Difference between revisions of "Part:BBa K5322030"

 
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==Usage and Biology==
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In the context of intestinal inflammation, the presence of CD8<sup>+</sup> T cells often leads to an immune hyperreactivity. To downregulate the immune response at the site of inflammation, we plan to utilize the PD-1/PD-L1 immune checkpoint to inhibit the activity of CD8+ cytotoxic T cells. For the purpose of facilitating further experiments, we selected mouse PD-L1 (BBa_K5322033) and conducted codon optimization, designing the plasmid pET29a-J23119-RBS-PD-L1 (Mus)-T7, as illustrated in the figure below.
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<img src="https://static.igem.wiki/teams/5322/wet-lab/pet29a-j23119-rbs-pd-l1-mus-t7-map.png" alt="pet29a-j23119-rbs-pd-l1-mus-t7-map" width="300">
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<p align="center"><b>Figure 1-1</b>  Plasmid pET29a-J23119-RBS-[PD-L1(Mus)]-T7</p>
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==Construction of the plasmid==
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===Usage and Biology===
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The sequence of J23119-RBS-PD-L1 (Mus)-T7 was sent to a company for synthesis. After two weeks, the company returned results indicating that random mutations occurred during the process of recombining the fragment with the vector and introducing it into <i>Escherichia coli</i>. Given the uncertainties associated with these random mutations, we decided to abandon this approach.
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<span class='h3bb'>Sequence and Features</span>
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==Sequence and Features==
 
<partinfo>BBa_K5322030 SequenceAndFeatures</partinfo>
 
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===Functional Parameters===
 
===Functional Parameters===
 
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Latest revision as of 12:11, 2 October 2024


Programmed Cell Death 1 Ligand 1 [Mus musculus] Expression System

Usage and Biology

In the context of intestinal inflammation, the presence of CD8+ T cells often leads to an immune hyperreactivity. To downregulate the immune response at the site of inflammation, we plan to utilize the PD-1/PD-L1 immune checkpoint to inhibit the activity of CD8+ cytotoxic T cells. For the purpose of facilitating further experiments, we selected mouse PD-L1 (BBa_K5322033) and conducted codon optimization, designing the plasmid pET29a-J23119-RBS-PD-L1 (Mus)-T7, as illustrated in the figure below.

pet29a-j23119-rbs-pd-l1-mus-t7-map

Figure 1-1 Plasmid pET29a-J23119-RBS-[PD-L1(Mus)]-T7

Construction of the plasmid

The sequence of J23119-RBS-PD-L1 (Mus)-T7 was sent to a company for synthesis. After two weeks, the company returned results indicating that random mutations occurred during the process of recombining the fragment with the vector and introducing it into Escherichia coli. Given the uncertainties associated with these random mutations, we decided to abandon this approach.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 356
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 314
    Illegal NheI site found at 939
    Illegal PstI site found at 356
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 205
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 356
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 356
    Illegal NgoMIV site found at 745
    Illegal AgeI site found at 577
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters