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	<h1>4. Results</h1>		<h1>4. Results</h1>
		+	<p>
		+	For our project, this binding casette was part of a folding plasmid. This was used to establish the FRET assay with the
		+	TetR-Oct1 simple staple (<a href=https://parts.igem.org/Part:BBa_K5237006>BBa_K5237006</a>) and simulated enhancer
		+	hijacking with the fgRNA and fusion dMbCas12a-dSpCas9 (<a
		+	href=https://parts.igem.org/Part:BBa_K5237000>BBa_K5237003</a>).
		+	</p>
	<p>Cloning success can be verified by sanger sequencing or nanopore sequencing.</p>		<p>Cloning success can be verified by sanger sequencing or nanopore sequencing.</p>
	<div class="thumb"></div>		<div class="thumb"></div>
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	<p>		<p>
−	For our project, this binding casette was part of a folding plasmid. This was used to establish the FRET assay with the	+
−	TetR-Oct1 simple staple (<a href=https://parts.igem.org/Part:BBa_K5237006>BBa_K5237006</a>) and simulated enhancer	+
−	hijacking with the fgRNA and fusion dMbCas12a-dSpCas9 (<a	+
−	href=https://parts.igem.org/Part:BBa_K5237000>BBa_K5237003</a>).	+
	</p>		</p>
	</section>		</section>

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Revision as of 08:35, 30 September 2024

BBa_K5237018

Oct1 Binding Casette

Binding casette containing 3x Oct1 recognition sites with Cas12a PAM sequences. Allows for Oct1 and Cas12a binding. The casette can be expanded through digestion and ligation. It was used to establish the FRET assay with tetR-Oct1 Simple staple, and simulated enhancer hijacking with fgRNA and fusion dMbCas12a-dSpCas9.

 

Contents

• 1 Sequence overview
• 2 Usage and Biology
• 3 Assembly and part evolution
• 4 Results
• 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 constructs 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

3. Assembly and part evolution

The designed cloning strategies allows for the easy assembly of repetetive repeats. It follows the procedure outlined by Sladitschek and Neveu (2015). Briefly, the oligos can be inserted into a vector digested with SalI and XhoI, yielding a vector with three binding repeats flanked by these restriction sites. The vector can be linearized with either SalI or XhoI, as both enzymes create compatible overhangs. The annealed oligos can then be ligated into the vector, resulting in six binding repeats, with the middle sequence losing its cleavage site compatibility. This process can be repeated to achieve the desired number of repeats by digesting the vector and re-ligating the oligos. For the experiments conducted, a folding plasmid with 12 repeats was created. Since the registry has some limitations regarding sequence depository, the binding casette is flanked by SalI and XhoI, and the top and bot oligos with the fitting overhangs annotated.

4. Results

For our project, this binding casette was part of a folding plasmid. This was used to establish the FRET assay with the TetR-Oct1 simple staple (BBa_K5237006) and simulated enhancer hijacking with the fgRNA and fusion dMbCas12a-dSpCas9 (BBa_K5237003).

Cloning success can be verified by sanger sequencing or nanopore sequencing.

Figure 2: Part of Sanger sequencing results of succesfull plasmid assembly with 12 Oct-1 binding sites.