Difference between revisions of "Part:BBa K3088001:Design"

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[[file:Succesful sequencing of pSB1C3 HE + OG.jpg|800px|thumb|centre|Figure 1. This the illustration of the plasmid (pSB1C3) containing part BBa K3088001 and BBa_K769001.]]
 
[[file:Succesful sequencing of pSB1C3 HE + OG.jpg|800px|thumb|centre|Figure 1. This the illustration of the plasmid (pSB1C3) containing part BBa K3088001 and BBa_K769001.]]
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<b> I.EnvZ/HBEGF Receptor Modelling </b>
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The chimeric receptor that we are working on is based from two different integral membrane proteins, EnvZ and HBEGF. EnvZ and HBEGF are basically a fully function protein receptor that can be found in Escherichia coli and human respectively. Our chimeric receptor will structurally consist of EnvZ structure as the base structure and HBEGF as part of periplasmic domain as well as the Diphtheria Toxin Binding Region.
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In order to model the EnvZ/HEBGF chimeric receptor, we should gather information about the amino acid sequence from both of the EnvZ and HBEGF. We use information from online database, Uniprot1, and gather the FASTA (https://www.uniprot.org/uniprot/Q99075 and https://www.uniprot.org/uniprot/P0AEJ4).
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To determine on which residue of EnvZ we should insert HBEGF in, we run secondary structure prediction of EnvZ via NetSurfP2. Then we predict its topography using TOPCONS webserver3. The topography data from both TOPCONS3 and Uniport1 database give a very similar result. Thus, using the topography data and the secondary structure information, we predict the sequence of EnvZ graphically as shown below.
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[[File:Gambar IGEM 1.png|700px|thumb|centre|Figure 2. On EnvZ, that consist of 450 amino acids, we now know that the periplasmic region is around 36th-159th residue. Therefore, if we want to insert the HBEGF, we have to insert it on this particular region. In order to determine the specific region, we do some literature researches and found that the region around 80th-146th residue is good to be swapped with insert. 4 On HBEGF, we determine the region for chimeric by doing literature research.5 We then come out with 27 residues from HBEGF including the important residue for Diphtheria Toxin (DT) binding, the 141th residue.1
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===Source===
 
===Source===

Revision as of 15:37, 20 October 2019


HBEGF/ENVZ Chimeric Complete


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Sequencing of this part (BBa_K3088001) combined with part (BBa_K769001) into pSB1C3 is confirmed and showed as follow:


AAAGATGGACTGACTGAATGCCTCAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCT TAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAG TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTT CGCTAAGGATGATTTCTGGAAGTGCCATTCCGCCTGACCTAGTTCAAGTGTCCGAGAAGAATTCGCGGCCGCTTCTAGAGAGGTACTGACTCATAAAAAA TTTATTTGCTTTGTGAGCGGATAACAATTATAATAACGATCGCGAAAGAGGAGAAATACGCGATCGAAGATGCGTCGTCTGCGTTTTAGTCCACGCAGCT CGTTTGCACGTACGCTTTTGTTAATCGTTACATTGCTTTTTGCTTCCCTGGTCACCACTTACTTAGTTGTATTAAACTTCGCAATCTTGCCAAGTCTGCA ACAGTTTAACAAGGTATTGGCTTACGAGGTTCGCATGCTTATGACCGACAAACTTCAATTGGAAGACGGGACTCAACTGGTTGTACCACCCGCATTTCGC CGTGAGAAGTACGTTAAGGAATTGCGCGCCCCCAGTTGCATCTGTCACCCCGGATACCATGGAGAGCGCTGCCACGGCTTGTCACTGGTGCCGTTGACAG AGATCCATCAGGGTGACTTTAGCCCGCTTTTCCGTTATACGTTGGCAATTATGTTACTTGCCATCGGTGGCGCATGGTTATTTATTCGCATTCAGAACCG TCCGTTGGTCGATTTAGAGCACGCTGCACTTCAAGTTGGTAAGGGCATCATCCCGCCACCATTGCGCGAGTACGGGGCCTCGGAAGTACGTAGTGTTACT CGTGCCTTCAATCATATGGCCGCCGGTGTTAAGCAGTTAGCAGACGACCGCACTCTGTTGATGGCTGGCGGGTCTCATGATTTGCGNACCCCCTGACNCG TATCCGCCTGGCTACTGAAATGATGTCGGAACAGGATGGAACTTGGCCGAGTCCTCAATAAAGACTTGAGGAATGAACGCTTTATTGAACGTTCTCCACT TTTGCTACCGCCCGAAANCCTTTGAANGGGACNNNATGGGGTTGGGGANAATTTTGCCAAANNGGGTCAACCGGAANNNAAAGGGCTTCCCCNNTTTTGG GGGAAAANNNNNCCCTTTTTTGNNNGGCCCTAAGGTTTAACCNNCCCCCGAAAAGGGGGTAAGGGNCCCNNNCCCCNCCNCCCGTTTTGGNNGGGAGGAC GNCCCCCCCCNA


Figure 1. This the illustration of the plasmid (pSB1C3) containing part BBa K3088001 and BBa_K769001.


I.EnvZ/HBEGF Receptor Modelling

The chimeric receptor that we are working on is based from two different integral membrane proteins, EnvZ and HBEGF. EnvZ and HBEGF are basically a fully function protein receptor that can be found in Escherichia coli and human respectively. Our chimeric receptor will structurally consist of EnvZ structure as the base structure and HBEGF as part of periplasmic domain as well as the Diphtheria Toxin Binding Region. In order to model the EnvZ/HEBGF chimeric receptor, we should gather information about the amino acid sequence from both of the EnvZ and HBEGF. We use information from online database, Uniprot1, and gather the FASTA (https://www.uniprot.org/uniprot/Q99075 and https://www.uniprot.org/uniprot/P0AEJ4). To determine on which residue of EnvZ we should insert HBEGF in, we run secondary structure prediction of EnvZ via NetSurfP2. Then we predict its topography using TOPCONS webserver3. The topography data from both TOPCONS3 and Uniport1 database give a very similar result. Thus, using the topography data and the secondary structure information, we predict the sequence of EnvZ graphically as shown below.


Figure 2. On EnvZ, that consist of 450 amino acids, we now know that the periplasmic region is around 36th-159th residue. Therefore, if we want to insert the HBEGF, we have to insert it on this particular region. In order to determine the specific region, we do some literature researches and found that the region around 80th-146th residue is good to be swapped with insert. 4 On HBEGF, we determine the region for chimeric by doing literature research.5 We then come out with 27 residues from HBEGF including the important residue for Diphtheria Toxin (DT) binding, the 141th residue.1

Source

This part was obtained by reverse translation from extensive protein modeling of HBEGF/ENVZ. The DNA sequence of both parts were obtained from National Center for Biotechnology Information (NCBI) & UniProt database

References