Abstract
DESIGNIntroduction
Our optogenetic toolbox enhancements can be divided into three subgroups: controllable protein import via Pex5, controllable protein import via Pex7 and controllable gene expression. Each of these sub-projects are of different design which will be illustrated in the following.
Pex5 import with LOV2
LOV2 is an optogenetic protein derived from Avena Sativa’s Phototrophin 1. In its dark state the J$_{\alpha}$-helix located at the C-terminus is bound to the core of the protein. Upon irradiation with blue light (~460 nm), a covalent bond between a cysteine residue on the LOV2 protein and a flavin mononucleotide chromophore causes the J$_{\alpha}$-helix to unfold, which in turn exposes the C-terminus [1]. This property is very useful, as short amino acid sequences can be attached to this end of the LOV-protein, for example a peroxisomal targeting sequence! The idea for this project was to attach PTS1 to the C-terminus and the protein of interest to the N-terminus. Upon irradiation with blue light, the fusion protein would be imported into our compartment. We used a mutated version of LOV2 whose C-terminus has an increased dark-state binding affinity. This is caused by the substitutions and T406A and T407A. These mutations greatly reduce the possibility of the J$_{\alpha}$-helix being exposed in the dark state. Our PTS1 sequence consists of the amino acids LQSKL. As a proof of concept for this construct we fused sfGFP to its N-terminus:
This was done in order to visualize our experiment’s results during microscopy: upon successful import of the fusion protein, one would observe GFP fluorescence localized to our compartment. Otherwise, the whole cell would be illuminated.
Pex7 import
The idea behind this project is to initially block the protein of interest’s PTS2 with a fluorescent protein which can be removed by an optogenetically activated TEV-protease. For this project we use the protein Phytochrome-B from Arabidopsis thaliana and its interaction partner PIF6. These two proteins, also derived from Arabidopsis thaliana, bind together upon irradiation by red light (660 nm) and separate upon irradiation with far-red light (780 nm). We used this property to activate a split version of a TEV-protease whose split halves were each fused to one of the two optogenetic proteins.
The TEV-protease was obtained from the Biobrick BBa_K1319004. This variant contains the anti self-cleavage mutation S219V. Using overhang-PCR we created a split version of the Biobrick protease based on work done by Wehr et al. [2] and the iGEM team Munich 2013. The split was made between amino acid 118 and 119.
Our construct for attaching proteins N-and C-terminally is highly variable: it consists only of a TEV-cleavage site, the PTS2 sequence and a short linker and was designed as a 3b-part for the yeast-toolbox(LINK). This means that we can attach any protein to its N- or C-terminus we desire. We planned on attaching different fluorescent proteins to each sides of the 3b-part in a Lv1-ligation(LINK). For our experiment we planned on using the pairs mTurquoise-mVenus and GFP-mRuby.
The two other constructs were planned as follows: Phytochrome B was fused to the C-terminal TEV-half, PIF6 was fused to the N-terminal TEV-half. The PhyB-TEV2 part and the TEV1-PIF6 part were supposed to be inserted into a shared plasmid via a LV2 Golden Gate ligation. Finally, the Lv2-plasmid and the remaining Lv1 construct were to be co transformed into S. Cerevisiae. Our experiment consisted of illuminating one sample with red light (660nm) while keeping another sample in the dark. Fluorescence microscopy would then be used to check whether the import was successful [3]. If cleavage and subsequent protein import was successful, fluorescence of one protein would be localized to the compartment while that of the other would be observed throughout the cell.
Optogenetically controlled gene expression
This project is based on work done by Weber et Al. [3]. Using the interaction between Phytochrome B and PIF6 they designed an optogenetic switch for enabling and disabling transcription of a chosen gene. It is based on the tetracycline operon and the transcription factor VP16. The tetO operator is located upstream of a minimal promoter which in turn is located upstream of the gene of interest. The tetR repressor binds to the tetO sequence. Fused to it is PIF6. Phytochrome B is fused to the transcription factor VP16. Upon illumination with red light, Phytochrome binds to the tetR-PIF6 complex. VP16 is now located in close proximity to the minimal promoter, which enables the RNA-polymerase-2 to start transcription of the gene of interest. We designed a promoter part for the Dueber toolbox(LINK) which consists of tetO and the minimal promoter region. This can be used as a promoter in a Lv1-ligation(LINK)for any desired gene of interest (a GFP-taggedPEX 11 in our example).
Transformation into S. Cerevisiae is accompanied by co-transformation of a Lv2 plasmid containing both the tetR-PIF6 and PhyB-VP16 constructs:
Verification methods depend on which gene is expressed. See size and number(LINK to size-number) and secretion(LINK to secretion) for details.
[3] Müller K., Zurbriggen MD., Weber W. (2014), Control of gene expression using a red- and far-red light-responsive bi-stable toggle switch. Nature Protocols 9, pp 622-632. doi:10.1038/nprot.2014.038
Results and discussion
Our GFP-LOV-PTS1 construct was successfully cloned and transformed into S. Cerevisiae. Following a lightbox experiment, GFP-fluorescence was observed throughout the cells in both the illuminated sample and the dark control, indicating unsuccessful import.
All three constructs of our Split-TEV PTS2 subproject have been successfully cloned to Lv1 in regards to the Dueber toolbox. A Lv2 plasmid containing the PhyB-TEV2 and TEV1-PIF6 constructs is required in order to transform all constructs into S. Cerevisiae. This has not been created so far.
The TetO-Pmin promoter construct has been brought to Lv1 with mRuby and Pex11 as genes of interest. The TetR-PIF6 construct has also been brought to Lv1. The PhyB-VP16 construct has not been successfully integrated into the Dueber toolbox.
Outlook
The variability of our compartment toolbox could be greatly increased by using optogenetics. We planned on using constructs suited for optogenetic control of protein import via both pathways as well as constructs designed for optogenetically controlled gene expression. Even though we did not get to finish our work on this sub project in this year’s project, we still want to underline its importance for future applications and improvements of our toolbox.
As mentioned on our project results page, we were not successful in demonstrating optogenetically controlled protein import via PTS1.
Our theory here is that the LOV2-variant obtained from Avena Sativa does not correctly function in S. Cerevisiae, possibly due to the different cytosolic conditions, such as pH, ion- or enzyme concentrations and so forth. Future tests would involve using the LOV2-variant from Arabidopsis Thaliana, as this has already been successfully used in yeast[SOURCE].
Another aspect we considered was the possibility of steric inhibition of the protein of interests function by the LOV2 attached to it. A future idea we came up for solving this problem would be to add a TEV-protease cleavage site between the protein of interest and the LOV2-protein. The corresponding TEV-protease could be fitted with a PTS, leading to cleavage of the fusion protein upon it being imported into the compartment.
Unfortunately we were unable to test our TEV-protease construct, as we did not finish the cloning process. However, we think that upon further development of the toolbox it is an aspect which should be considered, especially since there is only one more cloning step which needs to be completed in order for it to be eligible for transformation into S. Cerevisiae .
The optogenetic control of our secretion mechanism(LINK) via gene expression also still awaits testing due to unfinished cloning. If successful, it would enable secretion of our compartments content within a few hours after illumination. The system we worked on in the lab is not the only idea we thought about. Another approach we haven’t pursued yet is attaching the vSNARE-proteins we are using to PIF6 and insert Phytochrome B into the peroxisomal membrane via our Pex26 anchor(LINK ZU PMP). In theory, illumination with red light would then lead to instant secretion.
