Difference between revisions of "Part:BBa K5198009"

Revision as of 20:47, 30 September 2024

Gal4-5xUAS-miniCMV-CD19ecto

The ATLAS expression cassette for the extracellular secretion of CD19ecto antigen. Gal4-VP64-inducible expression, either via the synNotch system or a positive control activator system. This part is composed of 5 repeats of Gal4 Upstream Activating Sequence (UAS) (BBa_K4839011), the minimal version of the CMV promoter (BBa_K5198006), and the CD19ecto extracellular domain (BBa_K5198000). The expression of the antigen (or any payload) can be induced by activated synNotch receptors fused to Gal4-VP64 [1]. The CD19ecto contains its native signal peptide, which allows for extracellular export of the protein.

This construct is a key component of Duke iGEM's antigen amplifier circuit. The system is activated when the antiCD19-synNotch receptor recognizes surface-bound CD19, triggering the release of the Gal4-VP64 transcription factor. Gal4-VP64 then translocates to the nucleus, binds to the Gal4 UAS sequence of this part, driving the increased expression of the CD19 antigen ectodomain. The secreted CD19ecto can dimerize and activate nearby CAR-T cells, amplifying the therapeutic response [2].

Cloning

This part was constructed using two-piece Gibson assembly from the following fragments:

• pHR-Gal4UAS-miniCMV (Addgene plasmid # 79130 ; http://n2t.net/addgene:79130 ; RRID:Addgene_79130)
• CD19ecto (Addgene plasmid # 127889 ; http://n2t.net/addgene:127889 ; RRID:Addgene_127889)

We used the following primers for Gibson assembly:

pHR-5xUAS_fwd: 5’-GATCCTTGACTTGCGGCC-3’

pHR-5xUAS_rev: 3’-GATCCAACGAATGTCGAGAG-5’

CD19ecto_fwd: 5’- gacaccgggaccgatccagcctctcgacattcgttggatcATGGAGGTGCGCCCAGAAG-3’

CD19ecto_rev: 3’-gcatgttgcaggtgggagttgcggccgcaagtcaaggatcTTCCAGCCTCCTGTTCTGAG-5’

Both insert and backbone were amplified in a 25 µL PCR reaction with 1 ng of template DNA added. The PCR product was verified using gel electrophoresis, incubated with DpnI overnight, and purified using DNA Clean & Concentrator-5 provided by Zymo Research. The fragments were then added to the Gibson reaction and incubated at 50°C for 15 min. The product of Gibson reaction was transformed into DH5α E. coli cells and colonies were selected for sequencing.

Characterization

In order to test the expression limits of this part, our team analyzed mRNA relative expression to using real-time quantitative PCR (RT-qPCR). We targeted the CD19ecto transcript with the following primers:

qPCR CD19ecto fwd: 5’-AAC TGT ATG TAT GGG CCA AGG-3’

qPCR CD19ecto rev: 3’-CTG AGC AAC CAA TGC CAA AG-5’

These primers were selected during the optimization process to ensure the accuracy and reliability of the qPCR results [3]. Initially, primer combinations were chosen based on PCR amplification of a plasmid containing the CD19ecto element (Figure 1). Candidate pairs that produced visible bands ranging from 70 to 300 bp were selected for downstream optimization steps.