Difference between revisions of "Part:BBa K5302031"
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This work is derived from pBBR1MCS-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines INP(about 30kda) and V107(approximately 2kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express V107. The plasmid uses lac promotor and has kanamycin resistence. | This work is derived from pBBR1MCS-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines INP(about 30kda) and V107(approximately 2kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express V107. The plasmid uses lac promotor and has kanamycin resistence. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5302/images/part-registry-p-inp-v107-1.png" | ||
+ | width="60%" style="display:block; margin:auto;" alt="Jamboree Program" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption> | ||
+ | <b>Figure 1. </b> Colony PCR results of pBBR-INP-V107 | ||
+ | </caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | V107 is a small potent VEGF-binding peptide, which has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two V107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. | ||
+ | It was shown that V107 is strikingly amphipathic, and in the complex with the VEGF, it adopts a mixed conformation with the disordered N-tail (residues 1–4), type-I β-turn (residues 6–9), extended region (residues 9–12), and C-terminal α-helix (residues 13–19) Hydrophobic Ile7, Ala8, Met10, Trp11, Trp13, Phe16, and Leu19 are situated at the interface with the VEGF molecule | ||
+ | As for binding site, the hydrophobic side-chains of V107-Trp13, Phe16, and Leu19 contact VEGF residues Phe17, Met18, Tyr21, Lys48, Met81 and Ile83. The same cluster of residues on VEGF interacts with Flt-1D2 residues Pro143, Ile145, Leu204, and Leu221. | ||
+ | This peptide is composed of 19 amino acids, that is GGNECDAIRMWEWECFERL, and it shows great affinity with VEGF(Kd=0.53 μM).We used pBBR1MCS-2 plasmid as a backbone and transfered V107 into Escherichia coli Nissle 1917, and finally succeeded in expressing V107. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5302/images/part-registry-v107-1.png" | ||
+ | width="60%" style="display:block; margin:auto;" alt="Jamboree Program" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption> | ||
+ | <b>Figure 2. </b> VEGF-binding site of V107 | ||
+ | </caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5302/images/part-registry-v107-2.png" | ||
+ | width="60%" style="display:block; margin:auto;" alt="Jamboree Program" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption> | ||
+ | <b>Figure 3. </b> VEGF-binding site of V107 | ||
+ | </caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5302/images/part-registry-v107-4.png" | ||
+ | width="60%" style="display:block; margin:auto;" alt="Jamboree Program" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption> | ||
+ | <b>Figure 4. </b> Structure of v107 in the VEGF-bound state | ||
+ | </caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Latest revision as of 02:08, 2 October 2024
pBBR-INP-V107
This work is derived from pBBR1MCS-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines INP(about 30kda) and V107(approximately 2kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express V107. The plasmid uses lac promotor and has kanamycin resistence.
V107 is a small potent VEGF-binding peptide, which has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two V107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. It was shown that V107 is strikingly amphipathic, and in the complex with the VEGF, it adopts a mixed conformation with the disordered N-tail (residues 1–4), type-I β-turn (residues 6–9), extended region (residues 9–12), and C-terminal α-helix (residues 13–19) Hydrophobic Ile7, Ala8, Met10, Trp11, Trp13, Phe16, and Leu19 are situated at the interface with the VEGF molecule As for binding site, the hydrophobic side-chains of V107-Trp13, Phe16, and Leu19 contact VEGF residues Phe17, Met18, Tyr21, Lys48, Met81 and Ile83. The same cluster of residues on VEGF interacts with Flt-1D2 residues Pro143, Ile145, Leu204, and Leu221. This peptide is composed of 19 amino acids, that is GGNECDAIRMWEWECFERL, and it shows great affinity with VEGF(Kd=0.53 μM).We used pBBR1MCS-2 plasmid as a backbone and transfered V107 into Escherichia coli Nissle 1917, and finally succeeded in expressing V107.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4338
Illegal XbaI site found at 3189
Illegal PstI site found at 2016
Illegal PstI site found at 3177 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4338
Illegal PstI site found at 2016
Illegal PstI site found at 3177
Illegal NotI site found at 1057 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4338
Illegal BglII site found at 1803
Illegal BamHI site found at 3195 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4338
Illegal XbaI site found at 3189
Illegal PstI site found at 2016
Illegal PstI site found at 3177 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4338
Illegal XbaI site found at 3189
Illegal PstI site found at 2016
Illegal PstI site found at 3177
Illegal NgoMIV site found at 2467
Illegal NgoMIV site found at 2750
Illegal NgoMIV site found at 3616
Illegal NgoMIV site found at 5003
Illegal AgeI site found at 4843 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1379
Illegal SapI.rc site found at 2316
Illegal SapI.rc site found at 2526