reefs in tropical/subtropical has long been a global issue leading to devastation of marine environment and marine ecosystems. We have found that the survival of corals is strongly associate with viable zooxanthellae living symbiotically within the coral polyps. Healthy coral symbiosis is based on mutualistic relationships among coral, Zooxnathelae and other symbiont bacteria with nutritional exchange and metabolic interaction to an equilibrium state ^[1]. When the environmental stress by global climate change appears, this equilibrium will be broken and coral symbiosis be disassociated.
The engineering success of our project is based on our team work. With brainstorming, our team decided to focus our project on the concept of enriched nutrition for corals and their symbionts to resist bleaching caused by global climate change. We then 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.

Design and Build

Biological Parts

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⁺:

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.

Test and Result

Verification of the effectiveness of the engineered parts:

The full-length of homologous arm-Provgb:: vgb is 1555 bp which was obtained from the total gene synthesis (Figure 4. A). The whole sequence and pOJ260 were digested with the XbaI and EcoRI restriction enzyme (Figure 4. 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 4. 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 4. E, F) showed that we have successfully obtained the Modified Engineering Bacteria.

Figure 4. 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.

Fig. 5 indicates that Pseudomonas denitrificans construct with vgb gene will produce higher VB₁₂

Figure 5. The yield of VB₁₂ of CK and engineered Pseudomonas denitrificans

The images below shows comparison of healthy Zooxanthellae cells grown with enriched nutrients (in light brown color, upper left) and bleached Zooxanthellae without additional nutrition after heat shock (lower left), The right part is their fluorescence microscopic images.

The images (Fig. 7) below shows Fluorescence microscope observations of healthy corals with the presence of abundant Zooxanthella cells on the upper left. And bleached corals with bleached Zooxanthellae on the lower left. The right part is their fluorescence microscopic images.
Fig. 6 and 7 indicate the effect of nutrition on avoidance of coral bleaching.

Figure 3: The observation of zooxanthellae under microscope

In general, we isolated zooxanthellae from the host coral polyps (Figure 3. A, B) conduct heat shock experiments. It was observed that Zooxanthellae would be bleached if they were not nourished by vitamin B₁₂. If they did, Zooxantellae could live normally without bleaching.