Team:Hainan China/Contribution

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1.Contribution to biological parts

vgb gene:

We used vgb gene cloned from marine microalga Vitreoscilla sp. that lives in a low oxygen level. vgb gene is up-regulated at its maximum level when the environmental oxygen level is low. Traditional Pseudomonas denitrificans fermentation for vitamin B₁₂ production demands high oxygen supply which is not appropriate to our marine applications. Therefore, when Pseudomonas denitrificans is loaded with vgb gene by synthetic biology, it will make a disruptive change for the P. denitrificans construct to produce maximum vitamin B₁₂ with very low oxygen availability.

Promoter of vgb gene:

The promoter of the vgb gene is also oxygen-dependent. It will maximize gene expression in a micro-aerobic environment for the P. denitrificans construct to produce vitamin B₁₂. This promoter is relatively different from the promoter used by previous iGEM teams because it is relatively longer in sequence. Other iGEM teams could consider to use this promoter in the future.

Homologous sequence of Pseudomonas denitrificans:

Current iGEM registry does not contain this bacterium. The homologous recombination of Pseudomonas denitrificans plays an important role in the establishment of a disruptive process for VB₁₂ production at extremely low oxygen supply. Therefore, we made contributions to the future iGEM teams as we will upload this sequence into the iGEM registry.

pOJ260⁺:

We used pOJ260⁺ as our transformation vector. Original pOJ260 is a type of suicidal plasmid with amphotericin Apr prokaryotic resistance. We have modified it for the conjugation of vgb gene into Pseudomonas denitrificans. pOJ260⁺ is also not used in previous system. Future iGEM teams can consider this type of suicidal plasmid when choosing which plasmid to use.

Cellular chassis – Pseudomonas denitrificans:

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. Throughout our project, we have seen the effectiveness of Pseudomonas denitrificans as a chassis for synthesis of vitamin B₁₂. Therefore, future iGEM teams can use P. denitrificans as an effective VB₁₂ producer.

2.Contribution related to hardware

In order to make a long term observation on the Zooxanthellae growth in response to the variations in environmental stress and nutrition, we have designed a coral-in-a-chip which can be directly monitored under the microscope. We sincerely hope that our experience in the design of coral-in-a-chip can make a great contribution to future iGEM teams. They may use this technology in their projects for proof of concept at a single cell level.

3.Contribution related to biological experiment

Our logic and design of experiments could be followed. This series of experiments is designed to explore in vitro zooxanthella cell cultures with addition of nutritional elements such as P, F_e, N and VB₁₂ to test their tolerance of heat shock as environmental stress. Our aim is to understand which nutrients will enhance the resistance of zooxanthella against temperature elevation. Our study is of critical importance for the health and metabolic balance of marine coral micro-ecosystem under global climate change.

Future iGEM teams can use this method to establish a relationship between nutrients (or other substances) and microbial growth.