Figure 1. Our team presents projects of Finding Diphthy and ModVision in collaboration within iGEM 2018 for silver medal criteria. Discussion and surveys were done by our policy and practice division member iGEM UI Ainun Rahmania and Glory Lamria.

WHAT WE ASK?

After two months of lab session starting from June, we encountered such unexpected bad events in doing PCR amplification of the HT-1 and HT-2 fragments. These fragments are meant to be ligated linearly to form single basic biobrick part of HB-EGF/Tar since IDT^TM were not able to synthesize as one gBlocks. The cloning primer Fwd and Rev (personally synthesized primer) and for amplification fail to specifically anneal in the 5’ and 3’ of both fragments. The results of the PCR amplification are shown in following figure 2.

Figure 2. Gradient temperature PCR for both HT-1 and HT-2 fragments. Note that more than 2 bands could be found in HT-1 and HT-2 fragment. These amplified bands explain that the synthetically designed primers were not specific enough. HT-1 fragment size is 1105 bp, and HT-2 fragment size is 864 bp. The indicated arrows showed the expected amplified fragments.

As a means of collaboration, we desperately tried to contact iGEM NTU for help of ligation cloning of HT into commercial expression vector pcDNA3 on August. Coincidently, Angelysia went back to Jakarta, and we met each other for further discussion in cloning the respective gBlocks. The gBlocks are sent to NTU Singapore on the mid of August. We gave our highest gratitude for team iGEM NTU in helping us to clone the completed linear HT fragment into pcDNA3. The samples arrived back to IHVCB lab on the mid of September. Additionally, iGEM NTU also offers to clone several designed parts of our project related with FRET system into commercial vectors.

Figure 3. Representative of iGEM NTU ModVision and members of iGEM UI Finding Diphthy had a collaboration meeting. From left to right: Angelysia Cardilla (iGEM NTU Singapore), Andrea Laurentius (Wet lab Division iGEM UI), Brian Mendel (Advisor iGEM UI), Muhammad Iqbal Adi Pratama (Wet lab Division iGEM UI), and Valdi Ven Japranata (Team Leader iGEM UI).
We have started to do re-clone traditionally using EcoRI and PstI into pQE80L expression plasmids, and successfully obtained the pQE80L-HT (figure 4).

However, we tried to clone the HT into pSB1C3 and fail twice since both DNA have the same fragment size, and it would be difficult to be done in traditional cloning (figure 5). Therefore, iGEM NTU offers us help in cloning the leftovers HT complete fragment into pSB1C3 while our lab team could start to characterize the HT part along with BFP and DiphTox.

Figure 4. Gel analysis of PCR colonies on pQE80L-HT transformed BL21(DE3) strain E. coli. The subsequent 600s bp bands were found in all of the following colonies, indicating that the HT fragment was successfully inserted.

Figure 4. Gel analysis of PCR colonies on pSB1C3-HT transformed Top10 strain E. coli. Expected 600s bp bands were not found in all of the following colonies, although we could discover 300s bp bands. This concluded that the cloning of HT into pSB1C3 failed.