Difference between revisions of "Part:BBa K103006"

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<!-- Add more about the biology of this part here -->
 
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===Usage and Biology===
 
===Usage and Biology===
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*In our project Ompa-link is used as outer membrane anchor for our selection system
 
*In our project Ompa-link is used as outer membrane anchor for our selection system
 
*This brick contains our nonstandard restriction sites (NdeI and SacI) that allow 'scarless' cloning and easy creation of translation fusions
 
*This brick contains our nonstandard restriction sites (NdeI and SacI) that allow 'scarless' cloning and easy creation of translation fusions
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===Improvement by NEFU_China===
 
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Our team worked to improve the characterization of a signal peptide, BBa_K103006 , part from the iGEM Registry. This part originated from Univeristy of Warsaw 2008 iGEM team can be used as a outer-membrane-targeting anchor for selection system in E. coli.
 
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In order to make the production process and purification more efficiently, we added T7 promoter and His tag to the former of Lpp-ompa sequence and our another basic part (L-FABP) was fused to the back, constructing the composite parts (Lpp-ompa-L-FABP). Finally we linked the newly synthesized parts to the standard vector pSB1C3. The new plasmids can be used directly for IPTG induced expression in E. coli (DE3). See more details in composite part (BBa_K2302011)
 
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This biobrick (Lpp-ompA) is an improvement on the biobrick designed by the Warsaw 2008 iGEM team K103006 , and our improvement of this part is easy：
 
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1.The sequence that we have used is a synthetic gene with codon optimization, designed specifically for high level expression in E. coli. Thus it is shorter but with the same function as the other OmpA in the part registry.
 
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2. This part contains our nonstandard restriction sites (NdeI and Xhol) on the right and left separately of our newly Lpp-ompA that allows easy creation of translation fusion other interested protein.
 
 
| [[Image:Ompalinker.jpg|200px]]
 
| [[Image:Ompalinker.jpg|200px]]
 
This protein structure was predicted using threading and protein fold prediction and may differ from actual one.
 
This protein structure was predicted using threading and protein fold prediction and may differ from actual one.
 
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===Improvement by NEFU-China===
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Lpp-OmpA, a signal peptide can be used as an outer-membrane-targeting anchor in E. coli. Proteins fused to OmpA-link can be presented on cell surface.
 +
 +
Our team modified the signal peptide sequence to make it more conducive to direct our FABP to anchor to the outer membrane of E.coli. We reduced several amino acids at the N-end of the peptide chain so that the sequence was shorter than that in the Registry of iGEM, BBa_K103006, from University of Warsaw 2008 iGEM team, but the FABP with our signal peptide BBa_K2302003(https://parts.igem.org/Part:BBa_K2302003)
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was expressed in high level. In the meantime, we used codon optimization to further improve the expressed level of FABP. The result showed that expressed levels of FABP with our shorter signal peptides were higher than that in the Registry of iGEM.
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 +
===Result===
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E. coli was induced in 37℃ and membrane protein was separated by ultracentrifugation. The result of western blot showed the difference at the expressed levels of Lpp-ompA-L-FABP in E. coli transformed by the recombinant plasmid, indicated that the expressed levels of FABP with our shorter signal peptides were higher than that with BBa_K103006 in the Registry of iGEM.
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https://static.igem.org/mediawiki/parts/e/e4/NEFU-China_2017%2810%29.jpg
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==Toulouse_INSA-UPS’s 2022 Characterization of OmpA==
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===Crystallographic structure of OmpA ===
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<html>
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Toulouse_INSA_UPS_2022 contributed to the characterization of this part. The team added this year
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the Lpp-OmpA (AA 46 to 159) crystallized structure from the Protein Data Bank (PDB) (accession number: <a href="https://www.rcsb.org/structure/1G90">1G90</a>), thus providing new
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structural information to the whole protein prediction from Warsaw's 2008 iGEM team.
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<br/>
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<html><img src="https://static.igem.wiki/teams/4197/wiki/design/ompa-complet-v6.png" width="600px"/></html>
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<p>'''Figure 1: 3D structure of Lpp-OmpA (AA46 to 159) derived from <i>E. coli</i> (PDB: 1G90).'''
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The arrows represent 𝛽-sheets which here form a 𝛽-barrel
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across the outer membrane. The loops in red face the extracellular media while the blue-ones face the periplasm. </p>
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<br/>
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<html>
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<p>More information about the project for which the part was created: <a href="https://2022.igem.wiki/toulouse-insa-ups/index.html"> DAISY (INSA-UPS 2022)</a> </p>
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</html>
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==Link==
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【1】https://parts.igem.org/Part:BBa_K2302003
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【2】http://2017.igem.org/Team:NEFU_China
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 19:25, 10 October 2022

OmpA outer membrane protein A fused to linker; displays proteins on cell surface

One of our basic bricks used to create fusions attached to outer membrane

Usage and Biology

  • This part uses OmpA (outer membrane protein A [http://www.ncbi.nlm.nih.gov/pubmed/17559395?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]) fragment (AAs 46-159) N-terminally fused with lipoprotein signal peptide fragment (AAs 1-9)
  • Proteins fused to Ompa-link are presented on cell's surface
  • Unstructuralized linker (Gly Ser Gly) allows proper folding of both fusion partners
  • In our project Ompa-link is used as outer membrane anchor for our selection system
  • This brick contains our nonstandard restriction sites (NdeI and SacI) that allow 'scarless' cloning and easy creation of translation fusions
 Ompalinker.jpg

This protein structure was predicted using threading and protein fold prediction and may differ from actual one.

Improvement by NEFU-China

Lpp-OmpA, a signal peptide can be used as an outer-membrane-targeting anchor in E. coli. Proteins fused to OmpA-link can be presented on cell surface.

Our team modified the signal peptide sequence to make it more conducive to direct our FABP to anchor to the outer membrane of E.coli. We reduced several amino acids at the N-end of the peptide chain so that the sequence was shorter than that in the Registry of iGEM, BBa_K103006, from University of Warsaw 2008 iGEM team, but the FABP with our signal peptide BBa_K2302003(https://parts.igem.org/Part:BBa_K2302003) was expressed in high level. In the meantime, we used codon optimization to further improve the expressed level of FABP. The result showed that expressed levels of FABP with our shorter signal peptides were higher than that in the Registry of iGEM.

Result

E. coli was induced in 37℃ and membrane protein was separated by ultracentrifugation. The result of western blot showed the difference at the expressed levels of Lpp-ompA-L-FABP in E. coli transformed by the recombinant plasmid, indicated that the expressed levels of FABP with our shorter signal peptides were higher than that with BBa_K103006 in the Registry of iGEM.

NEFU-China_2017%2810%29.jpg

Toulouse_INSA-UPS’s 2022 Characterization of OmpA

Crystallographic structure of OmpA

Toulouse_INSA_UPS_2022 contributed to the characterization of this part. The team added this year the Lpp-OmpA (AA 46 to 159) crystallized structure from the Protein Data Bank (PDB) (accession number: 1G90), thus providing new structural information to the whole protein prediction from Warsaw's 2008 iGEM team.

Figure 1: 3D structure of Lpp-OmpA (AA46 to 159) derived from E. coli (PDB: 1G90). The arrows represent 𝛽-sheets which here form a 𝛽-barrel across the outer membrane. The loops in red face the extracellular media while the blue-ones face the periplasm.


More information about the project for which the part was created: DAISY (INSA-UPS 2022)

Link

【1】https://parts.igem.org/Part:BBa_K2302003

【2】http://2017.igem.org/Team:NEFU_China


Sequence and Features


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]