Part Collection
Collection of BioBrick parts, genetic constructs, and biological components used in this project.
Overview
This collection includes the biological parts and constructs developed for our CRISPR-dCas9 methylation system, featuring guide RNAs, fusion proteins, and optimized promoters for targeted epigenetic regulation.
| Code | Composition | Type | Derived from | Short Description |
|---|---|---|---|---|
| BBa_25MB7XQ4 | dnaAP2-promoter-specific-gRNA-1 | gRNA_gene [SO:0001264] | Specific gRNAs designed to guide the dCas9-dam system to the upstream regions of the dnaAP2 promoter region. | |
| BBa_25J0YX0Y | dnaAP2-promoter-specific-gRNA-2 | gRNA_gene [SO:0001264] | Specific gRNAs designed to guide the dCas9-dam system to the upstream regions of the dnaAP2 promoter region. | |
| BBa_25L670YA | dcas9-dam | Coding | dCas9 (dead Cas9) + E. coli Dam methylase | Encodes an improved dCas9-Dam fusion protein for enhanced methylation efficiency, featuring an extended, flexible (GGSS) linker (BBa_K1974030). |
| BBa_25IZOQVV | dnaAP2-opt-Methylation-Responsive Promoter | Promoter | E. coli dnaA operon | The dnaAP2-opt-Methylation-Responsive promoter is a modified E. coli dnaA promoter with a deleted P1 region and mutated DnaA boxes, designed to reduce background expression and regulatory feedback. It is designed to study methylation-dependent gene regulation in prokaryotic systems. Upon Adenine Methylation, there is an increase in the activity of the promoter. |
Basic Parts
dCas9 - Dam
The dCas9-linker-Dam fusion protein does site-specific adenine methylation at user-defined loci using CRISPR–dCas9 targeting. In our project, it was used to study methylation effects on the dnaAP2 promoter (BBa_25IZOQVV) upstream of a GFP reporter. By cloning the gRNA with the fusion construct through Golden Gate assembly, methylation was directed precisely to selected GATC sites, and changes in GFP fluorescence served as a readout of promoter activity. The system thus enables programmable, locus-specific methylation-based regulation of synthetic promoters in E. coli, offering improved efficiency due to the flexible 18-amino acid Glycine-Serine (GGSS) linker that minimizes steric hindrance and enhances target accessibility.
dnaAP2
The dnaAP2 optimised promoter is a synthetic variant of the native E. coli dnaA operon promoter. The dnaA operon contains two overlapping promoters, P1 and P2, responsible for initiating dnaA expression. To minimize background expression and isolate methylation-dependent effects, the P1 promoter was deleted, and DnaA binding sites (DnaA boxes) were mutated to disrupt autoregulation. NGG motifs were also introduced to increase potential CRISPR guide RNA binding sites for future editing or methylation targeting studies. This modified dnaA-P2 promoter was synthesized based on the E. coli dnaA operon sequence kindly provided by Dr. Bianca Sclavi and synthesized by Twist Biosciences. The construct was cloned upstream of a GFP reporter gene for functional testing in E. coli strains. The modified promoter is hypothesized to upregulate downstream gene expression upon adenine methylation of the GATC sites.
Composite Parts
Tet-on dCas9 Dam plasmid (Theoretical)
The Tet-on dCas9-Dam plasmid is a theoretical construct designed to provide inducible and reversible control over targeted DNA methylation in E. coli. It combines the CRISPR–dCas9-Dam fusion system with a tetracycline-inducible promoter, allowing precise temporal regulation of adenine methylation at user-defined loci.
In this design, expression of the dCas9–Dam fusion is triggered only in the presence of an inducer such as anhydrotetracycline (aTc). This ensures that methylation occurs exclusively when desired, minimizing off-target or background methylation that can arise from constitutive expression.
Tet-on dCas9 DNMT3A (Theoretical)
The Tet-On-dcas9-DNMT3A plasmid is a theoretical construct designed to enable inducible, locus-specific DNA methylation in mammalian cells. The part is based on the pKDR Tet-On.1 backbone, which contains the constitutively expressed hPGK promoter–driven rtTA (reverse tetracycline-controlled transactivator) module. The rtTA protein activates transcription from the Tet-responsive element (TRE) only in the presence of doxycycline, providing tight and reversible control of downstream gene expression.
To create a programmable epigenetic effector, we added a dCas9–DNMT3A fusion gene under the control of the TRE promoter. Upon doxycycline induction, rtTA binds TRE and drives expression of dCas9–DNMT3A, which can be directed to specific genomic loci via a co-expressed sgRNA to deposit CpG methylation and repress gene expression.
