Difference between revisions of "Part:BBa K5322031"

 
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===Usage and Biology===
 
  
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==Usage and Biology==
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5322031 SequenceAndFeatures</partinfo>
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The complete mouse PD-L1 protein has a large molecular weight and complex structure, making it challenging to express in the prokaryotic organism <i>Escherichia coli</i>. Therefore, we opted for a truncated version of the PD-L1 functional domain(<a href="https://parts.igem.org/Part:BBa_K5322034">BBa_K5322034</a>), designing an expression system for the PD-L1 functional domain: pET29a-J23119-RBS-PD-L1 (functional domain)-T7. The plasmid diagram is shown below.
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<img src="https://static.igem.wiki/teams/5322/wet-lab/pet29a-j23119-rbs-pd-l1-functional-domain-t7-map.png" alt="pET29a-J23119-RBS-PD-L1 Functional domain -T7" width="300">
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<p align="center"><b>Figure 1-1</b>  Plasmid pET29a-J23119-RBS-PD-L1 Functional domain -T7</p>
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==Construction of the plasmid==
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We ordered and synthesized the gene fragment J23119-RBS-PD-L1 (functional domain)-T7 from the company. After the gene arrived, we performed PCR to amplify the target fragment and the pET29a vector. The PCR products were analyzed by gel electrophoresis (30 min, 120 V), as shown in the figure. After purifying the products, we conducted homologous recombination and transformed them into <i>E. coli</i> DH5α, followed by incubating the plates at 37°C for 16 hours. Subsequently, we performed colony PCR on single colonies, and the results are shown in the figure.
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===Functional Parameters===
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<img src="https://static.igem.wiki/teams/5322/wet-lab/pcr-1.png" alt="PCR" width="300">
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<p align="center"><b>Figure 2-1</b>  PCR</p>
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<img src="https://static.igem.wiki/teams/5322/wet-lab/pcr-2.png" alt="Colony PCR" width="300">
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<p align="center"><b>Figure 2-2</b> Colony PCR </p>
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For the successful colony PCR, we inoculated the cultures at 37°C, 220 rpm, for 12-16 hours. After incubation, we extracted the plasmids and sent them for sequencing. The sequencing results are shown in the figure.
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<img src="https://static.igem.wiki/teams/5322/wet-lab/cexu-pet29a-j23119-rbs-pd-l1-functional-domain-t7-map.png" alt="Sequencing results" width="300">
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<p align="center"><b>Figure 2-3</b>Sequencing results</p>
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<p>The sequencing results confirmed the successful construction of the plasmid.</P>
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==Sequence and Features==
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<partinfo>BBa_K5322031 SequenceAndFeatures</partinfo>

Latest revision as of 13:31, 1 October 2024


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


Usage and Biology

The complete mouse PD-L1 protein has a large molecular weight and complex structure, making it challenging to express in the prokaryotic organism Escherichia coli. Therefore, we opted for a truncated version of the PD-L1 functional domain(BBa_K5322034), designing an expression system for the PD-L1 functional domain: pET29a-J23119-RBS-PD-L1 (functional domain)-T7. The plasmid diagram is shown below.

pET29a-J23119-RBS-PD-L1 Functional domain -T7

Figure 1-1 Plasmid pET29a-J23119-RBS-PD-L1 Functional domain -T7

Construction of the plasmid

We ordered and synthesized the gene fragment J23119-RBS-PD-L1 (functional domain)-T7 from the company. After the gene arrived, we performed PCR to amplify the target fragment and the pET29a vector. The PCR products were analyzed by gel electrophoresis (30 min, 120 V), as shown in the figure. After purifying the products, we conducted homologous recombination and transformed them into E. coli DH5α, followed by incubating the plates at 37°C for 16 hours. Subsequently, we performed colony PCR on single colonies, and the results are shown in the figure.

PCR

Figure 2-1 PCR

Colony PCR

Figure 2-2 Colony PCR

For the successful colony PCR, we inoculated the cultures at 37°C, 220 rpm, for 12-16 hours. After incubation, we extracted the plasmids and sent them for sequencing. The sequencing results are shown in the figure.

Sequencing results

Figure 2-3Sequencing results

The sequencing results confirmed the successful construction of the plasmid.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 377
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 519
    Illegal PstI site found at 377
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 277
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 377
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 377
  • 1000
    COMPATIBLE WITH RFC[1000]