Difference between revisions of "Part:BBa K5136034"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K5136034 short</partinfo>
 
<partinfo>BBa_K5136034 short</partinfo>
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==Biology==
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===SpyTag===
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SpyTag is a peptide tag of 13 residues developed by previous researchers, which could steadily bind to SpyCatcher by forming an isopeptide bond (1).
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===GFP===
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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 <i>E. coli</i> 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 that 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 (<partinfo>BBa_K5136034</partinfo>) to express His tag-SpyTag-GFP for subsequent use.
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==Characterization==
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===Agarose gel electrophoresis (AGE)===
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The basic part (BBa_K5136034) constructed was introduced into the backbone plasmid (pET-28a(+)) through Gibson assembly and transformed into <i>E. coli</i> 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).
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<br>
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a. <center><html><img src="https://static.igem.wiki/teams/5136/part/zxx/histag.png"width="400px"></html></center>
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<br>
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b. <center><html><img src="https://static.igem.wiki/teams/5136/part/zxx/224colony.png"width="200px"></html></center>
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<br>
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<center><b>Figure 1 Constructing His tag-SpyTag-GFP fused protein. a. the expression gene circuits for expressing SpyTag-GFP. b. DNA gel electrophoresis of the colony PCR products of BBa_K5136034_pET-28a(+) in <i>E. coli</i> BL21(DE3).</b></center>
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===SDS-PAGE===
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The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 25 °C, the 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.
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<br>
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<center><html><img src="https://static.igem.wiki/teams/5136/part/zxx/034colony.png"width="200px"></html></center>
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<br>
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<center><b>Figure 2 SDS-PAGE analysis of his tag-SpyTag-GFP.</b></center>
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===Fluorescence Intensity Determination===
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In order to verify the surface display effect of INPNC, plasmid pSB1C3 bearing composite part (<partinfo>BBa_K5136224</partinfo>) was transformed into <i>E.coli</i> BL21(DE3) for subsequent verification experiments, and<i> E.coli</i> 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 <i>E. coli</i> BL21(DE3), demonstrating that INPNC has excellent capacity to anchor target proteins on the cell membrane.
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<br>
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<center><html><img src="https://static.igem.wiki/teams/5136/part/zxx/zxxyingguang.png"width="400px"></html></center>
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<br>
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<center><b>Figure 3 Fluorescence intensity of samples after culturing 12 hours.</b></center>
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<br>
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<center><html><img src="https://static.igem.wiki/teams/5136/part/zxx/.png"width="400px"></html></center>
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<br>
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<center><b>Figure 4 Samples placed under blue light after culturing 12 hours.</b></center>
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==Reference==
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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).
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:46, 2 October 2024


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 that 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).

a.


b.


Figure 1 Constructing His tag-SpyTag-GFP fused protein. a. the expression gene circuits for expressing SpyTag-GFP. b. DNA gel electrophoresis of the colony PCR products of BBa_K5136034_pET-28a(+) in E. coli BL21(DE3).

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, the 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.


Figure 2 SDS-PAGE analysis of his tag-SpyTag-GFP.

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.


Figure 3 Fluorescence intensity of samples after culturing 12 hours.



Figure 4 Samples placed under blue light after culturing 12 hours.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 82
    Illegal AgeI site found at 630
  • 1000
    COMPATIBLE WITH RFC[1000]