Difference between revisions of "Part:BBa K1489004"
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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. | ||
| − | Expression Test | + | ===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. | 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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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. | 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=== | ||
| + | [[File: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. | ||
<!-- 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.
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
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal XhoI site found at 971 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]