Difference between revisions of "Part:BBa K5382120:Design"
 
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===Design Notes===
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===Design considerations===
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After constructing the plasmid, we transferred it into EcN for expression for subsequent operations, so that it can have a better expression level under the premise of ensuring the operation standard, so as to improve the binding rate and stability of the above membrane surface display system.
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Our design aims to utilize the ice nucleation protein InaK to display the Im7 protein on the surface of <i>Escherichia coli</i> Nissle1917(EcN) cells, that is, to express the InaK-linker-Im7 system on the cell membrane surface. Initially, we employed the <i>Escherichia coli</i> gene editing plasmid system pKD46 to integrate the T7 RNA polymerase gene, which is controlled by the lac-uv5 promoter, into the attB site of the EcN genome. Consequently, we successfully constructed an EcN strain that is compatible with the pET expression system. We then constructed a fusion protein expression plasmid for the ice nucleation protein InaK with Im7 (pET23a-InaK-Im7),introduced it into the engineered EcN strain, and induced protein expression with IPTG. Ultimately, the successful expression of the InaK-Im7 fusion protein was confirmed by SDS-PAGE electrophoresis (Figure 1).
  
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https://static.igem.wiki/teams/5382/part-pictures/im7.png<br>'''Figure 1.''' Induction of InaK-IM7 Fusion Protein Expression in EcN-Engineered Bacteri.<br>
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===Experimental result===
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Lanes 1, 3, 5: Pre-induction; Lanes 2, 4, 6: Post-induction with IPTG.
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We transferred pET23a-CL7-sfGFP into Escherichia coli BL21 and purified it to meet the use requirements. The results were as follows:
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<br>
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It can be seen that the purified protein concentration and purity are high, and the obtained InaK-Im7 fusion protein can be used for subsequent binding with CL7-sfGFP fusion protein and fluorescence confocal experiments to verify the construction of the membrane surface display system(For details, refer to the engineering section of our wiki).
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It can be seen that the purified protein concentration and purity are high, and subsequent incubation binding and fluorescence confocal experiments can be conducted to verify the binding of Inak-Im7 and CL7-sfGFP (see the engineer and result section of wiki for the results).
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===Source===
 
===Source===
  
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Inak is an ice nucleated protein from Pseudomonas syringae KCTC1832.
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The InaK is an ice nucleated protein from <i>Pseudomonas syringae</i> KCTC1832.<br>
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linker is composed of Gly and Ser.
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The linker is composed of Gly and Ser.<br>
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Im7 is an immune protein derived from E. coli.
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The Im7 is an immune protein derived from <i>E. coli</i>.<br>
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The source of the composite parts is artificially constructed plasmids containing Inak and Im7 genes (pCold-Inak-Im7).
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The source of the composite parts is artificially constructed plasmids(pET23a-InaK-Im7).
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===References===
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Latest revision as of 12:47, 2 October 2024


InaK-linker-Im7_Cell membrane anchoring protein and immune protein complex


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1800
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 722
    Illegal NgoMIV site found at 962
    Illegal NgoMIV site found at 1130
    Illegal AgeI site found at 517
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 692
    Illegal BsaI.rc site found at 1182
    Illegal SapI.rc site found at 326


Design considerations

Our design aims to utilize the ice nucleation protein InaK to display the Im7 protein on the surface of Escherichia coli Nissle1917(EcN) cells, that is, to express the InaK-linker-Im7 system on the cell membrane surface. Initially, we employed the Escherichia coli gene editing plasmid system pKD46 to integrate the T7 RNA polymerase gene, which is controlled by the lac-uv5 promoter, into the attB site of the EcN genome. Consequently, we successfully constructed an EcN strain that is compatible with the pET expression system. We then constructed a fusion protein expression plasmid for the ice nucleation protein InaK with Im7 (pET23a-InaK-Im7),introduced it into the engineered EcN strain, and induced protein expression with IPTG. Ultimately, the successful expression of the InaK-Im7 fusion protein was confirmed by SDS-PAGE electrophoresis (Figure 1).

im7.png
Figure 1. Induction of InaK-IM7 Fusion Protein Expression in EcN-Engineered Bacteri.
Lanes 1, 3, 5: Pre-induction; Lanes 2, 4, 6: Post-induction with IPTG.
It can be seen that the purified protein concentration and purity are high, and the obtained InaK-Im7 fusion protein can be used for subsequent binding with CL7-sfGFP fusion protein and fluorescence confocal experiments to verify the construction of the membrane surface display system(For details, refer to the engineering section of our wiki).

Source

The InaK is an ice nucleated protein from Pseudomonas syringae KCTC1832.
The linker is composed of Gly and Ser.
The Im7 is an immune protein derived from E. coli.
The source of the composite parts is artificially constructed plasmids(pET23a-InaK-Im7).