Part:BBa_K3143339
csgA-GS linker-SpyTag-INP-GS-linker-SpyTag
CsgA is a major curlin subunit of E.coli. INP is an outer membrane-bound protein from Pseudomonas syringae[1,2]. A SpyTag is fused to csgA & INP so that any protein fused to SpyCatcher can be fixed on cell membrane through SpyTag-SpyCatcher system. See more in csgA-SpyTag BBa_K3143338 and INP-SpyTag BBa_K3143338.
Figure 1: The SpyTag/SpyCatcher system
Characterization
We designed an experiment to measure the capture ability of CsgA–SpyTag, INP–SpyTag and CsgA–INP-SpyTag. We used sfGFP–SpyCatcher protein expressed and lysed from E.coli BL21 as the indicator.
We first expressed SpyCatcher-GFP protein in B Cell, and then collected the SpyCatcher-GFP protein in the supernatant by cell disruption and high-speed centrifugation. Then we added a certain amount of SpyCatcher-GFP protein to the bacterial culture medium of CsgA–SpyTag, INP–SpyTag and CsgA–INP-SpyTag, reacting for two hours.
Then, we centrifuged the reaction system and measure the fluorescence of the supernatant. The decrease of florescence indicated the amount of sfGFP – SpyCatcher protein the A cell surface captured.
As shown in the results in Fig 2a, we saw that the expression band of SpyCatcher-GFP was clearly visible on the PAGE gel. In Fig 2b, we can see that after the reaction, the fluorescence values of different groups were decreased, and CsgA–INP-SpyTag was the most significant one.
Figure 2: A PAGE Gel showed that SpyCatcher-GFP was succefully expressed and collected B Florescence measurement of csgA, INP, csgA+INP group
Reference
1. Olsén A, Arnqvist A, Hammar M, et al. The RpoS sigma factor relieves H‐NS‐mediated transcriptional repression of csgA, the subunit gene of fibronectin‐binding curli in Escherichia coli[J]. Molecular microbiology, 1993, 7(4): 523-536.
2. Kim D, Ku S. Bacillus cellulase molecular cloning, expression, and surface display on the outer membrane of Escherichia coli[J]. Molecules, 2018, 23(2): 503.
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 984
- 1000COMPATIBLE WITH RFC[1000]
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