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<p>Inspired from certain parts in the database and a recent literature which demonstrated the application of a blue light-induced system in <em>P.pastoris</em>, we established blue light control system. A constitutive or inducible promoter and its downstream gene that encoded light-activated transcriptional factor protein EL222, SV40-VP16-EL222. EL222 is expressed as monomer, and turn to dimer as the blue light (465 nm) was on. Then, activated EL222 binds to the (C120)<sub>5</sub> region and initiate the expression of our target gene, <em>PTS</em> (Figure.1).</p>
<p>Inspired from certain parts in the database and a recent literature which demonstrated the application of a blue light-induced system in <em>P.pastoris</em>, we established blue light control system. A constitutive or inducible promoter and its downstream gene that encoded light-activated transcriptional factor protein EL222, SV40-VP16-EL222. EL222 is expressed as monomer, and turn to dimer as the blue light (465 nm) was on. Then, activated EL222 binds to the (C120)<sub>5</sub> region and initiate the expression of our target gene, <em>PTS</em> (Figure.1).</p>
<h4>Figure 1. Design of the optogenetic plasmid and gene circuit of SV40-VP16-EL222 (<a href="https://parts.igem.org/Part:BBa_K4263009">BBa_K4263009</a>).</h4>
<h4>Figure 2. Gel electrophoresis result of colony PCR to detect the insertion of BBa_K4263009 and BBa_K4263010 into engineered <em>P.pastoris</em> yeast 2OZPP. (The engineered yeast 2OZPP has the ability to express the lycopene synthetic genes from Corynebacterium glutamicum ATCC13032 with the PAOX1 promoter into <em>P.pastoris</em> strain GS115.) A target band around 2399 bp should appear to indicate a successful construction, which was shown as marked.</h4>
<h4>Figure 2. Gel electrophoresis result of colony PCR to detect the insertion of BBa_K4263009 and BBa_K4263010 into engineered <em>P.pastoris</em> yeast 2OZPP. (The engineered yeast 2OZPP has the ability to express the lycopene synthetic genes from Corynebacterium glutamicum ATCC13032 with the PAOX1 promoter into <em>P.pastoris</em> strain GS115.) A target band around 2399 bp should appear to indicate a successful construction, which was shown as marked.</h4>
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<h4>Figure 3. Titer of lycopene and patchulol of each parallel.</h4>
<h4>Figure 3. Titer of lycopene and patchulol of each parallel.</h4>
The development of biotechnology for the industry pushed it to increase the productivity and safety of the biotechnological processes. One of those breakthroughs were the inducible promoters. The principle is based on the recruitment of transcriptional regulators using small-molecule responsive DNA-binding proteins (Janicki et al., 2004). However, chemically-induced systems present many drawbacks such as an uncontrolled spatiotemporal resolution, the speed of the cellular uptake, and the release of the inducer.
In order to meet these requirements, it is possible to employ a clever optogenetic system that allows positive regulation of the expression of the gene of interest (GOI). Previously described photosensitive transcription factor (TF) that consists of a nuclear localization signal (NLS), the VP16 transactivation domain, and the bacterially-derived protein EL222 (Motta-Mena et al.., 2014; Nash et al., 2011). EL222 has the capacity to self-dimerize upon light stimulation thanks to its light oxygen and voltage (LOV) domain. EL222 has a DNA binding domain and the artificial promoter C120 contains 5 repeated binding sites for the fixation of EL222 and one sequence of a minimal promoter. When exposed to blue light (450nm), this system allows the transcription machinery recruitment via VP16, and hence the transcription activation of the gene positioned downstream of the artificial promoter C120 (BBa_K3570023) (fig. 1-2).
Fig. 1: Non-activated optogenetic system. GOI: gene of interest.
Fig. 2: Activated optogenetic system upon blue light illumination. GOI: gene of interest.
..
Biology
The EL222 is a light-sensitive transcription factor that englobes two functional domains. A light-oxygen-voltage-sensing (LOV) domain that responds to illumination at 450nm, which leads to a homodimerization of two entities of EL222. The second one is an HTH (helix-turn-helix)- DNA binding domain, that would recognize and bind specifically to C120 repeat. The cofactor used by this optogenetic system is FMN (flavin mononucleotide) EL222 protein originated from Erythrobacter litoralis.
Experiments
Team iGEM Toulouse 2020 did not have sufficient time to complete the cloning and hence, to test this part functionality.
References
Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, Y., Bertrand, E., Singer, R. H., & Spector, D. L. (2004). From Silencing to Gene Expression. Cell, 116(5), 683–698. https://doi.org/10.1016/s0092-8674(04)00171-0
Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., & Gardner, K. H. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature Chemical Biology, 10(3), 196–202. https://doi.org/10.1038/nchembio.1430
Nash, A. I., McNulty, R., Shillito, M. E., Swartz, T. E., Bogomolni, R. A., Luecke, H., & Gardner, K. H. (2011). Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proceedings of the National Academy of Sciences, 108(23), 9449–9454. https://doi.org/10.1073/pnas.1100262108
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 256
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 256
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 256
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 256
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 256
1000
COMPATIBLE WITH RFC[1000]
Improve From SCUT-China 2022
K4263009
SV40-VP16-EL222
Usage
Inspired from certain parts in the database and a recent literature which demonstrated the application of a blue light-induced system in P.pastoris, we established blue light control system. A constitutive or inducible promoter and its downstream gene that encoded light-activated transcriptional factor protein EL222, SV40-VP16-EL222. EL222 is expressed as monomer, and turn to dimer as the blue light (465 nm) was on. Then, activated EL222 binds to the (C120)5 region and initiate the expression of our target gene, PTS (Figure.1).
Figure 1. Design of the optogenetic plasmid and gene circuit of SV40-VP16-EL222 (BBa_K4263009).
Figure 2. Gel electrophoresis result of colony PCR to detect the insertion of BBa_K4263009 and BBa_K4263010 into engineered P.pastoris yeast 2OZPP. (The engineered yeast 2OZPP has the ability to express the lycopene synthetic genes from Corynebacterium glutamicum ATCC13032 with the PAOX1 promoter into P.pastoris strain GS115.) A target band around 2399 bp should appear to indicate a successful construction, which was shown as marked.
Biology
The photosensitive protein EL222 from Erythrobacter litoralis is one of the systems based on light-induced homodimerization. It is composed of 222 amino acids (aa). The N-terminal of EL222 is a light-oxygen-voltage (LOV) domain, and the C-terminal is a helix-turn-helix (HTH) DNA binding domain. The Jα helix links the LOV and HTH. When EL222 is irradiated by blue light, the combination of flavin mononucleotide (FMN) and LOV causes the Jα helix to wobble away from LOV, thereby releasing HTH, which makes EL222 homodimerize and bind to the regulatory element termed clone 1-20 bp (C120).
Experiment
We synthesized and constructed the parts on the same vector pPIcza. Four groups of our host, three parallel tests each, are set with difference in the promoter which control the expression of EL222 in 250ml shake flasks. All of the flasks are cultured for 48h, and their OD were checked every 24h, then methanol was added.
Blue light was sprayed by LEDs (5W, 465 nm) that are hanging above the flasks. The OD value and the titer from each parallel is examined by GC and HPLC, and we were aware that lycopene and patchulol were produced when blue light on, which means our blue light-induced system is able to work effectively. Besides, the expression level was related to the dose of the blue light. The longer exposure to blue light, the higher titer of patchulol. (Figure.3)
Beside, Sequence optimization of our dynamically regulated light-control part BBa_K4263009 was done to allow it to better play a regulatory role in P.pastoris.(reference to the existed part BBa K3570021[2])
