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	<h1>4. Results</h1>		<h1>4. Results</h1>
−	<p>In spite of unsuccessful cloning of anti-EGFR nanobody into the CDS of intimin, protein expression was tested in <i>E.coli</i> 10-beta as they will used as the donor strain for our upcoming conjugation tests. The <i>E.coli</i> 10-beta cells were transformed with pNeae2, and protein expression was induced with 50 µMIPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin (~76 kDa) after induction (Figure 1). </p>	+	<p>Despite unsuccessful cloning of anti-EGFR nanobody into the CDS of intimin, protein expression was tested in <i>E.coli</i> 10-beta as they will used as the donor strain for our upcoming conjugation tests. The <i>E.coli</i> 10-beta cells were transformed with pNeae2, and protein expression was induced with 50 µMIPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin (~76 kDa) after induction (Figure 1). </p>
	<p>Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR <i>in vitro</i> and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells. </p>		<p>Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR <i>in vitro</i> and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells. </p>
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Revision as of 10:51, 30 September 2024

BBa_K5237015

Anti-EGFR adhesin

This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on E.coli. With this part, we sought to enhance bacterial binding to mammalian cells and potentially improve the chances of DNA delivery by the Type IV secretion system (T4SS).

 

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

Despite unsuccessful cloning of anti-EGFR nanobody into the CDS of intimin, protein expression was tested in E.coli 10-beta as they will used as the donor strain for our upcoming conjugation tests. The E.coli 10-beta cells were transformed with pNeae2, and protein expression was induced with 50 µMIPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin (~76 kDa) after induction (Figure 1).

Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR in vitro and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells.

Figure 1: Fluorescence western blot scan showing expression of myc-tagged intimin by E.coli 10-beta after IPTG induction. E.coli 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the E.coli lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced E.coli lysate.