Team:NYMU-Taipei/results/optomagnetic-design1

Then, after we have the fundamental material of Mms13, we did the next step of our construct.(See Figure 3)

Fig. 3:We used several steps of recombinant PCR to assemble (1)mms13-rLuc； (2)YN-mms13-rLuc； (3)Y-mms13-rLuc, why we chose recombinant PCR is because of two reasons: (1) the less time it took and (2)we found better precision in recombinant PCR than in ligation process.

The characterized and checked results shown in Figure 4.

Fig. 4:Checked YFP-Mms13-rluc part.

For parts mms13-rLuc, links to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624007 BBa_K624007]; YN-mms13-rLuc, links to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624008 BBa_K624008]; EYFP-mms13-rLuc fusion, links to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624006 BBa_K624006].

Construct CHAMP Design

As for the CHAMP part, we use ligation process to get the whole sequences of CHAMP peptides. However, we still use the recombinant PCR procedure to anchor either YC or YFP in our process. Several electrophoresis results for CHAMP constructs are shown below.

Fig. 6:Total CHAMP Ligation, and its part links to[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624010 BBa_K624010].

Fig. 5:YFP with CHAMP Ligation sequences' N-terminus by recombinant PCR, and its associated parts-(1)CHAMP-YC, links to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624026 BBa_K624026], (2)CHAMP-EYFP, links to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624025 BBa_K624025].

Whole Construct and Experimental Results

After we have both materials of Mms13's construction and CHAMP peptides' construction, we use ligation procedure to fulfill six constructs we design in our optomagnetic-design.

Fig. 7: The main design construct. Ori is the origin of replication of AMB-1. Pmsp1 is the promoter for AMB-1.

The correlated part of construct in Fig. 7 is pYMB essentials+[YN-13-R]+[YC-CHAMP], links to link:http://partsregistry.org/wiki/index.php?title=Part:BBa_K624018.

Fig. 8: A construct without YC on the CHAMP is our negative control in our design. It would not light up no matter there is magnetic field applied or not.

The related part of construct in Fig. 8 is pYMB essentials+[YN-13-R], links to http://partsregistry.org/wiki/index.php?title=Part:BBa_K624024.

Fig. 9:. We design this construct with the whole YFP on the N-terminus of Mms13 and the CHAMP design to induce the BRET phenomenon. We suppose that with the CHMAP design, the construct will light up only when magnetic force is applied.

Fig. 10: This design is constructed with the whole YFP on the N-terminus of Mms13 but “without” the CHAMP design. The construct should be a positive control with constant light no matter whether force is applied or not.

Fig. 11: The design construct is similar with construct in Fig. 5. Why we construct this design with the whole YFP on the C-terminus of the CHAMP design instead of Mms13’s N-terminus is that we try to avoid the interferences of DNA transformation with sequences added in front of the natural construct. The construct is supposed to light up when force pull the Mms13 and turn off if no magnetic field is applied.

Fig. 12: In this last construct, only r-luciferase anchored on Mms13’s C-terminus. It will light up in yellow light (with 580 nm wavelength) due to the luciferase reaction, instead of the green light induced from BRET phenomenon (540 nm wavelength).