Enhancer/Promoter Library Construction

Technical Information

Enhancer/promoter library construction strategies

Chip-based oligo synthesis

Chip-based oligo synthesis is ideal for generating pre-designed enhancer/promoter variants with various sizes and sequences. Our chip-based DNA synthesis can usually go up to 300 nt with a low error rate.

Obtained from genomic DNA

Variable fragments of an enhancer/mutation library can be obtained from genomic DNA or bacterial artificial chromosome (BAC) through probe hybridization. Pre-designed probes are employed to capture and enrich putative regulatory elements from the genomic sequence or BAC. Additionally, an unbiased shotgun-type approach can be utilized to obtain potential regulatory elements from the genomic DNA. Subsequently, these fragments will be cloned into vector backbones and subjected to screening for further characterization.

Directed evolution from existing regulatory elements

The construction of enhancer/promoter libraries can be based on existing sequences of these regulatory elements. By employing various mutagenesis strategies, such as error-prone PCR, utilization of degenerate codons, or DNA shuffling, novel variants are generated. These variants can subsequently undergo screening processes to identify those with desired traits, facilitating the targeted evolution of regulatory elements.

Key components

Vector design for enhancer library

Figure 1. Key vector components for an enhancer library.

Enhancer: The enhancer sequence to be screened can be inserted either upstream or downstream of the reporter gene. When the enhancer is positioned downstream (A) of the reporter gene and before the poly(A) signal, it is transcribed alongside the reporter gene and, therefore, can be measured directly by mRNA-seq. When positioned upstream (B) of the reporter gene, a barcode sequence is commonly incorporated in the 3’ UTR region of the reporter gene and can be transcribed with the reporter gene. The barcode serves as the proxy of the enhancer and can be sequenced by mRNA-seq.

Minimal promoter: A user-selected minimal promoter sequence is placed here. This will drive transcription of the downstream reporter if an enhancer element is present to activate the transcription. In the absence of such enhancer, the minimal promoter will be almost completely inactive.

Click to view more information about commonly used minimal promoters  

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Reporter: A visually detectable bright fluorescent protein gene (such as GFP) or a chemiluminescent protein gene (such as luciferase). This allows highly sensitive detection of enhancer activity.

Barcode: Enhancer library barcodes are unique, short sequences within individual plasmids. After screening, the read count of each barcode is determined through NGS analysis. By correlating the unique barcode sequences with the specific enhancer variants, the transcriptional activity of particular enhancers can be identified.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Vector design for promoter library

Figure 2. Key vector components for a promoter library.

Promoter: The promoter sequence to be screened can be inserted here to drive the transcription of the reporter.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Reporter: A visually detectable bright fluorescent protein gene (such as GFP) or a chemiluminescent protein gene (such as luciferase). This allows highly sensitive detection of promoter activity.

Barcode: Promoter library barcodes are unique, short sequences within individual plasmids. After screening, the read count of each barcode is determined through NGS analysis. By correlating the unique barcode sequences with the specific promoter variants, the transcriptional activity of particular promoters can be identified.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Experimental data

Figure 3. A cone-specific promoter library construction and screening. (A) Experimental workflow. Novel promoter variants were generated and cloned into the AAV backbone, driving the expression of a GFP reporter. The AAV promoter library was injected into the subretinal region of mice and subsequent screening and analysis were performed. (B) Subretinal expression pattern of the library. The AAV promoter library driving GFP was co-injected with another AAV carrying ProA1 promoter driving Td Tomato. ProA1 is a known cone-cell-specific promoter. Co-localization of the red and green fluorescence indicated the presence of cone-specific promoters in the promoter library.