Team:NYMU-Taipei/results/optomagnetic-design1
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Six Constructs and Experimental Results
We now follow the steps we construct our design to examine and illustrate what we have done so far.
The total parts we construct and their detailed information will be performed and recorded in our part registry.
Fig. 1:This is the total constructs of our optomagnetic design, the notation for each columns with different colors can be seen in the following paragraphs.
The Beginning
As soon as we have the AMB-1 bacteria colony, we do the AMB-1 colony PCR to get the Mms13's DNA sequences as our parts to do the following steps.
The correcterized gel electrophoresis result is shown below.(See Figure 2) The detail of Mms13's information, please link [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624005].
Fig. 2:The correcterized gel electrophoresis result of Mms13.
Construct Mms13
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 correcterized and checked results shown in Figure 4.
Fig. 4:Checked YFP-Mms13-rluc part.
For parts mms13-rLuclink:http://partsregistry.org/wiki/index.php?title=Part:BBa_K624007, YN-mms13-rLuclink:http://partsregistry.org/wiki/index.php?title=Part:BBa_K624008
Fig. 3: The main design construct. Ori is the origin of replication of AMB-1. Pmsp1 is the promoter for AMB-1.
Fig. 4: 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.
Fig. 5:. 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. 6: 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. 7: 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. 8: 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).
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