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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===Experimental result===
 
===Experimental result===
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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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We transferred pET23a-linker-Im7 into <i>Escherichia coli</i> BL21 and purified it to meet the use requirements. The results were as follows:
 
https://static.igem.wiki/teams/5382/part-pictures/im7.png<br>'''Figure 1.''' EcN engineered bacteria induced expression of InaK-Im7 fusion protein. 1, 3, 5 are before induction, 2, 4, 6 are after induction with IPTG.<br>
 
https://static.igem.wiki/teams/5382/part-pictures/im7.png<br>'''Figure 1.''' EcN engineered bacteria induced expression of InaK-Im7 fusion protein. 1, 3, 5 are before induction, 2, 4, 6 are after induction with IPTG.<br>
 
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).
 
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(pCold-Inak-Im7).
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===References===
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Revision as of 11:34, 1 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

Experimental result

We transferred pET23a-linker-Im7 into Escherichia coli BL21 and purified it to meet the use requirements. The results were as follows: im7.png
Figure 1. EcN engineered bacteria induced expression of InaK-Im7 fusion protein. 1, 3, 5 are before induction, 2, 4, 6 are after induction with IPTG.
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).

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(pCold-Inak-Im7).