Part:BBa_K5198009

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.

Figure 1. Gel electrophoresis of candidate primer pairs for qPCR analysis of CD19ecto. Two primer pairs (red boxes) were selected for further optimization. The first lane was loaded with a 1kb Hyperladder.

The amplified sequences were purified according to the protocol described on our Experiments page. A standard curve was created by serially diluting the purified product (10¹⁰ - 10⁵ DNA molecules) and the dilutions were run in a SYBR-Green qPCR reaction. For primer pair 31 (sequence reported above), the resulting fluorescence plot (Figure 2) shows reliable amplification across a range of concentrations.

Figure 2. Amplification plot of CD19ecto standard curve with the reported qPCR primers. The assay shows robust amplification across a range of different DNA concentrations.

The Ct values obtained from the standard curve amplification were analyzed using slope analysis and fitted to a linear regression model. The resulting slope ranged from -3.1 to -3.6 (Figure 3), which suggests efficient amplification of the target sequence.

Figure 3. Slope analysis of qPCR primer efficiency. Ct values from the standard curve amplification were plotted against the number of template copies and fitted to a linear regression model. The resulting slope indicates efficient and accurate amplification of the target sequence. The error bars indicate standard deviation of technical duplicates.

The reported qPCR primers demonstrated a unique melting temperature with no signs of unspecific binding (Figure 4). This optimization enabled our team to have confidence that the selected set of qPCR primers can be used across different biological samples to determine accurate levels of transcripts produced by the CD19ecto construct.

Figure 4. Melt curve plot for the reported set of primers. The primers anneal at a unique temperature with very little of nonspecific product at lower temperatures, which suggests target-specific binding.

The optimized set of primers was used to measure abundance of CD19ecto mRNA in two cases:

1. Baseline expression in the absence of Gal4-VP64 activator - transfection with BBa_K5198009 only
2. Maximum expression when Gal4-VP64 is in excess - co-transfection with BBa_K5198009 and constitutively active Gal4-VP64 activator (pcDNA5-Gal4-VP64)

The constructs were transfected into the HEK293T cell line and incubated for 48 hours. Following incubation, RNA was extracted, purified, and reverse-transcribed using the Zymo Quick-RNA Microprep Kit. The condition with the constitutively-active Gal4-VP64 activator exhibited a significant increase in expression (6.88 ± 1.36 SD mean fold change) compared to the baseline condition (Figure 5). These values were determined using the ΔΔ-Ct method [3]. These results demonstrate the expression limits of the secretion cassette and its responsiveness to the Gal4 transcriptional activator.

Figure 5. Relative fold difference in CD19ecto mRNA expression in HEK293T cells transfected with the secretion cassette and constitutively active Gal4-VP64. The control condition is a transfection with the secretion cassette only. The Gal4-VP64 condition shows a significant increase in CD19ecto expression (6.88 ± 1.36 fold) compared to the control (p < 0.05). The measurements were normalized to GAPDH gene expression. Error bars represent the standard deviation (SD) of duplicate biological samples.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 469
  Illegal SapI.rc site found at 305
  

References

[1] L. Morsut et al., “Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors,” Cell, vol. 164, no. 4, pp. 780–791, Feb. 2016, doi: 10.1016/j.cell.2016.01.012.

[2] Z. L. Chang, M. H. Lorenzini, X. Chen, U. Tran, N. J. Bangayan, and Y. Y. Chen, “Rewiring T-cell responses to soluble factors with chimeric antigen receptors,” Nat Chem Biol, vol. 14, no. 3, pp. 317–324, Mar. 2018, doi: 10.1038/nchembio.2565.

[3] K. J. Livak and T. D. Schmittgen, “Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method,” Methods, vol. 25, no. 4, pp. 402–408, Dec. 2001, doi: 10.1006/meth.2001.1262.

[4] J. Huggett and S. A. Bustin, “Standardisation and reporting for nucleic acid quantification,” Accred Qual Assur, vol. 16, no. 8, pp. 399–405, Aug. 2011, doi: 10.1007/s00769-011-0769-y.