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From 2014.igem.org

Contents 1 Introduction 1.1 Controlling Inteins 1.2 The As LOV2 domain 1.3 Photocaging 1.4 S11 split inteins 2 Cloning and Methods 3 Results 3.1 Screening 3.2 Fucking Data … 4 Discussion 5 Outlook 5.1 References

Introduction

Protein splicing allows for the introduction of major changes to a proteins sequence and structure even after translation. Although this mechanism alone offers a huge amount of possible applications highlighted in our toolbox, it would be advantageous to have control over the time and location of the splicing reaction especially for in vivo applications. However, the fact that inteins are attached to the protein they splice off from makes induction in whatever way rather difficult.

Controlling Inteins

Different ways exist for controlling the intein splicing reaction. In the literature this is mostly referred to as conditional trans splicing (CTS) or conditional protein splicing (CPS). Probably the longest known method of inducing protein splicing is to chemically control the activity of inteins by changing the pH or redox state of their environment. This has been achieved for a multitude of inteins such as Ssp DnaB [#References|[5]] or . Yet the major drawback is obvious:These methods are only effectively applicable in vitro and usually depend on complexly purified reagents.

Within the past decades, more advanced systems were discovered and designed involving the binding of different ligands or the fusion of inteins to various protein domains Ways of controlling inteins (Conditional Trans-Splicing!) -> splicing reaction, chemically: pH, redox-state -> ligand or light dependant (PhyB/PIF3, Calmodulin?) protein domain “assembling”/complementation? -> This only works for less active inteins, because otherwise they are more likely to attach than the -> new way needed

We therefore came up with a new method to inhibit an inteins function light-dependently: LOV2 domain photocaging.

The As LOV2 domain

As LOV2 comes is the second Light-Oxygen-Voltage domain from the Avena sativa phototropin 1. In its host organism, the common oat, phototropin 1 is a blue-light receptor involved in the response of growth to environmental light conditions and may be responsible for the opening of stomata and the movement of chloroplasts [4]. LOV domains absorb light through a flavin mononucleotide cofactor inside the proteins chromophor absorb light through a flavin mononucleotide (FMN) cofactor, which leads to the formation of a covalent bond between the chromophore and a reactive cysteine residue of the apo protein. This triggers a signal in the attached serine/threonine kinase, leading to protein autophosphorylation4

In the context of cell biology and engineering signalling pathways

Normal use -> allosteric? effect/spatially blocking active centre (would be second way of controlling as seen above…) -> but …

Photocaging

photocaging offers better way Definition -> small peptide in Jalpha Helix

Problem: small Inteins needed

S11 split inteins

WHY the hell? Where from, where split, name reference!

Cloning and Methods

Results

During the planning phase two preparatory experiments were run to test the conditions for light induction: Please visit conditions testing to read more about the procedures and results.

The cloning was performed on the basis of our two-gate assembly part. However, due to the need for fast cloning, all constructs were made CPEC. For each split fluorescent protein, the constructs were assembled directly into pSBX1K3:

• split N-protein and N-intein
• split C-intein and C-protein
• split non-splicing N-protein and N-intein
• split non-splicing C-intein and C-protein
• LOV domain with caged C-Intein position 6
• LOV domain with caged C-intein position 12
• (only for mRFP: LOV domain with BsaI cloning site for further customized caging positions)
• mutated positive controls

For split sfGFP all N splicing-partner and control constructs were taken from the split fluorescent protein assay.

Cloning scheme:

plasmids --- PCR ---> PCR products --- CPEC ---> constructs

Screening

Fucking Data …

Discussion

Outlook

reference to Cas9 + possible applications (Philipps idea)

References

references, especially thesis? -> search, ask Barbara!

[1] Aranko, A. S., Oeemig, J. S., Kajander, T. & Iwaï, H. Intermolecular domain swapping induces intein-mediated protein alternative splicing. Nat. Chem. Biol. 9, 616–22 (2013).

[2] Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–84 (2012).

[3] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). [4] Deblasio, S. L., Luesse, D. L. & Hangarter, R. P. A Plant-Specific Protein Essential for Blue-Light-Induced Chloroplast Movements 1. 139, 101–114 (2005).�