<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 establish blue light control system, including a minimal constitutive promoter with (C120)<sub>5</sub> region where EL222 binds to. 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.</h4>
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<h2>Biology</h2>
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<p>It is a blue light-induced promoters, which consist of (C120)<sub>5</sub> and a minimal TATA-box constutive promoter (minP). (C120)<sub>5</sub> acts as a regulative region that respond to the binding of EL222 dimers, and activated the expression of downstream genes.</p>
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<h2>Experiment</h2>
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<p>We synthesize and construct 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.</p>
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<p>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 are aware that lycopene and patchulol are produced when blue light is on, which means our blue light-induced system is able to work effectively. Besides, the expression level is related to the dose of the blue light. The longer exposure to blue light, the higher titer of patchulol (Figure.1). </p>
<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4263010 parameters</partinfo>
Latest revision as of 16:12, 30 September 2022
K4263010
(C120)5-PminCYC
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 establish blue light control system, including a minimal constitutive promoter with (C120)5 region where EL222 binds to. 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.
Biology
It is a blue light-induced promoters, which consist of (C120)5 and a minimal TATA-box constutive promoter (minP). (C120)5 acts as a regulative region that respond to the binding of EL222 dimers, and activated the expression of downstream genes.
Experiment
We synthesize and construct 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 are aware that lycopene and patchulol are produced when blue light is on, which means our blue light-induced system is able to work effectively. Besides, the expression level is related to the dose of the blue light. The longer exposure to blue light, the higher titer of patchulol (Figure.1).
Figure 2. Titer of lycopene and patchulol of each parallel.
