A 450nm light control system in S. cerevisiae

Usage

BIT-China team's 2023 IGEM project "Light's control iT" proposes to control the ratio of sclareol and santalol arbitrarily under light control, so we designed an light control circuit, in which the photo-sensitive transcription factor being the most critical part.

Biology

In normal yeast cells, P_tdh3 is a constitutive promoter, and the downstream el222 gene is expressed to generate the photosensitive transcription factor EL222.

Under 450 nm light illumination, the photosensitive transcription factor EL222 undergoes dimerization and then binds to the P_C120 promoter, activating the transcription and expression of the downstream target gene gal4.GAL4 protein acts as an amplifier to further start the transcript of genes downstream of P_gal1-s promoter.

The optical control system includes four basic components, including P_TEF1(BBa_K4616002),P_c120 (BBa_K4616005), el222(BBa_K4616138) and gal4 (BBa_K4616231). For details, please check the specific information of the relevant components.

Figure 1.PCR result. M-marker.1-4: P_TEF1_VP16-EL222^A79Q _T_CYC1_P_c120_gal4_T_ACT1(composite part K4616666,4843 bp).

Experiments

We verified through experiment that this system is sensitive to blue light at 450nm, so we further integrated this system into S.cerevisiae to produce different proportions of products by controlling different light cycles. At the same time, considering that the P_c120 promoter in the gene line has different starting strength in different kinds of chassis, in order to make the gene circuit better adapt to the yeast cell, we introduced a set of gal transcription factor regulation system based on the original gene circuit, which amplified the signal and improved the expression controllability. Comparing to the final circuit, we replaced the biosynthetic genes with fluorescent proteins, to better visualize and quantify the expression level of the two cassettes. The second gene circuit is shown below.

Figure 2.Light control system in S.cerevisiae

In the initial construction of engineered cells, the effect of fragment ligation using OE-PCR (overlap-extension PCR) technology was not satisfactory due to the excessive number of short gene fragments with different lengths. We switched to Gibson assembly to construct DNA fragments and plasmids.

Figure 3.Constructed light regulated plasmid.（A）Plasmid EZ-L580 was purchased from Addgene[1].（B）light controlled green fluorescent protein particles were constructed on the backbone of EZ-L580

Figure 4.Verification of success plasmid construction by PCR. (A)M-marker. 1-P_gal1-s (part K4616168,719 bp). (B)M-marker. 1-egfp(750 bp)

To verify that this circuit can achieve our expected effect in yeast, we introduced the plasmid into yeast cells for expression and test.

Figure 5.Yeast expressing fluorescent proteins regulated by light. (A) Yeast producing green fluorescent protein was induced under blue light. (B)Yeast without blue light induction.

We plan to use fluorescence intensity to express the yield of compounds, but due to time reasons, we only imported green fluorescent protein fragments into the light regulation line, which does not affect our detection of yeast yield changes under periodic pulsed light. We performed periodic light induction on S.cerevisiae and detected it.

Figure 6.Temporal control of gene expression by blue light based light control system in a single cell. OFF–ON–OFF–ON cycle for every 8 h over a period of 52 h.

Results

In general, the OD of yeast will reach a relatively stable plateau after 24h. Therefore, in order to reduce the impact of yeast growth on fluorescence intensity, we choose to start intermittent light detection after 24h.

As shown in the figure, we conducted intermittent light culture at an interval of 8h, and sampling and detection at an interval of 4H. That is,28-36h is blue light culture,36-44h is dark culture, and 44h-52h is blue light culture again.

It can be seen that when blue light illumination is started, the fluorescence intensity of the yeast will decrease first and then increase, which may be due to the influence of blue light on some pathways in yeast, which will reduce the content of fluorescent protein in the yeast, and at this time, the new fluorescent protein has not been synthesized, so the fluorescence intensity will decrease; When the blue light is continuously irradiated, it will induce the promoter to function, increase the green fluorescent protein, and then increase the fluorescence intensity of the bacteria.

When the cell is in the dark, the promoter in the metabolic pathway of green fluorescent protein production stops functioning, resulting in the inability to synthesize green fluorescent protein, and then the fluorescence intensity of the cell will continue to decline.The second time of blue light irradiation, the fluorescence intensity also showed a first decline and then rise, the same as the first time, which also verified our speculation.

The experimental group plans to carry out promoter enhancement engineering, by strengthening the strength of P_gal promoter and enhancing its response to transcription factors, to weaken the problem of yield decline when light penetration decreases. Through literature research, the team decided to select a specific promoter P_gal1-s for gene circuit construction[2], and the new gene circuit is shown in the figure below.

Figure 7.Light control system after promoter modification

Reference

[1] Premkumar, et al. "Blue light-mediated transcriptional activation and repression of gene expression in bacteria. " Nucleic Acids Research (2016).

[2] Zhao, Evan M.,et al. "Optogenetic Amplification Circuits for Light-Induced Metabolic Control."American Chemical Society 5(2021).

Sequence and Features

Assembly Compatibility: