Part:BBa_K5136034
His tag-SpyTag-gfp
Biology
SpyTag
SpyTag is a peptide tag of 13 residues developed by previous researchers, which could steadily bind to SpyCatcher by forming an isopeptide bond (1).
GFP
GFP is a kind of green fluorescence protein, whose excitation wavelength is 488 nm and emission wavelength is 525 nm. When expressed in engineered bacteria, GFP will emit green fluorescence under light of 488 nm wavelength.
Usage
We aim to display metallothioneins MT2A and MT3 on the surface of E. coli by INPNC, which could improve the adsorption rate of heavy metal ions for the harmless treatment of deinking wastewater. However, it is tricky to verify whether a protein has been successfully displayed on the surface of bacteria. So, we introduce the Spy system. In our project, SpyTag is fused with GFP, and his tag is added at the C-terminal of the fused protein SpyTag-GFP for the subsequent protein purification. Then, the engineered bacteria which express the fused protein INPNC-His tag-SpyCatcher are treated with purified his tag-SpyTag-GFP. By monitoring the fluorescence intensity of bacteria, we can verify if INPNC could anchor target proteins on the cell surface. Here, we constructed the basic part (BBa_K5136034) to express His tag-SpyTag-GFP for subsequent use.
Characterization
Agarose gel electrophoresis (AGE)
The basic part (BBa_K5136034) constructed was introduced into the backbone plasmid (pET-28a(+)) through Gibson assembly and transformed into E. coli BL21 (DE3). The positive clones were selected, verified and cultivated, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (953 bp) can be observed at the position around 1000 bp (Figure 1).
SDS-PAGE
The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 25 °C, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Figure 2), the target protein (29 kDa) can be observed at the position around 30 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.
Fluorescence Intensity Determination
In order to verify the surface display effect of INPNC, plasmid pSB1C3 bearing composite part (BBa_K5136224) was transformed into E.coli BL21(DE3) for subsequent verification experiments, and E.coli BL21(DE3) carrying the I0500_pSB1C3 vector was set as the control group. After being induced with 0.2%(w/v) arabinose for 12 hours, the culture solution of the experimental group(expressing INPNC-his tag-SpyCatcher) and the culture solution of the control group were incubated with His tag-SpyTag-GFP at 37 °C in the shaker. Cultures were taken after 12 hours, and then were centrifuged to get the precipitate. The fluorescence intensity of the samples was measured. It was clearly seen from Figure 3 that the fluorescence intensity of the experimental group was significantly higher than the fluorescence intensity of the control group. In addition, we placed the precipitation containing the control group and the experimental group under blue light. We clearly saw the green fluorescence of the experimental group excited by blue light, while the control group was contrary (Figure 4). This fact indicated the combination between His tag-SpyTag-GFP and the INPNC-His tag-SpyCatcher displayed on the surface of E. coli BL21(DE3), demonstrating that INPNC has excellent capacity to anchor target proteins on the cell membrane.
Reference
1. B. Zakeri et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 82
Illegal AgeI site found at 630 - 1000COMPATIBLE WITH RFC[1000]
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