Difference between revisions of "Part:BBa K5382130:Design"
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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> | 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> | ||
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). | 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/ | + | https://static.igem.wiki/teams/5382/part-pictures/20241001211624.jpg<br>'''Figure 1.''' SDS-PAGE electrophoretic result of CL7-sfGFP obtained by nickel column elution<br> |
Loading sequence: supernatant, precipitation, flow through, 20mM, 50mM, 80mM, 100mM, 150mM, 200mM, Marker, 300mM, 500mM<br><br> | Loading sequence: supernatant, precipitation, flow through, 20mM, 50mM, 80mM, 100mM, 150mM, 200mM, Marker, 300mM, 500mM<br><br> | ||
The protein size of CL7-sfGFP is 45KDa, which is consistent with the band about 45KDa in the protein glue<br> | The protein size of CL7-sfGFP is 45KDa, which is consistent with the band about 45KDa in the protein glue<br> | ||
Revision as of 13:18, 1 October 2024
CL7-linker-sfGFP_Green fluorescent protein and CL7 complex
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 158
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 158
Illegal NheI site found at 1247 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 158
Illegal BglII site found at 106 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 158
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 158
Illegal AgeI site found at 56
Illegal AgeI site found at 70 - 1000COMPATIBLE 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).
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).