On the basis of previous research, we designed and constructed a useful vector pPIGBACK for microalgae transformation. pPIGBACK is a powerful construct with NSII, AmpR, double terminator, ORI, and PrbcL, and it is able to embark on gene double-crossover homologous recombination in Synechococcus elongatus PCC 7942 genome. (see more detail: Pigments - Backbone Design)

Cultivation of Microalgae

After several failed experiences and many efforts, we found some tricky steps (such as BG11 preparation etc.) and got the hang of microalgae cultivation. All of the key points were recorded in our protocols.

Constructs & Transformants

We sent 13 parts to iGEM and successfully transformed pigments into Synechococcus elongatus PCC 7942.

Yellow Microalgae with CrtZ

We have successfully used our microalgae transformation platform to transform CrtZ into Synechococcus elongatus PCC 7942 and the transformant are more yellow than wild type! (see more detail: Pigments - Functional Test)

Endolysin-Holin-NrtA

We successfully transformed NrtA gene from cyanobacteria Synechocystis sp. PCC 6803 to E.coli. The engineering E.coli which expresses NrtA protein can catch nitrate (NO₃^-) and nitrite (NO₂^-) to induce algae undergoing nitrogen starvation. Due to biosafety concern, we also improved a suicide mechanism endolysin-holin and successfully constructed it. Moreover, we even grouped endolysin-holin and NrtA into a powerful part. (see more detail: Nitrogen Starvation - Functional Test)

Modeling

We established 13 models to check and predict the results of the experiments. These models are extremely important to our project! (see more detail: Modeling)

Unique and Detailed Protocols for the Public

We provided unique and detailed protocols of our experiments to the public. Students, teachers, scientists all over the world and future iGEM teams can repeat our experiments or design their own experiments with these protocols. (see more detail: Notebook - Protocols)

Sharing Genetic Engineering Technology

Chung Cheng University – Fusion PCR

In 2016, NYMU-Taipei team helped students from National Chung Cheng University to form their iGEM team. Excitedly, they are going to participate iGEM competition this year as team name “CCU_Taiwan”. In 2017, we showed them how the Photo-Bioreactor functions, taught them how to run Fusion PCR through webcam, and informed them some team operations and matters.

Taipei America School – Site Directed Mutagenesis

TAS Taipei help us by testing our holin-endolysin-NrtA construct, and we teach them how to conduct site-directed mutagenesis as well as assay biofilm production. (See more detail: Collaborations)

Assistance to Establish New iGEM Teams

We helped students in Chung Hsing University and Taipei Wego Senior High School to establish their own iGEM teams. We are looking forward to seeing them at Boston in 2018!

Experimental Technology

In addition to our comprehensive microalgae transformation platform, to achieve our goals, we also applied lots of experimental technologies. Although most of our team members are freshman in college, we tried hard to learn relatively complicated experimental methods. Moreover, we realized that, with more experimental technologies and knowledge, we can be closer to our goals and do more things for the world. Therefore, we strongly encourage future iGEM teams to explore new technologies. Deeper thinking and thrive on challenge that come along with the journey of learning are as important as the technology itself.

Fusion PCR
We used fusion PCR in NrtA construct, pPIGBACK construct and fusion of pigments and the promoter prbcL.

Sticky End PCR
We used sticky end PCR in holin, endolysin, NrtA and CrtZ construct.

3 Piece Overlap PCR
We used 3 piece overlap PCR in lycopene and pPIGBACK construct.

Site Directed Mutagenesis
We used site directed mutagenesis in pPIGBACK and PrbcL construct.

Electroporation
We used electroporation in IndC construct.

Making Competent Cell
We made competent cell in electroporation experiments.

French Pressure Cell Press NrtA
We used French pressure cell press in NrtA functional test.

Microalgae DNA Extraction
We extracted our engineered Synechococcus elongatus PCC 7942 genomic DNA to confirm the correctness of transformation.

Team:NYMU-Taipei/Contribution