Part:BBa_K3143336
csgA-GS linker-spytag
CsgA is a major curlin subunit of E. coli. Curlin is the structural subunit of the curli fimbriae. [1,2]. A SpyTag is fused to CsgA so that any protein fused to SpyCatcher can be fixed on cell membrane through SpyTag-SpyCatcher system. See more in INP-SpyTag BBa_K3143338 and csgA-SpyTag-INP-SpyTag BBa_K3143339.
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. Hammar M, Arnqvist A, Bian Z, et al. Expression of two csg operons is required for production of fibronectin‐and congo red‐binding curli polymers in Escherichia coli K‐12[J]. Molecular microbiology, 1995, 18(4): 661-670.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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