Part:BBa_K5322031
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.
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.
Figure 2-1 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.
Figure 2-3Sequencing results
The sequencing results confirmed the successful construction of the plasmid.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 377
- 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 277
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 377
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 377
- 1000COMPATIBLE WITH RFC[1000]
| None |