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	In contrast to GCN4 that binds the CRE target sequence with the N-terminal region, rGCN4 was engineered as a reverse		In contrast to GCN4 that binds the CRE target sequence with the N-terminal region, rGCN4 was engineered as a reverse
	variant, binding a modified DNA target sequence with the C-terminal region. The described binding affinity of rGCN4		variant, binding a modified DNA target sequence with the C-terminal region. The described binding affinity of rGCN4
−	compares favorably to the wild-type GCN4 binding affinity to its native target sequence. In our project we first	+	to it's optimal target sequence INVii (5' GTCAtaTGAC 3', upper case letters indicate direct interaction between protein and DNA), compares favorably to the wild-type GCN4 binding affinity to its native target sequence.
		+	In our project we first
	wanted		wanted
	to analyze the DNA binding affinity of rGCN4 and then fuse it to GCN4 to create a		to analyze the DNA binding affinity of rGCN4 and then fuse it to GCN4 to create a

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Revision as of 01:13, 29 September 2024

BBa_K5237008

Staple subunit: rGCN4

rGCN4 is an engineered, reverse, variant of the yeast transcription factor GCN4, featuring a basic region and a leucine zipper dimerization domain. Unlike GCN4, rGCN4 binds its target DNA INVii (5' GTCAtaTGAC 3') at its C-terminus.

 

Contents

• 1 Sequence overview
• 2 Usage and Biology
• 3 Assembly and part evolution
• 4 Results
  • 4.1Protein expression and purification
  • 4.2Electrophoretic Mobility shift assay
• 5 References

The PICasSO Toolbox

Figure 1: Example how the part collection can be used to engineer new staples

The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox based on various DNA-binding proteins to address this issue.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts

At its heart, the PICasSO part collection consists of three categories. (i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling and can be further engineered to create alternative, simpler and more compact staples. (ii) As functional elements, we list additional parts that enhance the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's with our interkingdom conjugation system.

(iii) As the final component of our collection, we provide parts that support the use of our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.

The following table gives a complete overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation

Our part collection includes:

Sequence and Features

4. Results

4.1 Protein expression and purification

The FLAG-rGCN4 protein could be readily expressed in E. coli BL21 (DE3). The protein was purified using an anti-FLAG resin. Fractions taken during purification were analyzed by SDS-PAGE. Purified protein was quantified using a Lowry assay, 3.4 mg/mL were obtained, resulting in 422 µM of monomeric FLAG-GCN4.

Figure 2: SDS-PAGE analysis of FLAG-GCN4 purification Fractions analysed are the raw lysate, flow through and eluate. Depicted is GCN4 (this part), rGCN4 (BBa_K5237008), and bGCN4 (BBa_K5237009). Protein size is indicated next to construct name and purified band with protein of interest highlighted by a red box.

4.2 Electrophoretic Mobility shift assay

rGCN4 was tested for its DNA-binding capabilities using an electrophoretic mobility shift assay (EMSA). The protein was incubated with a DNA probe containing the rGCN4 binding site (INVii). The formation of a protein-DNA complex was analyzed by native PAGE. To further analyze DNA binding, quantitative shift assays were performed for GCN4 (BBa_K5237008) and rGCN4. 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:

Θ_app = Θ_min + (Θ_max - Θ_min) × (K_a² [L]_tot²) / (1 + K_a² [L]_tot²) Equation 1

Here [L]_tot describes the total protein monomer concentration, K_a corresponds to the apparent monomeric equilibration constant. The Θ_min/max values are the experimentally determined site saturation values (For this experiment 0 and 1 were chosen for min and max respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation constant K_k of (0.2930.033)×10^-6 M, which is almost identical to the rGCN4 binding affinity to INVii a _d of (0.2980.030)×10^-6 M.

Figure 3: Quantitative EMSAQuantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted for each data point. Values are presented as mean +/- SD.

The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10^-6 M) and rGCN4 ((0.2980.030) × 10^-6 M) are approximately a factor 10 higher then those described in literature ((96) × 10^-8 M for GCN4 and (2.90.8) × 10^-8 M for rGCN4) (Hollenbeck et al., 2001). The differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos. Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity) bands in the SDS-PAGE analysis, indicating the co-purification of small amounts of unspecific proteins.

The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed

5. References

Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis, 10(5–6), 366–376. https://doi.org/10.1002/elps.1150100515

Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nature Protocols, 2(8), 1849–1861. https://doi.org/10.1038/nprot.2007.249

Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 binds with high affinity to DNA sequences containing a single consensus half-site. Biochemistry, 39(21), 6380–6389. https://doi.org/10.1021/bi992705n

Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity. Biochemistry, 40(46), 13833–13839. https://doi.org/10.1021/bi0106916

McKnight, S. L., & Tjian, R. (1988). Analysis of transcriptional regulatory proteins of the human genome. Science, 241(4870), 1306–1313. https://doi.org/10.1126/science.2847199

Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes. Scientific Reports, 5, 10907. https://doi.org/10.1038/srep10907