Difference between revisions of "Part:BBa K1489004"

 
 
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<partinfo>BBa_K1489004 short</partinfo>
 
<partinfo>BBa_K1489004 short</partinfo>
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A fusion protein of OmpA (BBa_K1489002) fused to Bovine Galectin-1 (BBa_K1489001). Used to bind sugars at the outer cell membrane. Under pBAD regulation.
 
A fusion protein of OmpA (BBa_K1489002) fused to Bovine Galectin-1 (BBa_K1489001). Used to bind sugars at the outer cell membrane. Under pBAD regulation.
  
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===Expression Test===
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BBa_K1489004 was transformed into chemically competent DH5-alpha cells and plated overnight with chloramphenicol challenge. One colony was incubated in 5 mL LB media + 34 ug/mL chloramphenicol and grown to mid-log phase at 37oC. Arabinose was added to a final concentration of 0.2% to initiate protein production and cells were re-incubated at 37 oC for 21 hours. Cells were then pelleted and lysed with 10% SDS solution. Cell lysate was centrifuged at 13200 rpm in a tabletop centrifuge for 1 hour to separate soluble and insoluble fractions. Both fractions were loaded on an SDS-PAGE gel for western blot analysis using anti-His antibodies.
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[[File:OmpA_BovineExpression.jpg]]
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The presence of bands in both the induced and uninduced cells indicates leaky expression of the protein. The molecular weight corresponds with the weight of OmpA-Bovine.
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===Structure Prediction===
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[[File:OmpA-Bovine.png]]
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The structure of OmpA-BtGal was predicted using the RaptorX online server (Källberg et. al). The base structure of OmpA (right) is not predicted to be affected by the addition of BtGal (left); the two parts are connected by the unstructured linker (GGGSGGGS).
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References
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Morten Källberg, Haipeng Wang, Sheng Wang, Jian Peng, Zhiyong Wang, Hui Lu & Jinbo Xu. Template-based protein structure modeling using the RaptorX web server. Nature Protocols 7, 1511–1522, 2012.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 23:57, 16 October 2014

pBAD-RBS-OmpA-Bovine Galectin-1-B0012 Terminator

A fusion protein of OmpA (BBa_K1489002) fused to Bovine Galectin-1 (BBa_K1489001). Used to bind sugars at the outer cell membrane. Under pBAD regulation.

Expression Test

BBa_K1489004 was transformed into chemically competent DH5-alpha cells and plated overnight with chloramphenicol challenge. One colony was incubated in 5 mL LB media + 34 ug/mL chloramphenicol and grown to mid-log phase at 37oC. Arabinose was added to a final concentration of 0.2% to initiate protein production and cells were re-incubated at 37 oC for 21 hours. Cells were then pelleted and lysed with 10% SDS solution. Cell lysate was centrifuged at 13200 rpm in a tabletop centrifuge for 1 hour to separate soluble and insoluble fractions. Both fractions were loaded on an SDS-PAGE gel for western blot analysis using anti-His antibodies.

OmpA BovineExpression.jpg

The presence of bands in both the induced and uninduced cells indicates leaky expression of the protein. The molecular weight corresponds with the weight of OmpA-Bovine.

Structure Prediction

OmpA-Bovine.png

The structure of OmpA-BtGal was predicted using the RaptorX online server (Källberg et. al). The base structure of OmpA (right) is not predicted to be affected by the addition of BtGal (left); the two parts are connected by the unstructured linker (GGGSGGGS).

References Morten Källberg, Haipeng Wang, Sheng Wang, Jian Peng, Zhiyong Wang, Hui Lu & Jinbo Xu. Template-based protein structure modeling using the RaptorX web server. Nature Protocols 7, 1511–1522, 2012. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal XhoI site found at 971
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]