Difference between revisions of "Team:NYMU-Taipei/Demonstrate"
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<p>pET32a FPF-skel1 is a pET32a whose original MCS is replaced with our newly designed MCS -- FPF-Skel1. This new MCS allows one fluorescent protein and another protein(in our case, a DKK1-binding protein)to be inserted. The inserted proteins would be expressed as fusion protein by a T7 promoter with fluorescent protein attached to the C-terminal of the other protein and his tag attached to the C-terminal of fluorescent protein(called Binding-Fluorescent type, BF type). </p> | <p>pET32a FPF-skel1 is a pET32a whose original MCS is replaced with our newly designed MCS -- FPF-Skel1. This new MCS allows one fluorescent protein and another protein(in our case, a DKK1-binding protein)to be inserted. The inserted proteins would be expressed as fusion protein by a T7 promoter with fluorescent protein attached to the C-terminal of the other protein and his tag attached to the C-terminal of fluorescent protein(called Binding-Fluorescent type, BF type). </p> | ||
| − | <img src=""> | + | <img src="https://static.igem.org/mediawiki/2018/9/95/T--NYMU-Taipei--exp1-1.jpg" style="width:600px"> |
<p>pET32a FPF-skel2 functions similarly. However, in the protein expressed by this plasmid, the fluorescent protein is attached to the N-terminal of the other protein and his tag is attached to the N-terminal of the fluorescent protein(called Fluorescent-Binding type, FB type). In this case, the sequence of protein is inverted. </p> | <p>pET32a FPF-skel2 functions similarly. However, in the protein expressed by this plasmid, the fluorescent protein is attached to the N-terminal of the other protein and his tag is attached to the N-terminal of the fluorescent protein(called Fluorescent-Binding type, FB type). In this case, the sequence of protein is inverted. </p> | ||
| − | <img src=""> | + | <img src="https://static.igem.org/mediawiki/2018/d/d7/T--NYMU-Taipei--exp1-2.png" style="width:600px"> |
<p>We made this additional plasmid because FRET efficiency is highly related to the distance between the FRET pairs. Having one of the FRET pair at the farther end of a single domain of a protein, instead of the closer end, might result in drastic difference in FRET efficiency. It is also worth mentioning that modeling protein conformation might still be different from reality. Hence, to make sure that we can find the best protein conformation for FRET, we intend to express both BF and FB type fusion protein.</p> | <p>We made this additional plasmid because FRET efficiency is highly related to the distance between the FRET pairs. Having one of the FRET pair at the farther end of a single domain of a protein, instead of the closer end, might result in drastic difference in FRET efficiency. It is also worth mentioning that modeling protein conformation might still be different from reality. Hence, to make sure that we can find the best protein conformation for FRET, we intend to express both BF and FB type fusion protein.</p> | ||
Revision as of 20:36, 17 October 2018
FPF system
Success in constructing a FRET Protein Fusion(FPF) system
To simplify the construction of FRET fusion proteins, we have designed a system to standardize the process. The system consists of two plasmids-- pET32a FPF-skel1 and pET32a FPF-skel2.
pET32a FPF-skel1 is a pET32a whose original MCS is replaced with our newly designed MCS -- FPF-Skel1. This new MCS allows one fluorescent protein and another protein(in our case, a DKK1-binding protein)to be inserted. The inserted proteins would be expressed as fusion protein by a T7 promoter with fluorescent protein attached to the C-terminal of the other protein and his tag attached to the C-terminal of fluorescent protein(called Binding-Fluorescent type, BF type).
pET32a FPF-skel2 functions similarly. However, in the protein expressed by this plasmid, the fluorescent protein is attached to the N-terminal of the other protein and his tag is attached to the N-terminal of the fluorescent protein(called Fluorescent-Binding type, FB type). In this case, the sequence of protein is inverted.
We made this additional plasmid because FRET efficiency is highly related to the distance between the FRET pairs. Having one of the FRET pair at the farther end of a single domain of a protein, instead of the closer end, might result in drastic difference in FRET efficiency. It is also worth mentioning that modeling protein conformation might still be different from reality. Hence, to make sure that we can find the best protein conformation for FRET, we intend to express both BF and FB type fusion protein.
With the FRET protein fusion system, we are able to construct plasmids below with ease. That the backbone’s effect on protein conformation has also been confirmed to be minimal as it contains little unnecessary sequence and the expressed proteins has functioned normally as can be seen in fluorescence graphs below.
- pET32a FPF-Skel1 YPet-Ubc9
- pET32a FPF-Skel1 YPet-H7
- pET32a FPF-Skel1 YPet-E1E2
- pET32a FPF-Skel1 YPet-E3E4
- pET32a FPF-Skel1 YPet-E1
- pET32a FPF-Skel1 YPet-E3
- pET32a FPF-Skel1 CyPet-SUMO1
- pET32a FPF-Skel1 CyPet-VHH G5
- pET32a FPF-Skel1 CyPet-VHH H7
- pET32a FPF-Skel1 CyPet-E3E4
- pET32a FPF-Skel1 CyPet-E3