Part:BBa_K2516003

Membrane-bound SuperNova (for C. reinhardtii)

This is a membrane-bound version of SuperNova protein, originally introduced by iGEM 2014 Carnegie Mellon team. Link: BBa_K1491017

SuperNova is a photosensitizing protein, which produces Reaction Oxygen Species (superoxide and singlet oxygen) under irradiation of 500-600nm wavelength light. It is a monomeric version of another photosensitizing protein called KillerRed. There are several advantages over KillerRed, SuperNova does not oligomerize inside the cell and does not interfere with mitotic cell division. This advantage allows performing targeted protein inactivation inside the cell by fusion to the target protein without loss of target protein function. [1]

The second application that seems very simple and powerful is light controlled safety system. SuperNova can be utilized as a killer switch for genetically modified organisms. Under irradiation of 500-600nm wavelength light, SuperNova will generate ROS and kill the cell. Its phototoxicity was proven in bacterial and mammalian cells. [1]

In case of KillerRed, it was proven that membrane-bound version is more toxic and kills the cell more rapidly. [2]

[Improvement]

Codon optimized for C. reinhardtii.

SuperNova protein was made membrane-bound by adding 4 sequences:

1) Signal peptide from Peptidyl serine alpha-galactosyltransferase gene (SGT1) (1-26 aa) [3].

It correctly predicted by SignalP 4.1 server [4] (predicts signal peptides) (Figure 1):

Figure 1. Results of SignalP 4.1 server.

S score – probability of signal peptide Y score – probability of signal peptidase site C score – combined S and Y scores

2) (GGGGS)2 flexible linker (27-36 aa).

3) Transmembrane domain and C-terminus from SGT1 protein (37-74 aa). [3]

C-terminus was also added because it gave an optimal level of confidence in the presence of transmembrane domain both by TMHMM server 2.0 and Philius transmembrane prediction server.

Transmembrane domain and signal peptide were correctly predicted by TMHMM server v2.0 (predicts transmembrane domains) [5](Figure 2):

Figure 2. Results of TMHMM server 2.0.

4) (EAAAK)2 rigid linker (75-84 aa).

Total signal peptide, transmembrane domain and linkers were predicted by TMHMM server v2.0 and Philius transmembrane prediction server [6] with high confidence values: Type confidence = 0.91 and topology confidence = 0.93 (Figure 3):

Figure 3. Results of Philius transmembrane prediction server.

3D model of membrane-bound SuperNova

Figure 4. 3D model of membrane bound SuperNova.

The Modeller 9.0 software was used to develop a homology model of the Transmembrane domain and Rigid linker (Figure 5) [7] The Supernova crystal structure coordinates (PDB ID:3WCK) were linked to this homology model. The Charmm-GUI membrane embedding code was used to embed this structure into a lipid bilayer composed of phospholipids and mono- and diacyl glycerols . [8] The distance from the chromophore to the lipid bilayer was measured to be 23 Angstroms well within the range of the radius of effectivity (whereas 3-4 nm is a half radius of action). [9]

Reference list:

1. Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., ... & Ayabe, T. (2013). SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Scientific reports, 3, 2629.

2. Bulina, M. E., Lukyanov, K. A., Britanova, O. V., Onichtchouk, D., Lukyanov, S., & Chudakov, D. M. (2006). Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed. Nature protocols, 1(2), 947-953.

3. The UniProt Consortium. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 45: D158-D169 (2017). http://www.uniprot.org/uniprot/H3JU05

4. Nielsen, H. (2017). Predicting Secretory Proteins with SignalP. Protein Function Prediction: Methods and Protocols, 59-73.

5. Krogh, A., Larsson, B., Von Heijne, G., & Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of molecular biology, 305(3), 567-580.

6. Reynolds, S. M., Käll, L., Riffle, M. E., Bilmes, J. A., & Noble, W. S. (2008). Transmembrane topology and signal peptide prediction using dynamic bayesian networks. PLoS computational biology, 4(11), e1000213.

7. Martí-Renom, M. A., Stuart, A. C., Fiser, A., Sánchez, R., Melo, F., & Šali, A. (2000). Comparative protein structure modeling of genes and genomes. Annual review of biophysics and biomolecular structure, 29(1), 291-325.

8. Vieler, A., Wilhelm, C., Goss, R., Süß, R., & Schiller, J. (2007). The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC. Chemistry and physics of lipids, 150(2), 143-155.

9. Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., ... & Ayabe, T. (2013). SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Scientific reports, 3, 2629.

The following characterization is done by 2019 DUT_China_B

Characterization:
Culture engineered Chlamydomonas reinhardtii and wild algae to logarithmic phase at 25 ℃, with 5.9 klx continuous illumination, and 150 rpm. Inoculate the algae strains into TAP liquid medium respectively, with the initial concentration of the cells being 1.0 ×105 cells/mL. Detect the absorbance of algae cells at 750 nm every 12 h by microplate reader. The growth curve was plotted based on the absorbance-cell density curve, see protocol 2.

Figure 1. the absorbance-cells density curve of Chlamydomonas reinhardtii

We found that the cell growth of wild and engineered Chlamydomonas reinhardtii after illumination at 585 nm, 5.9 klx for 1 hour have no significant difference. It’s reported that C. reinhardtii cells generally cope very well with high rates of generation of superoxide, H2O2, and even singlet oxygen. The phototoxicity of supernova maybe weak in C. reinhardtii cells.

Reference:

[1]Foyer, C. H . Redox Homeostasis and Antioxidant Signaling: A Metabolic Interface between Stress Perception and Physiological Responses[J]. THE PLANT CELL ONLINE, 2005, 17(7):1866-1875.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 606
  Illegal BsaI.rc site found at 691