Difference between revisions of "Part:BBa K5237012"

Revision as of 08:29, 29 September 2024

Caged NpuN Intein

The Caged NpuN Intein is derived from the naturally split intein DnaE of the cyanobacterium Nostoc punctiforme, designed to facilitate controlled protein trans-splicing. By caging the N- and C-terminal intein fragments (NpuN and NpuC), splicing is inhibited until removal of the cages, allowing precise regulation of protein linkage. The caged NpuN intein fragment was codon optimized for expression in human cells, with modifications to the NpuC sequence for enhanced functionality. The system enables the conditional assembly of proteins, such as the oligomerization of dead Cas9, via cathepsin B-mediated cleavage, providing a versatile tool for synthetic biology applications.

1 Sequence Overview
2 Usage and Biology
3 Assembly and Part Evolution
4 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 parts 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:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000	fgRNA Entryvector MbCas12a-SpCas9	Entryvector for simple fgRNA cloning via SapI
BBa_K5237001	Half-Staple: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9
BBa_K5237002	Half-Staple: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
BBa_K5237003	Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS	Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
BBa_K5237004	Half-Staple: Oct1-DBD	Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005	Half-Staple: TetR	Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006	Simple-Staple: TetR-Oct1	Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007	Half-Staple: GCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008	Half-Staple: rGCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009	Mini-Staple: bGCN4	Assembled staple with minimal size that can be further engineered
Functional elements: Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications.
BBa_K5237010	Cathepsin B-Cleavable Linker (GFLG)	Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits, to make responsive staples
BBa_K5237011	Cathepsin B Expression Cassette	Cathepsin B which can be selectively express to cut the cleavable linker
BBa_K5237012	Caged NpuN Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237013	Caged NpuC Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237014	fgRNA processing casette	Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems.
BBa_K5237016	FRET-Donor: mNeonGreen-Oct1	Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237017	FRET-Acceptor: TetR-mScarlet-I	Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237018	Oct1 Binding Casette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237019	TetR Binding Cassette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020	Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64	Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker.
BBa_K5237021	NLS-Gal4-VP64	Trans-activating enhancer, that can be used to simulate enhancer hijacking.
BBa_K5237022	mCherry Expression Cassette: UAS, minimal Promotor, mCherry	Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.
BBa_K5237023	Oct1 - UAS binding casette	Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.
BBa_K5237024	Minimal promoter Firefly luciferase	Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking.

1. Sequence Overview

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Inteins are protein sequences that splice themselves out of a polypeptide chain through an autocatalytic cleavage reaction. This process ligates the flanking polypeptides, termed exteins (Mills, Johnson & Perler, 2014; Wang et al., 2022). Some inteins are naturally split in two parts – termed N- and C-terminal intein fragments. Trans-splicing of two split intein fragments can covalently link two different proteins (Ventura & Mootz, 2019).
The naturally split intein DnaE from the cyanobacterium Nostoc punctiforme (Npu) was previously utilized to link different protein fragments in prokaryotic and eukaryotic systems (Gramespacher et al., 2017). DnaE consists of the NpuN and NpuC intein fragments. Caging of NpuN and NpuC with truncated fragments of the opposite intein fragment inhibits protein trans-splicing. This allows for the controlled induction of protein trans-splicing upon removal of the intein cages (Gramespacher et al., 2017). Here, we utilized NpuN and NpuC to induce linkage of dead Cas9 (dCas9) proteins upon removal of intein cages by cathepsin B cleavage.

3. Assembly and Part Evolution

The sequence for NpuN was taken from Gramespacher et al. (2017) and optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.).
The protein sequence of NpuC_1-30 was taken from Gramespacher et al. (2017). Two modifications were made to the sequence of NpuC_1-30 by Gramespacher et al. (2017): a repeat of NpuC_1-30 was used and three point mutations (K2E, R6E, K11E) were introduced in each repeat. The nucleotide sequence was codon optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.).

4. References

Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. https://doi.org/10.1021/jacs.7b02618

Mills, K. V., Johnson, M. A., & Perler, F. B. (2014). Protein Splicing: How Inteins Escape from Precursor Proteins. Journal of Biological Chemistry, 289(21), 14498-14505. https://doi.org/10.1074/jbc.R113.540310

Ventura, B. D., & Mootz, H. D. (2019). Switchable inteins for conditional protein splicing. Biological Chemistry, 400(4), 467-475. https://doi.org/doi:10.1515/hsz-2018-0309

Wang, H., Wang, L., Zhong, B., & Dai, Z. (2022). Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology [Mini Review]. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.810180

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      <!-- Part summary -->
-     <section id="1">
+     <section>
        <h1>Caged NpuN Intein</h1>
        <p>The Caged NpuN Intein is derived from the naturally split intein DnaE of the cyanobacterium <i>Nostoc punctiforme</i>, designed to facilitate controlled protein <i>trans</i>-splicing. By caging the N- and C-terminal intein fragments (NpuN and NpuC), splicing is inhibited until removal of the cages, allowing precise regulation of protein linkage. The caged NpuN intein fragment was codon optimized for expression in human cells, with modifications to the NpuC sequence for enhanced functionality. The system enables the conditional assembly of proteins, such as the oligomerization of dead Cas9, via cathepsin B-mediated cleavage, providing a versatile tool for synthetic biology applications.</p>
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      <ul>
        <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
-             overview</span></a>
+             Overview</span></a>
        </li>
        <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
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        </li>
        <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
-             and part evolution</span></a>
+             and Part Evolution</span></a>
        </li>
-       <li class="toclevel-1 tocsection-8"><a href="#4"><span class="tocnumber">4</span> <span
+       <li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span
              class="toctext">References</span></a>
        </li>
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    <section id="1">
-     <h1>1. Sequence overview</h1>
+     <h1>1. Sequence Overview</h1>
    </section>
 </body>
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    </section>
    <section id="3">
-     <h1>3. Assembly and part evolution</h1>
+     <h1>3. Assembly and Part Evolution</h1>
      <p>The sequence for NpuN was taken from Gramespacher <i>et al.</i> (2017) and optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.).<br>
 The protein sequence of NpuC<sub>1-30</sub> was taken from Gramespacher <i>et al.</i> (2017). Two modifications were made to the sequence of NpuC<sub>1-30</sub> by Gramespacher <i>et al.</i> (2017): a repeat of NpuC<sub>1-30</sub> was used and three point mutations (K2E, R6E, K11E) were introduced in each repeat. The nucleotide sequence was codon optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.).</p>