Engineering iterations of the Light Control System
Design & Build
After determining the direction of the project, we selected Saccharomyces cerevisiae as the chassis, and built a light control system on it for the production of blended fragrance. As E.coli is a better system for protein engineering and there were studies on this system in prokaryotes[1] through a large number of literature research and information retrieval, so we selected E.coli for verification of function of the light control system. Considering the advantages of E.coli, such as shorter culture cycle, faster growth rate, and easy transformation and expression of heterologous genes, the team hopes to simplify the experimental operation, shorten the experimental time, and improve the effect of iteration number. We replaced the biosynthetic genes for terpenoid fragrances with fluorescent proteins, deleted signal amplification circuit involving GAL4, and replaced yeast promoters with bacterial ones, to generate the first designed gene circuit, as shown below.
We constructed plasmids according to sequences shown from the literature[1] and transformed them into E.coli TOP10 competent cells, as a chassis, for test. The construction of the engineered bacteria was verified by colony PCR, where primers specific for the three cassettes (PrrnBp1_rbs34_VP16_EL222, PBlindv1_ rbs34_eGFP, and PBLrep_rbs34_mCherry) were used. The amplification of DNA fragment corresponding to the expected size indicates successful construction of the strain.
Test
In order to verify the performance of the light control system, we cultured the engineered E.coli and illuminated the culture with pulsed light. After inspecting cells under fluorescence microscope, we verified that both GFP and RFP were expressed, indicating that the light control system worked as expected.
Since there are clear data in the literature[1], the literature data are used to model and guide the design of subsequent lighting experimental conditions. (More information please view our modeling page ->)
Learn & Redesign
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 Pc120 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.
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.
Bulid & Test
To verify that this circuit can achieve our expected effect in yeast, we introduced the plasmid into yeast cells for expression and test.
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 Saccharomyces cerevisiae and detected it.
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 is evident that upon the initiation of blue light exposure, the fluorescence intensity of the yeast initially decreases before subsequently rising. This observed phenomenon could be attributed to the impact of blue light on certain yeast pathways, resulting in a reduction in the fluorescent protein content within the yeast cells. During this phase, the newly synthesized fluorescent protein has not yet reached sufficient levels, leading to the initial decrease in fluorescence intensity. As the blue light exposure continues, it triggers the activation of the promoter, resulting in an increase in green fluorescent protein production, subsequently leading to an augmentation in the overall bacterial fluorescence intensity.
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.
Learn
From the results of Test, it can be seen that our light control components can achieve the light control effect well, so we decided to take the next step of design. On one hand, Professor Zhao from the school of chemistry and chemical engineering, Beijing Institute of Technology gave the following suggestions for the particularity of the fermenter we need: for large-scale industrial equipment, light cannot completely penetrate the entire medium, but this characteristic can be used to calculate and design, so that the yeast in the upper part of the culture medium in the constructed container can be exposed to light to produce biomass a, and the lower part cannot be exposed to light to produce biomass B. This breaks our original idea that every yeast is exposed to light. For specific amplification design, please refer to relevant HP links.On the other hand, the experimental group plans to carry out promoter enhancement engineering, by strengthening the strength of Pgal 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 Pgal1-s for gene circuit construction [2], and the new gene circuit is shown in the figure below.
Redesign
Through literature research, we combined the production of sclareol and santalol with light control, so that the cell factory can produce a specific proportion of mixed fragrance. The circuit is still under construction, with all the biosynthetic genes amplified (shown in Figure 13 and 14) and we hope we can show you the result in the near future.
In addition, in order to verify that the product will not affect the cell growth, we also carried out the toxicity experiment of the products of sclareol and santalol on cells[3,4]. View the experiment page for details.
Bulid & Test
(1)The toxicity test results of the product on cells are shown in the figure below, indicating that the growth of yeast was not negatively affected by santalol or sclareol under their relatively high concentrations'.
(2)We have amplified the gene fragment of the synthetic product by PCR, and the result is shown in the figure below. The target fragment was obtained, but it was not integrated into the yeast genome due to time reasons.
(3) In order to determine the proportion of products we synthesized under specific light conditions, we used GC-MS for detection and quantification. We have analyzed a series of standard samples with known concentrations, and by integrating the peak areas and plot against their concentrations, we built standard curves for the two products in our project, as shown in the figure below. After the synthesis, we would perform GC-MS analysis under the same conditions, and calculate the product concentration using standard curve.
Prospect
Our work can provide guiding help for industry to produce relevant products through light control system, and provide ideas for industrial production.
In the actual production in the future, our engineered cells can reduce the environmental pollution and related labor and equipment costs of traditional production methods. In the future, we will continue to improve our yeast strain, which is controlled by light to produce mixed fragrance, and improve the yield and control accuracy. Thereafter, we hope to expand the product category and apply it to more substances produced in proportion. We also plan to use PYB-PIF3 type red light inducible promoter together with existing lines to build three product controllable gene production lines.
