Part:BBa_K3425044
ehaA β-domain
Native beta domain of autotransporter ehaA, surface protein from enterohemorrhagic E. coli O157:H7. This domain is exported and anchored to the outer membrane, making this protein a useful display system in E. coli [1]. In this case, it is used in the display of a Mycobacterium tuberculosis biomarker-binding single domain camellid antibody BBa_K3425043.
This particular part is designed to be used together with two other parts (see table below) as the agglutination construct for the NANOFLEX system, as a way to test nanobody functionality. In this case the nanobody BBa_K3425043 specific to the Mycobacterium tuberculosis biomarker HSP 16.3 BBa_K3425041 is displayed.
| Part number | Name | Description |
|---|---|---|
| BBa_K3425042 | pelB | signal peptide |
| BBa_K3425043 | B-F10 | HSP Nanobody |
| BBa_K3425044 | ehaA | Transmembrane domain |
Custom Type IIS design
This construct requires a signal peptide (pelB) at the N-terminal, the transmembrane domain (ehaA) at the C-terminal and the detection module (nanobody) inbetween. The idea behind this design is that, while pelB and ehaA are the same, the nanobody can be exchanged by a nanobody which binds the target you are interested in.
Each of these parts is designed to be cloned with iGEM Type IIS standard into the Level 0 backbone pSB1C00. The three parts together form one CDS, so except the first and last fusion sites, which are part of the standard, the others are custom and annotated in the sequences. The fusion sites are detailed in the table below.
By maintaining the three parts in separate pSB1C00 plasmids you can maintain more than one nanobody and assemble the construct to Level 1 backbones (such as pSB1K01) together with promoter, RBS and terminator in a 6-DNA parts one-step reaction (instead of 4-DNA parts).
| 5' Fusion site | Part | 3' Fusion site |
|---|---|---|
| FS_c (AATG) | pelB | pelB_FS |
| pelB_FS | B-F10 | ehaA_FS |
| ehaA_FS | ehaA | FS_e (GCTT) |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 555
Illegal AgeI site found at 144 - 1000COMPATIBLE WITH RFC[1000]
References
[1] Salema, V., Marín, E., Martínez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodelón, C. & Fernández, L. Á. (2013). Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies. PloS one, 8(9), e75126.
| None |