Difference between revisions of "Part:BBa K5382130:Design"
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===Design Notes===
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===Design Considerations===
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In the expression of CL7-Linker-sfGFP, the detection and purification of its expression must be considered, otherwise the binding of CL7 and Im7 may be affected, thus affecting the membrane surface anchorings of the entire system, and the purity of sfGFP may be affected, thus affecting the subsequent dual fluorescence verification. This may affect the judgment of anchoring (film surface display system) validation. The process of double fluorescence verification here can be simply summarized as follows: The vesicle membrane was stained with Dil orange film dye, and CL7-linker-sfGFP was incubated with vesicles to anchor sfGFP on the membrane surface. After the samples were treated, the anchoring could be verified by the orange fluorescence of Dil and the green fluorescence of sfGGP under a confocal fluorescence microscope. After the verification, it can be proved that the membrane surface system is successfully constructed, and sfGFP can be changed into targeting elements such as single-chain antibodies and nano-antibodies to provide targeting.
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Our design aims to link sfGFP in series with CL7 so that sfGFP can be anchored to the surface of the cell membrane through the strong biological orthogonal interaction of Im7 and CL7.<br>
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Therefore, we constructed a fusion protein expression plasmid of green fluorescent protein and CL7 (pET23a-CL7-sfGFP), transferred it into <i>Escherichia coli</i> BL21, added IPTG to induce expression, broke up the bacteria to get the protein and finally purified it by nickel column eluting. The SDS-PAGE electrophoretic result of the eluent is as follows (Figure. 1).
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https://static.igem.wiki/teams/5382/part-pictures/sfgfp.jpg<br>'''Figure 1.'''  SDS-PAGE electrophoretic result of CL7-sfGFP obtained by nickel column elution<br>
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Loading sequence: supernatant, precipitation, flow through, 20mM, 50mM, 80mM, 100mM, 150mM, 200mM, Marker, 300mM, 500mM<br><br>
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The protein size of CL7-sfGFP is 45KDa, which is consistent with the band about 45KDa in the protein glue<br>
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It can be seen that the purified CL7-sfGFP protein has high concentration and purity.We were then able to bind it to the InaK-Im7 cell membrane anchor protein and perform fluorescence confocal experiments to verify the construction of the membrane surface display system (for details, refer to the engineering part of our wiki).
 
===Source===
 
===Source===
  
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Derived from wild-type GFP in the jellyfish Aequorea victoria, sfGFP has improved its folding properties through a series of mutations, allowing it to fold quickly and correctly and fluorescein brightly under a variety of environmental conditions.
 
Derived from wild-type GFP in the jellyfish Aequorea victoria, sfGFP has improved its folding properties through a series of mutations, allowing it to fold quickly and correctly and fluorescein brightly under a variety of environmental conditions.
 
The source of composite parts is artificially constructed plasmid containing CL7 and sfGFP genes (PET23a-CL7-sfGFP).
 
The source of composite parts is artificially constructed plasmid containing CL7 and sfGFP genes (PET23a-CL7-sfGFP).
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===Experimental results===
 
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We transferred pET23a-CL7-sfGFP into <i>Escherichia coli</i> BL21 and purified it to meet the use requirements. The results were as follows:<br>
 
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https://static.igem.wiki/teams/5382/part-pictures/sfgfp.jpg<br>'''Figure 1.'''  CL7-sfGFP protein debacteria-breaking supernatant was incubated with nickel column, imidazole eluent with gradient concentration above 80mM, and purified protein solution was concentrated and purified by ultrafiltration SDS-PAGE electrophoresis<br>
 
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Loading sequence: supernatant, precipitation, flow through, 20mM, 50mM, 80mM, 100mM, 150mM, 200mM, Marker, 300mM, 500mM<br><br>
 
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The protein size of CL7-sfGFP is 45KDa, which is consistent with the band about 45KDa in the protein glue<br>
 
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It can be seen that the purified protein concentration and purity are both high.
 

Revision as of 13:10, 1 October 2024


CL7-linker-sfGFP_Green fluorescent protein and CL7 complex


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 158
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 158
    Illegal NheI site found at 1247
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 158
    Illegal BglII site found at 106
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 158
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 158
    Illegal AgeI site found at 56
    Illegal AgeI site found at 70
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Considerations

Our design aims to link sfGFP in series with CL7 so that sfGFP can be anchored to the surface of the cell membrane through the strong biological orthogonal interaction of Im7 and CL7.
Therefore, we constructed a fusion protein expression plasmid of green fluorescent protein and CL7 (pET23a-CL7-sfGFP), transferred it into Escherichia coli BL21, added IPTG to induce expression, broke up the bacteria to get the protein and finally purified it by nickel column eluting. The SDS-PAGE electrophoretic result of the eluent is as follows (Figure. 1).


sfgfp.jpg
Figure 1. SDS-PAGE electrophoretic result of CL7-sfGFP obtained by nickel column elution
Loading sequence: supernatant, precipitation, flow through, 20mM, 50mM, 80mM, 100mM, 150mM, 200mM, Marker, 300mM, 500mM

The protein size of CL7-sfGFP is 45KDa, which is consistent with the band about 45KDa in the protein glue
It can be seen that the purified CL7-sfGFP protein has high concentration and purity.We were then able to bind it to the InaK-Im7 cell membrane anchor protein and perform fluorescence confocal experiments to verify the construction of the membrane surface display system (for details, refer to the engineering part of our wiki).

Source

The CL7 is a protein tag obtained by engineering CE7 to remove its DNA-binding and catalytic activity, preserving its high affinity binding ability to the corresponding inhibitory protein Im7. The linker is composed of Gly and Ser. Derived from wild-type GFP in the jellyfish Aequorea victoria, sfGFP has improved its folding properties through a series of mutations, allowing it to fold quickly and correctly and fluorescein brightly under a variety of environmental conditions. The source of composite parts is artificially constructed plasmid containing CL7 and sfGFP genes (PET23a-CL7-sfGFP).