Revision as of 07:47, 30 September 2024 (view source) Marik (Talk | contribs) ← Older edit		Revision as of 08:37, 30 September 2024 (view source) Marik (Talk | contribs) Newer edit →
Line 207:		Line 207:
	</tr>		</tr>
	<tr>		<tr>
−	<td><a href="https://parts.igem.org/Part:BBa_K52370012" target="_blank">BBa_K5237012</a></td>	+	<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
	<td>Caged NpuN Intein</td>		<td>Caged NpuN Intein</td>
	<td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple		<td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
Line 213:		Line 213:
	</tr>		</tr>
	<tr>		<tr>
−	<td><a href="https://parts.igem.org/Part:BBa_K52370013" target="_blank">BBa_K5237013</a></td>	+	<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
	<td>Caged NpuC Intein</td>		<td>Caged NpuC Intein</td>
	<td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple		<td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
Line 219:		Line 219:
	</tr>		</tr>
	<tr>		<tr>
−	<td><a href="https://parts.igem.org/Part:BBa_K52370014" target="_blank">BBa_K5237014</a></td>	+	<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
	<td>fgRNA processing casette</td>		<td>fgRNA processing casette</td>
	<td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>		<td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
	</tr>		</tr>
	<tr>		<tr>
−	<td><a href="https://parts.igem.org/Part:BBa_K52370015" target="_blank">BBa_K5237015</a></td>	+	<td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
	<td>Intimin anti-EGFR Nanobody</td>		<td>Intimin anti-EGFR Nanobody</td>
	<td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large		<td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
Line 322:		Line 322:
	target="_blank">BBa_K5237016</a>).		target="_blank">BBa_K5237016</a>).
	</p>		</p>
−	<div class="thumb">
−	<div class="thumbinner" style="width:80%;">
−	<img alt="" src="https://static.igem.wiki/teams/5237/figures-corrected/basic-staple-fret.svg"
−	style="width:99%;" class="thumbimage">
−	<div class="thumbcaption">
−	<i>
−	<b>Figure 2: Overview of a Simple Staple use-case in FRET measurement</b>
−	</i>
−	</div>
−	</div>
−	</div>
−
−
	</section>		</section>
	<section id="3">		<section id="3">
Line 349:		Line 336:
	<p>Oct1 was N-terminally fused to the His6-mNeonGreen. The fusion protein was expressed from a T7 based expression		<p>Oct1 was N-terminally fused to the His6-mNeonGreen. The fusion protein was expressed from a T7 based expression
	plasmid and subsequently		plasmid and subsequently
−	purified using metal affinity chromatography with Ni-NTA beads.(Figure 3, left)	+	purified using metal affinity chromatography with Ni-NTA beads.(Figure 2, left)
	DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different		DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different
	buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, 1.8		buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, 1.8
Line 355:		Line 342:
	0.1 % (v/v) IGEPAL&#174; CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250:		0.1 % (v/v) IGEPAL&#174; CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250:
	Na<sub>2</sub>HPO<sub>4</sub>, 150 mM		Na<sub>2</sub>HPO<sub>4</sub>, 150 mM
−	NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Figure 3, right)	+	NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Figure 2, right)
	</p>		</p>
	<div class="thumb">		<div class="thumb">

---

Revision as of 08:37, 30 September 2024

BBa_K5237004

Half-Staple: Oct1-DBD

Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor, it can be readily fused with other DNA-bindig proteins to form a functional staple for DNA-DNA proximity. We used this part as a component for our Simple staple (BBa_K5237006) resulting in a bivalent DNA binding staple, and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).

 

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

4. Results

Oct1 was N-terminally fused to the His6-mNeonGreen. The fusion protein was expressed from a T7 based expression plasmid and subsequently purified using metal affinity chromatography with Ni-NTA beads.(Figure 2, left) DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂HPO₄, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: Na₂HPO₄, 150 mM NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Figure 2, right)

Figure 3: Expression and DNA binding analysis of His₆-mNeonGreen-Oct1-DBD.
Left image:Lane 1: raw lysate of E. coli expression culture after steril-filtration; Lane 2: Flow through of first wash (10 bed volumes of NaP10 (Na₂HPO₄, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na₂HPO₄, 150 mM NaCl, 20 mM Imidazol)); Lane 4: Elution of purified protein.
1 µL of each fraction was loaded after mixing and heating with 4x Laeemli buffer, on a 4-15% TGX-Gel. The expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.
Right image Purified mNeonGreen-Oct1 fusion-protein (1000 nM, 100 nM or 10 nM) were equilibrated with 0.5 µM DNA, containing three Oct1 binding sites, in different buffer compositions. (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: 50 mM NaH2PO4, 150 mM NaCl, 250 mM Imidazol) Bands were visualized with SYBR-Safe staining.