iGEM 2023 — Team bit-china


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# Engineering

2023\_bit-china

[Lights Control iT!](#banner)

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Content:

* [**Overview**](#title_1)
* [**Light Control System**](#title_2)
* [**Microbial Chassis**](#title_3)
* [**Reference**](#title_4)

![](https://static.igem.wiki/teams/4616/wiki/elf/to-top.png)

## Overview

Synthetic biology uses the Design-Build-Test-Learn (DBTL) cycle, which combines frequent design,
genetic engineering, resultant testing, and data analysis, in order to learn and solve bottlenecks
in biomanufacturing, to achieve better results in the actual production process.

![](https://static.igem.wiki/teams/4616/wiki/engineering/circle.png)

The goal of our project is to construct a set of gene circuits for light-controlled production of
mixed fragrances in yeast. In order to achieve the goal, we divided the project into two parts,
construction and verification of gene circuits, and optimization of chassis. We further break the
tasks down into smaller parts and underwent multiple rounds of optimization.
For construction of gene circuits, we divided the circuit into three cassettes, the photosensitive
transcription regulator, the light activated gene(s), and the light repressed gene(s), and
constructed them separately. In order to achieve the desired function, we first tested the light
control system in a simpler chasiss, *E.coli*, then we tested the system in yeast using
fluorescent proteins in place of biosynthetic enzymes, and finally attempted to construct the actual
circuit for intermixed terpenoid production. Through iterations and optimizations, complex function
is realized in our designed system.

![](https://static.igem.wiki/teams/4616/wiki/engineering/overview-2.png)

## 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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-1.png)

Figure 1. Gene circuits in *E. coli*

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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-2.png)

Figure 2. *E.coli* transformation experimental verification result. M-marker.
1-rrnBp1\_rbs34\_VP16\_EL222 (composite part K4616010, 937 bp). 2-PBlindv1\_
rbs34\_eGFP(composite part K4616011, 794 bp).

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-3.png)

Figure 3. *E.coli* transformation experimental verification result. M-marker.
1-PBLrep\_rbs34\_mCherry (composite part K4616012, 765 bp).

---

### 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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-4.png)

Figure 4. *E.coli* engineering bacteria expressing fluorescent protein under light
and dark conditions.（A） Green fluorescent protein was induced under blue light.（B）
Red fluorescent protein is produced in the dark.（C）*E.coli* engineering bacteria in
the open field.

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 ->)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model_4%27%29%20%7D%7D)

---

### 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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-5.png)

Figure 5. Light control system in *Saccharomyces 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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-6.png)

Figure 6. Constructed light regulated plasmid.（A）Plasmid ez-l580 was purchased from
addgene [2].（B）Finally, light controlled green fluorescent protein particles were
constructed.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-7.png)

Figure 7. PCR result. （A）M-marker. 1-Pgal1-s (part K4616168, 719 bp). （B）M-marker.
1-egfp(750 bp).

---

### 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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-8.png)

Figure 8. *Saccharomyces cerevisiae* expressing fluorescent proteins regulated by
light.（A）*Saccharomyces cerevisiae* producing green fluorescent protein was induced
under blue light.（B）*Saccharomyces cerevisiae* 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 *Saccharomyces cerevisiae* and detected
it.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-9.png)

Figure 9. 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.

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D).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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-10.png)

Figure 10. Light control system after promoter modification

---

### 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.

![](https://static.igem.wiki/teams/4616/wiki/description/total.jpg)

Figure11. Gene circuit

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.](https://2023.igem.wiki/bit-china/experiments#toxic)

---

### 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'.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-12.png)

Figure 12. Growth chart of cytotoxicity test

(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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-13.png)

Figure 13. Genes related to the synthesis of sclareol PCR result. M-marker.
1-tps\_lpps (part K4616888, 4132 bp).

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-14.png)

Figure 14. Genes related to the synthesis of santalol PCR result. （A）M-marker.
*1-sass* (part K4616003, 1757 bp). *2-cprs* (part K4616009, 2157bp). （B）M-marker.
*1-cyp76* (part K4616004, 1546 bp).

(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.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-15.png)

Figure 15. The standard curve of sclareol

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-16.png)

Figure 16. The standard curve of santalol

---

### 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.

## Iteration on microbial chassis (Yeast.) for optimizing production effects

### Design & construction

In order to reduce the basic metabolic pathways in yeast cells to consume extra energy and
material, and thus to increase the productivity of our designed production and regulation lines,
we analyzed the metabolic flow of yeast, applied OptKnock to select the metabolic pathways that
do not affect the growth of yeast for genome knockdown,[5,6] and used homologous recombination
technology to knock down some of the genes as shown in the following Fig. 17 Fig. 18, and
constructed a first version of chassis cells in the final form as shown in Fig. 19.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-17.png)

Figure 17. Metabolic pathways related to products formation and candidate genes to knockout.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-18.png)

Figure 18. Metabolic pathways related to products formation and candidate genes to knockout.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-19.png)

Figure 19. Metabolic pathways that should be on the relevant metabolic pathway after knockout

---

### Test

We want to verified that the knocked-out gene did not affect the normal growth of the cells, so we
designed an experiment to detect the change of OD600, which reflects the growth of the cells, over
time, and used the software (Origin) to fit the growth curves using the  **logistic growth
model**, which
proved that the growth of the yeast (strain Δ236W) was affected by the knocked-out gene, which was unsatisfactory
in comparison with our expected effect.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-20.png)

Figure 20. Growth rate curves

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-21.png)

Figure 21. Growth curves

---

### Learn

Through communication with our instructor, we believe that we need to consider the effect of NADPH in
the metabolic flow reaction and that the original model should be modified to re-screen the genes
that need to be knocked out.

---

### Redesign & Test

Considering the effect of NADPH, we redesigned the Optknock model and came up with genes of metabolic
pathways that need to be knocked out. In addition, considering the effect of GAL80 and GAL4 genes
present in yeast, we also decided to knock them out. The pathways that need to be knocked out and
the metabolic pathways after knockout are shown below.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-22.png)

Figure 22. Updated metabolic pathways related to products formation and new candidate gene to knockout

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-23.png)

Figure 23. Metabolic pathways related to products formation after gene knockout

We knocked out the above genes *(gal4, gal80, spe4)*, cultured the knocked-out chassis cells, measured their growth curves,
still fitted them using the logistic growth model, and used Origin for graphing, and the results are
shown below.

![](https://static.igem.wiki/teams/4616/wiki/engineering/engineering-24-2.jpg)

Figure 24. Growth curves fitted to OD600 data

As shown in the figure, the growth was not significantly affected or even better. Hence the
envisioned
results were achieved. Constructing our gene line on this chassis could increase the yield of our
expected product.

[More information please view our modeling page ->](model_2)

The actual experimental work is often slow and tortuous, and we inevitably encountered
various problems
in our work, and the results were not always as good as we expected, requiring repeated thinking and
attempts. Through the process of engineering iterative DBTL, which combines the depth of modeling and
experimentation, we have achieved the optimization of metabolic flow, the optimization of chassis
selection, the satisfaction of production conditions, and the analysis of production estimation, which
provides the basis for the product to be put into production in the future.

## Reference

1. [1]Jayaraman P, Devarajan K, Chua TK, Zhang H, Gunawan E, Poh CL. Blue
   light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic
   Acids Res. 2016 Aug 19;44(14):6994-7005. doi: 10.1093/nar/gkw548. Epub 2016 Jun 28. PMID: 27353329;
   PMCID: PMC5001607.
2. [2]Zhao EM, Lalwani MA, Chen JM, Orillac P, Toettcher JE, Avalos JL. Optogenetic Amplification
   Circuits for Light-Induced Metabolic Control. ACS Synth Biol. 2021 May 21;10(5):1143-1154. doi:
   10.1021/acssynbio.0c00642. Epub 2021 Apr 9. PMID: 33835777; PMCID: PMC8721662.
3. [3]Wang Y, Gong X, Li F, Zuo S, Li M, Zhao J, Han X, Wen M. Optimized biosynthesis of santalenes and
   santalols in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2021 Dec;105(23):8795-8804. doi:
   10.1007/s00253-021-11661-9. Epub 2021 Nov 5. PMID: 34738171.
4. [4]Cao X, Yu W, Chen Y, Yang S, Zhao ZK, Nielsen J, Luan H, Zhou YJ. Engineering yeast for
   high-level production of diterpenoid sclareol. Metab Eng. 2023 Jan;75:19-28. doi:
   10.1016/j.ymben.2022.11.002. Epub 2022 Nov 9. PMID: 36371032.
5. [5]Lu H, Li F, Sánchez BJ, Zhu Z, Li G, Domenzain I, Marcišauskas S, Anton PM, Lappa D, Lieven C,
   Beber ME, Sonnenschein N, Kerkhoven EJ, Nielsen J. A consensus S. cerevisiae metabolic model Yeast8
   and its ecosystem for comprehensively probing cellular metabolism. Nat Commun. 2019 Aug
   8;10(1):3586. doi: 10.1038/s41467-019-11581-3. Erratum in: Nat Commun. 2020 Oct 22;11(1):5443. PMID:
   31395883; PMCID: PMC6687777.
6. [6]Heirendt L, Arreckx S, Pfau T, Mendoza SN, Richelle A, Heinken A, Haraldsdóttir HS, Wachowiak J,
   Keating SM, Vlasov V, Magnusdóttir S, Ng CY, Preciat G, Žagare A, Chan SHJ, Aurich MK, Clancy CM,
   Modamio J, Sauls JT, Noronha A, Bordbar A, Cousins B, El Assal DC, Valcarcel LV, Apaolaza I, Ghaderi
   S, Ahookhosh M, Ben Guebila M, Kostromins A, Sompairac N, Le HM, Ma D, Sun Y, Wang L, Yurkovich JT,
   Oliveira MAP, Vuong PT, El Assal LP, Kuperstein I, Zinovyev A, Hinton HS, Bryant WA, Aragón Artacho
   FJ, Planes FJ, Stalidzans E, Maass A, Vempala S, Hucka M, Saunders MA, Maranas CD, Lewis NE, Sauter
   T, Palsson BØ, Thiele I, Fleming RMT. Creation and analysis of biochemical constraint-based models
   using the COBRA Toolbox v.3.0. Nat Protoc. 2019 Mar;14(3):639-702. doi: 10.1038/s41596-018-0098-2.
   PMID: 30787451; PMCID: PMC6635304.

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# Experiments

2023\_bit-china

[Lights Control iT!](#banner)

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Content:

* [**Fragment acquisition**](#title_1)
* [**Chassis construction**](#title_2)
* [**Strain construction**](#title_3)
* [**Testing of raw materials and their
  products**](#title_4)
* [**Reference**](#title_5)

![](https://static.igem.wiki/teams/4616/wiki/elf/to-top.png)

## Fragment acquisition

### PCR—general purpose

The *tl* and *tpps* genes were a gift from Professor Yongjin Zhou,and the plasmid skeleton was purchased from Addgene Company for plasmid EZ-L580. Other expression vectors
were purchased from Jiutian Gene Company.

We used Snapgene software to design primers, and the Tm values of the designed upstream and downstream
primers were set to be similar, with each primer a very large range bp. For amplification of long
fragments (2 Kb and above), PrimeSTAR Max DNA polymerase was selected, and PCR reactions were performed
using bacterial genomes or plasmids as templates to obtain the target fragment. Enzyme and template DNA
should be placed on ice and taken for 200 μ Prepare the reaction system using the EP tube of L. Add each
component in order of volume from most to least. After vortex mixing, amplify according to the PCR
reaction program preset in the instruction manual.

The reaction program of Phanta polymerase: Cycle from program 1 (denaturation) to program 3 (extension),
typically lasting 30-35 cycles, to obtain sufficient PCR amplification products. Analyze the primers and
set the annealing temperature according to the Tm value for PCR. The extension time of PCR depends on
the length of the amplified DNA fragment. The extension speed is 5-10 seconds/Kb, and the extension time
is set based on the length of the target product.

The reaction system and procedure are as follows:

Table 1. PCR reaction system of Phanta DNA Polymerase

| Composition | Volume（μL） |
| --- | --- |
| Template | x |
| Upstream primer（10 μM） | 1 |
| Downstream primers（10 μM） | 1 |
| Phanta Max Super-Fidelity DNA Polymerase | 1 |
| dNTP Mix(10 mM each) | 1 |
| ddH2O | 44-x |
| Total volume | 50 |

NOTE: a.2 × The Phanta Max Buffer already contains Mg2+with a final concentration of 2 mM.

b. The optimal reaction concentration varies among different templates, as shown in the table below:

Table 2. 50 μ Recommended template usage for the l system

| Template Type | Template Starting Quantity |
| --- | --- |
| Genomic DNA | 50 - 400 ng |
| Plasmid or virus DNA | 10 pg - 30 ng |
| cDNA | 1 - 5 μl(not exceeding 1/10 of the total volume of PCR reaction) |

Table 3. PCR reaction procedure of Phanta Polymerase

| Program | temperature | time | cycle |
| --- | --- | --- | --- |
| Pre denaturation | 95℃ | 30 sec/3min |  |
| Denaturation | 95℃ | 15 sec | 25-35 |
| Annealing | 56-72℃ | 15 sec | 25-35 |
| Extend | 72℃ | 30-60 sec/1 kb DNA | 25-35 |
| Post extension | 72℃ | 5 min |  |
| Insulation | 4℃ | ∞ |

---

The reaction program of PrimeSTAR Max DNA polymerase: Cycle from program 1 (denaturation) to program 3
(extension), typically lasting 30-35 cycles, to obtain sufficient PCR amplification products. Analyze
the primers and set the annealing temperature according to the Tm value for PCR. The extension time of
PCR depends on the size of the amplified DNA fragment. The extension speed is 5-10 seconds/Kb, and the
extension time is set based on the length of the target product.

The reaction system and procedure are as follows:

Table 4.PCR reaction system of PrimeSTAR Max DNA Polymerase

| Composition | Volume（μL） |
| --- | --- |
| Template | DNA 1 |
| Upstream primer（10 μM） | 1 |
| Downstream primers（10 μM） | 1 |
| 2×PrimeSTAR Max | 25 |
| ddH2O | 22 |
| Total volume | 50 |

Table 5. PCR reaction procedure of PrimeSTAR Max DNA Polymerase

| Program | temperature | time | cycle |
| --- | --- | --- | --- |
| Pre denaturation | 98℃ | 2 min | 30-35 |
| Denaturation | 98℃ | 15 sec |  |
| Annealing | Tm-2℃ | 15 sec |  |
| Extend | 72℃ | 5-10 sec/1 kb DNA |  |
| Post extension | 72℃ | 5 min |  |
| Insulation | 4℃ | ∞ |

---

### OverlapExtension PCR (OE-PCR)

Overlap extension polymerase chain reaction (OE-PCR) refers to the method of splicing multiple
amplified short DNA fragments through primer design to obtain a complete long DNA fragment.

There are homologous complementary sequences (20-30 bp) between each adjacent fragment, and PrimeSTAR Max
DNA polymerase is used for PCR reaction using first and last primers. Therefore, except for the reaction
procedure, other steps are the same as above.

In the reaction system, it is necessary to add at least two DNA templates with overlapping sequences, as
well as primers at both ends, and add each component in order of volume from most to least. The PCR
reaction system is 50 μ L. After the sample preparation is completed, it should be thoroughly mixed
before reacting.

Table 6. Composition of PCR reaction solution (50 μ L system)

| Composition | Volume（μL） |
| --- | --- |
| Fragment 1 | 1 |
| Fragment 2 | 1 |
| Upstream primer（10 μM） | 1 |
| Downstream primers（10 μM） | 1 |
| 2×PrimeSTAR Max | 25 |
| ddH2O | 22 |
| Total volume | 50 |

---

### Plasmid extraction

Selection of reagent kit: Tiangen's yeast genomic DNA extraction kit.

Before use, please add anhydrous ethanol to the rinsing solution PW. Please refer to the label on the
bottle for the added volume.

* 1. column balance step: Add 500 to the adsorption column CP3 (the adsorption column is placed in the
  collection tube) μ Centrifuge the equilibrium solution BL of I at 12000 rpm (~13400Xg) for 1 minute,
  pour out the waste liquid in the collection pipe, and place the adsorption column back into the
  collection pipe. (Please use the columns that were processed that day)
* 2. Take 1-5 ml of overnight cultured bacterial solution and add it to a centrifuge tube. Use a
  conventional desktop centrifuge to centrifuge at 12000 rpm (~13400 X g) for 1 minute, and try to
  remove the supernatant as much as possible (when there is a large amount of bacterial solution, the
  bacterial sediment can be collected into a centrifuge tube through multiple centrifugations).
* 3. Add 250 to the centrifuge tube with bacterial sediment in 3 directions μ Solution P1 (please
  check if RNase A has been added first), and thoroughly suspend bacterial precipitation using a
  pipette or vortex oscillator.
* **Attention:** If there are incomplete mixed bacterial blocks, it will
  affect the lysis, leading to low extraction amount and purity.
* 4.Add 250 to the 4-way centrifuge tube μ I solution P2, gently flip up and down 6-8 times to fully
  lyse the bacterial body.
* **Attention:** Mix gently and do not shake violently to avoid interrupting
  genomic DNA and causing genomic DNA fragments to be mixed in the extracted plasmid. At this point,
  the bacterial solution should become clear and viscous, and the time taken should not exceed 5
  minutes to avoid damage to the plasmid.
* If it does not become clear, it may be due to excessive
  bacterial growth and incomplete lysis, and the bacterial population should be reduced.
* 5.Add 350 to the 5-way centrifuge tube μ I solution P3, immediately gently flip up and down 6-8
  times, thoroughly mix, and white flocculent precipitates will appear at this time. Centrifuge at
  12000 rpm (~13400Xg) for 10 minutes.
* **Attention:** After adding P3, it should be mixed immediately
  to avoid local precipitation. If there are still small white precipitates in the supernatant, it can
  be centrifuged again and taken from the supernatant.
* 6. Transfer the supernatant collected in the previous step to the adsorption column CP3 using a
  pipette (the adsorption column is placed in the collection tube), taking care not to suck out
  sediment as much as possible. Centrifuge at 12000 rpm (~13400Xg) for 30-60 seconds, discard the
  waste liquid from the collection tube, and place the adsorption column CP3 into the collection tube.
* 7. Optional steps: Add 500 to the adsorption column CP3 μ I deproteinized solution PO, centrifuged
  at 12000 rpm (~13400Xg) for 30-60 seconds, poured out the waste liquid in the collection tube, and
  placed the adsorption column CP3 back into the collection tube.
* If the host bacteria are end A
  ten host bacteria (TG1, BL21, HB101, JM series, ET12567, etc.), and these host bacteria contain a
  large amount of nuclease, which is easy to degrade the plasmid ONA, this step is recommended.
* 8.If the host bacteria are endA host bacteria CDH5a, TOP10, etc.), this step can be omitted.
* Add
  600 to the 8-way adsorption column CP3 μ I rinse solution PW (please check if anhydrous ethanol has
  been added first), centrifuge at 12000 rpm (~13400Xg) for 30-60 seconds, pour out the waste liquid
  in the collection tube, and place the adsorption column CP3 into the collection tube.
* 9. Repeat step 8.
* 10. Place the adsorption column CP3 into a collection tube and centrifuge at 12000 rpm (~13400Xg)
  for 2 minutes to remove the residual rinsing solution from the adsorption column.
* **Note:** The residual ethanol in the rinse solution will affect subsequent enzyme
  reactions (such
  as enzyme digestion, PCR, etc.) experiments. To ensure that downstream experiments are not affected
  by residual ethanol, it is recommended to open the cover of the adsorption column CP3 and leave it
  at room temperature for a few minutes to thoroughly trace the residual rinsing solution in the
  adsorption material.
* 11.Place the adsorption column CP3 in a clean centrifuge tube, add 50-100ul of elution buffer EB
  dropwise to the middle of the adsorption membrane, and let it stand at room temperature for 2
  minutes. Centrifuge at 12000 rpm (~13400Xg) for 2 minutes to collect the plasmid solution into the
  centrifuge tube.
* **Note:** The volume of elution buffer should not be less than 50 μ I. The small
  size affects the recovery efficiency. The pH value of the eluent has a significant impact on the
  elution efficiency. If subsequent sequencing is carried out, ddH20 needs to be used as the eluent to
  ensure its pH value is within the range of 7.0-8.5. A pH value as low as 7.0 will reduce the elution
  efficiency. And DNA products should be stored at 20 ° C to prevent DNA degradation. To increase the
  recovery rate of the plasmid, the obtained solution can be re added to the adsorption column, placed
  at room temperature for 2 minutes, centrifuged at 12000 rpm (~13400Xg) for 2 minutes, and the
  plasmid solution collected into a centrifuge tube.
* **Note:** Low copy or large plasmid (>10 kb) extraction

If the extracted plasmid is a low copy plasmid or a large plasmid with a size of 10 kb, the amount of
bacterial body used should be increased. 5-10 ml of overnight culture should be used, and the amount of
P1, P2, and P3 should be increased proportionally. The elution buffer EB should be preheated in a water
bath at 65-70 ° C, and the adsorption and elution time can be appropriately extended to increase
extraction efficiency. The other steps are the same.

---

### DNA recovery by agarose gel electrophoresis:

Selection of reagent kit: Magen's gel recovery micro reagent kit.

Use the agarose gel recovery kit for gel recovery, and the steps are as follows:

* (1) Put the electrophoresis gel under the blue light gel cutter, cut the target DNA fragment into
  1.5 mL EP tube, and weigh it.
* (2) Add 400 to the centrifuge tube containing agarose gel μ L of GSB solution is placed in a 55 ℃
  metal bath or water bath pan until the gel is fully melted. During this period, shake up and down to
  accelerate uniform melting.
* (3) Place it at room temperature for 1 minute, cool it, and transfer all the liquid using a pipette
  to an adsorption column placed on the collection tube. Let it stand for 2 minutes and centrifuge at
  12000 rpm for 1 minute.
* (4) In order to improve the efficiency of rubber recycling, the previous step can be selectively
  repeated, and then the waste liquid can be poured out.
* (5) Add 600 to the adsorption column μ L solution WB (with anhydrous ethanol added), centrifuge at
  13000 rpm for 1 minute, and discard the waste liquid.
* (6) Repeat the previous step.
* (7) Centrifuge at 13000 rpm for 2 minutes. Take out the adsorption column and place it in a clean
  1.5 mL centrifuge tube. Open the lid and let it dry for 5 minutes to allow the ethanol to fully
  evaporate.
* (8) Add 15-30 to the center of the adsorption column μ Preheat EB solution at 65 ℃, let it stand for
  1 minute, and centrifuge at 13000 rpm for 1 minute to wash. Can be repeated 1-2 times to improve
  recycling efficiency.
* (9) Bring the recovered DNA fragments to the spectrophotometer for concentration detection, and
  store the fragments with higher concentrations for subsequent work.
* (10) Store the recovered DNA fragments in a -20 ℃ refrigerator.

**Note:**

* (1) To increase the recovery efficiency, the collected solution can be added again to the centrifuge
  column and centrifuged at 13000 rpm for 1 minute;
* (2) The elution buffer should not be less than 30 µ l, and the volume is too small, which affects
  the recovery efficiency;
* (3) The pH value of the eluent has a significant impact on the elution efficiency. If water is used
  to ensure its pH value is between 7.0 and 8.5

## Chassis construction

### Colony PCR

We analyzed the Tm value of the designed identification primer using SnapGene and set the
annealing temperature, usually Tm-4 ℃. The elongation rate of Taq DNA polymerase is 5-10 sec/1
kb. The extension time of the PCR reaction depends on the length of the amplified target DNA
fragment.

Colony PCR can usually be used to verify positive clones, which is time-saving and convenient.
During the PCR reaction process, high-temperature denaturation causes the bacteria to undergo
thermal lysis, exposing the DNA to the reaction system, and the primers bind to the
complementary DNA single strand for replication. We designed upstream and downstream primers on
the target gene or plasmid.

The reaction system should be prepared on ice and reagents should be added in order of volume
from most to least. M5 Taq HiFi PCR Mix is used for colony PCR reactions. This DNA polymerase is
not a high-fidelity enzyme, but it reacts quickly and is commonly used for short fragment
amplification and for verification purpose.
Due to the fact that colony PCR reactions are usually conducted in large batches for screening,
the overall reaction system can be uniformly formulated, thoroughly mixed, and then evenly
distributed to 200 μL EP tubes, each reaction system is 20 μ L.

Use a sterile pipetman to pick up single colonies on the plate and dissolve them in 10 μ In L
sterile ddH2O, mix well before adding 2 μL bacterial culture to the reaction system.

Mark the PCR tube and store the remaining bacterial culture in a 4 ℃ refrigerator for a short
period of time. If a positive colony is successfully verified, the remaining bacterial solution
can be used for inoculation, purification, and Sanger sequencing to further verify the
construct.

The PCR program of M5 Taq DNA polymerase: After pre-denaturation, cycle from step 2
(denaturation) to step 4 (extension), typically lasting 25-30 cycles, to obtain sufficient PCR
products.

Table1.The reaction system and program of M5 Taq HiFi PCR Mix

| Composition | Volume（μL） |
| --- | --- |
| Template | Specific DNA |
| Upstream primer（10 μM） | 1 |
| Downstream primers（10 μM） | 1 |
| 2×M5 Taq HiFi PCR Mix 10 | 10 |
| ddH2O | 6 |
| Total volume | 20 |

Table 2.Colony PCR reaction procedure of M5 Taq DNA polymerase

| Program | temperature | time | cycle |
| --- | --- | --- | --- |
| Pre denaturation | 95℃ | 5 min |  |
| Denaturation | 95℃ | 30 sec | 30-60 |
| Annealing | Tm-2℃ | 15 sec | 30-60 |
| Extend | 72℃ | 5-10 sec/1 kb DNA | 30-60 |
| Post extension | 72℃ | 5 min |  |
| Insulation | 4℃ | ∞ |

---

### Homologous recombination

Homologous recombination is the use of homologous fragments to replace target genes and achieve
the goal of gene knockout or other genetic manipulations.

![](https://static.igem.wiki/teams/4616/wiki/wet-lab/shiyan2tu01.png)

**Figure 1.** Knockout and insertion sites of genes

Use PCR to amplify the upstream and downstream 500bp of the knockout gene as the homologous arm.
Connect the upstream and downstream sequences to the substitute gene through Gibson assembly,
transfer this gene into the yeast cell, and the cell replaces the knockout gene through the
principle of gene recombination. When designing primers, there is an overlap zone at the
connection point, which can only be assembled during the recombination.

![](https://static.igem.wiki/teams/4616/wiki/wet-lab/shiyan2tupian02.png)

**Figure 2.** Left and right arms and their insertion sites

Introducing genes into the genome can be done using similar principle. After selecting insertion
sites on the genome, PCR was used to remove the left and right arms of the insertion sites.
Then, the genes constructed on the left arm were connected to the right arm through Gibson
assembly, and an overlay region was added to the construct during primer design. The constructed
fragment is shown in the figure below. The target fragment is introduced into yeast and
undergoes homologous recombination under the action of cells, achieving the goal of inserting
the target gene.

![](https://static.igem.wiki/teams/4616/wiki/wet-lab/shiyan2tupian03.png)

**Figure 2.** Recombined gene fragments

---

### Determining growth curves

1.To plot the growth curve, first activate the strain.
Remove the corresponding frozen bacteria from the -80 ℃ ultra-low temperature refrigerator, or
select the positive transformants of plasmids from the transformation plate, and mark them on
the Malt Agar Medium. Incubate overnight at 30 ℃ for colony growth.

2. Pick single colonies from the plate and inoculate them into 5 mL LB culture medium. Shake and
cultivate overnight at 30 ℃ and 220 rpm in a shaker.Select 3 single bacterial colonies from each
type of bacteria and inoculate them with seed solution as parallel groups.

Table 3.Each type of bacteria

| Dilution ratio |
| --- |
| Gal4-Δ —— Gal4-Δ |
| --- |
| Gal4-Δ —— Gal4-Δ and Gal80-Δ |
| Gal4-Δ and Gal80-Δ —— Gal4-Δ |
| Gal4-Δ and Gal80-Δ —— Gal4-Δ and Gal80-Δ |
| Gal4-Δ and Gal80-Δ and SPE4-Δ —— Gal4-Δ |
| Gal4-Δ and Gal80-Δ and SPE4-Δ —— Gal4-Δ and Gal80-Δ |

3. Transfer to 200μL seed solution to a 50 mL conical flask containing 20 mL of sterile LB
culture medium (1:100 inoculation), and incubate in a shaker at 30 ℃ and 220 rpm.

4. Take a sample every 1 hour and use a UV spectrophotometer to measure the OD600 of the
bacterial solution. If the OD600 is too high, it is necessary to use ddH2O for dilution and
measurement to ensure the accuracy of the values.

## Strain construction

### Gibson Assembly

Selection of reagent kit: NovoRec from Novoprotein Company ® PlusOnestepPCRCloningKit (NR005)
Kit

Table 1. Product Composition of the Used Reagent Kits

| Components | NR005-01A(20rxns) | NR005-01B(96rxns) |
| --- | --- | --- |
| NovoRec® Plus Recombinase | 20μl | 100μl |
| 5×Reaction Buffer | 200μl | 1ml |
| NovoRec® pUC19 (Linearized, 50ng/μl) | 20μl | 20μl |
| 800bp PCR Fragments (Positive Control, 50ng/μl) | 20μl | 20μl |
| Novoprotein 2×SpecificTM Taq Master Mix | 1ml | 1ml×5 |

**1. Carrier preparation**

Select the appropriate site for cloning. Single enzyme digestion, double enzyme digestion, or
PCR amplification can be used, and the 5 'protruding end, 3' protruding end, or flat end are
all suitable for this Kit. Due to the poor degree of single enzyme tangency and to improve
the positive rate, we suggest that you use a dual enzyme cleaved vector. Final vector
concentration should be >15ng/μl. A high concentration of vector is beneficial for improving
efficiency.

**2. Primer design**

When using a one-step direct cloning kit, primer design is very important, and the
overall principle is to introduce homologous recombination sequences through the 5
'end of the primer. The amplified products, as well as between the amplified
products and the linearized cloning vector, have completely identical sequences that
can be homologous recombination with each other. Under the premise of following the
basic principles of primer design, only 15-20 bp vector homologous sequences need to
be added to the upstream and downstream primers. Please refer to the following
figure for details:

![](https://static.igem.wiki/teams/4616/wiki/wet-lab/shiyan3tu01.png)

**Figure 1.** The vector is digested to form flat ends

![](https://static.igem.wiki/teams/4616/wiki/wet-lab/shiyan3tu02.png)

Figure2.After the vector is digested, the 5 'end protrudes:

![](https://static.igem.wiki/teams/4616/wiki/wet-lab/shiyan3tu03.png)

Figure3.After the vector is digested, the 3 'end protrudes:

**Note:** The horizontal lines in Figures 1, 2, and 3 represent Homologous sequences and
clearance sites

**3. Acquisition of target fragments**

The target fragment is usually obtained through PCR. To ensure the
specificity and sensitivity of PCR amplification, use high fidelity enzymes
whenever possible. The length of each PCR primer should be at least 40-45bp,
including 15-20bp homologous to the vector at the 5 'end and 20-25bp
specific sequence of the target fragment (note: if constructing an
expression vector clone, please check if the reading frame is correct after
the primer design is completed).

**Note:**: If there are any heterobands after PCR amplification, they need to be
cut and recycled, otherwise the heterobands will affect the recombination
reaction.

**4. Recombination of target genes and vectors**

1) Add the linearized carrier and the target fragment in a certain
molar ratio to a centrifuge tube for recombination reaction on an
ice box.

Table 2. Novoprotein Fast Pfu DNA Polymerase Reaction
System

| Reagent | 2-3 fragment connection | 4-6 fragment connection |
| --- | --- | --- |
| Carrier dosage | X\* | X\* |
| Insert Fragment Dose | Y | Y |
| Molar ratio of carrier to inserted fragment | 1:2 | 1:1 |
| 5× Reaction buffer | 4μl | 4μl |
| NovoRec ® Plus Recombinase | 1μl | 1μl |
| ddH2O | Add to 20μL | Add to 20μL |
| Reaction time | 50℃，10min | 50℃，30-60min |

\* When the amount of carrier added is 100-200ng, high recombination
efficiency can be achieved.

\*\* It is recommended that the primer homologous arm length of each
fragment should be greater than 20 bp, and the molar ratio of each
fragment to the carrier should be 1:1. The reaction time can be extended
to 30 minutes, and the maximum should not exceed 1 hour.

Formula for calculating the dosage of each component:

1/2 (2-3 fragments) or 1/1 (4-6 fragments)=[Carrier mass X (ng)] × Target
fragment size (bp)/Carrier size (bp) × Target fragment quality Y
(ng).

2) After the reaction is completed, it can be directly transformed. If
not transformed, it needs to be stored at 4 ℃ or -20 ℃.

**5. Reaction product conversion and coating**

We suggest that the efficiency of the receptive cells used should
reach or exceed 1 × 107 cfu/μG. The conversion steps are as
follows:

(1) Melting a tube of 100 on ice μ DH5 of l α Sensory cells,
gently bouncing the tube wall causes the cells to resuspend.
Join 10 μ L's
The reaction solution was introduced into the receptive cells
and subjected to a light bounce count, followed by an ice bath
for 30 minutes.

(2) Heat shock in a 42 ℃ water bath for 90 seconds and quickly
place it on ice for 5 minutes.

We suggest that the efficiency of the receptive cells used should
reach or exceed 1 × 107cfu/μG. The conversion steps are as
follows:

(3) Join 500 μ Incubate in SOC or LB liquid medium at 37 ℃ for
45-60 minutes.

(4) Centrifuge at 5000rpm for 3 minutes to collect bacteria, and
evenly coat a certain amount of bacteria on the antibiotic
containing surface as needed
On the board.

**Note:** In order to verify the complete cleavage of
the carrier, it is recommended to create an empty vector as a
control while performing the transformation. When the verify is
well treated, there should be no clone growth in the control!

**6. Positive clone identificatio**

Generally, we recommend using colony PCR for positive
clone identification, and Novoprotein 2 is recommended ×
Specific Taq Master Mix (directory number E010 in
Novoprotein).

Selection of identification primers: To avoid false
positive results, we suggest that one primer be carrier
specific and the other primer be target fragment
specific.

---

### Preparation of *E. coli* electrocompetent cells

Preparation of receptive state: Pick the bacteria and
inoculate them in LB liquid medium. Shake and incubate
at 37 °C for about 12 hours until the later stage of
logarithmic growth. Inoculate the bacterial suspension
in a 1% ratio in a 50mLLB liquid medium, shake at 37C
for 2-3 hours until OD600=0.5, and prepare 0.1M CaCl2
(M=110.98) to weigh 0.555g 50ml

1. Transfer the culture medium into a centrifuge tube,
place it on ice for 10 minutes, and then centrifuge at
3000rpm at 4 ° C for 10 minutes

2. Discard the supernatant and gently suspend the cells
in 5ml of pre-cooled 0.05mol/L CaCl2 solution. After 30
minutes on ice, centrifuge at 3000rpm for 10
minutes.

3. Discard the supernatant and add 2mL of pre cooled
0.1mol/L CaCl2 solution (containing 15% glycerol, 10ml
aliquot, 8.5+1.5 killed glycerol). Gently suspend the
cells and let them stand on ice for a few minutes to
form a receptive cell suspension.

4. Place 100uL of competent cells in a refrigerator at
-80 ° C.

---

### Heat shock transformation of *E. coli* competent cells

1.Place the *E. coli* JM109 competent cells
extracted from the -80 ℃ ultra-low temperature
refrigerator on ice and slowly freeze thaw.

2. Add 10 ng plasmids to each receptive state, or 5-10 μ
L-linked products, or 100 ng PCR products,
Carefully blow and mix with a pipette, and let stand on
ice for 30 minutes.

3. Place the centrifuge tube in a 42 ℃ water bath and
heat shock for 90 seconds. After removal, immediately
place it on ice for 5 minutes.

4. Add 200 μ L sterile and non antibiotic LB liquid
culture medium, carefully mix well.

5. Incubate in a shaking table at 37 ℃ and 220 rpm for 60
minutes.

6. Take an appropriate amount of bacterial solution and
apply it to the LB plate containing the corresponding
antibiotics, and mark the date, name, strain, plasmid,
and other information on the plate. Then, invert and
cultivate overnight in a constant temperature incubator
at the corresponding cultivation temperature.

---

### Cytotoxicity test

**1. Concentration gradient setting:**

Accordingly , the final yields of sclareol and santalol in engineered yeast can reach 11.4g/L [1] and 68.8mg/L [2], respectively. Therefore, we ultimately set the concentration gradients as follows:

Table 3. Final concentration gradient setting| Santalol | 0mg/L | 50mg/L | 100mg/L | 200mg/L |
| --- | --- | --- | --- | --- |
| Sclareol | 0mg/L | 200mg/L | 350mg/L | 500mg/L |

**2. Selection of solvents:**

Because sclareol is solid at room temperature, we need to consider what solvent should be used to dissolve it. After reviewing the literature and consulting with our supervisor, we decided to use methanol as the solvent for the next step of the experiment.

**3. Preparation of solid medium:**

YPD solid medium (yeast extract 10g/L, glucose 20g/L, peptone 20g/L, agar powder 20g/L) was utilized for strain growth in the study. We have been exploring for a long time on how to accurately add different concentrations of sclareol and santalol into the culture medium. The final plan is as follows:

(1) methanol was used to dilute different amounts of santalol or sclareol to the same volume.

(2) Separately package different concentrations of santalol and sclareol into several small tubes, and then sterilize them through the sterilizing filters into the clean tubes in the clean bench.

(3) To eliminate the interference of other relevant factors and ensure the accuracy of the terpenoid concentrations, we controlled the volume of the solid medium in each plate to 20mL. After sterilization of the YPD solid medium, different concentrations of the sterile santalol and sclareol were quickly added after the culture medium was cooled to 40-50℃, and then the mixtures were poured it into the responding plates for standing.

(4) After the plate has cooled, we take a drop of yeast solution and place it on three different locations on the medium. After the solution has seeped into the plates, the plates were carefully placed in a 30℃ incubator for 48-hour cultivation.

---

## Testing of raw materials and their products

### FPP detection method

**Reagent:**

Farnesyl diphosphate triammonium salt, E; E-farnesol, ergosterol, a mixture of phosphatase
inhibitors (Cat. No. P5726, a mixture of sodium orthovanadate, sodium molybdate, and imidazole),
and alkaline phosphatase (Cat. No. P6774) were purchased from Sigma.

The bead cell breaker and 0.5-mm zirconia/silica beads are from Biospec Products.

The anion exchangers Macro Prep High Q and AG 1X-2 (100-200 mesh) in the form of chlorides and
disposable rotating columns come from Bio Rad. Use the Bio Rad Protein Assay kit to measure
proteins using IgG as the standard.

**Strain:**

Kali 571 (erg9D: HIS3) brewer's yeast is derived from SW23B # 74 by restoring nutrient
deficient forms. A stock solution of 10 mg/ml ergosterol was prepared in 1:1 (v/v) ethanol:
Triton X-100 (from Sigma).

Gas chromatography-mass spectrometry: Gas chromatography is performed on a Hewlett Packard
5890 chromatograph equipped with a 5970 mass selective detector. The column is Rtx-5 ms
(0.25mm i.d. \* 15m, 1um film thickness) from Restek. The detector and injector are both at
300 ° C. Set the oven to 85 ° C for 1 minute, then increase the temperature to 300 ° Cat 15
° C/minute.

**The conditions for converting FPP to E, E-farnesol:**

(1) The determination of FPP hydrolysis was carried out in a total volume of 0.1ml
containing 51 g FPP and 0.1M Bis Tris propane/HCl (pH 7) or 0.1M glycine/NaOH (pH 10.4).
The amount of alkaline phosphatase in each measurement ranges from 0.28 to 0.56
units.

(2) Include other components as needed. Incubate the test mixture at 37 ° C for 20
minutes and extract with 0.3ml hexane.
The hexane layer obtained after centrifugation was analyzed using GC-MS using an
external standard of E, E-farnesol.

**Preparation of cell-free extract:**

(1) Culture the mutant Cali 57-1 at 30 ° C in 50 ml YPDE medium (10 g/L yeast
extract, 20 g/L peptone, 20 g/L glucose, and 5 mg/L ergosterol) until the early
stable stage.

(2) Wild type strain ATCC 28383 was cultured in YPD without the addition of
ergosterol. Shake the flask and ferment the sample as described. Centrifuge the
cells and wash the precipitate twice with water.

(3) Then, the cells were suspended in a buffer solution (50 mM Bis Tris
propane/HCl, pH 7, or 0.1M 2-amino-2-methylpropanol/NaOH, pH 10.35) with
approximately 2 ml buffer solution/ml.

(4) In some experiments, reserve solutions of Triton X100 (5% w/v), MgCl2 (1 M),
and a mixture of phosphatase inhibitors were added to the final concentrations
of 0.025%, 15 mM, and 1% (v/v), respectively.

(5) Add zirconia silica beads (0.5mm) to 50% of the total volume. Homogenize
cells using a cell bead crusher at 4 ° C (stirring 5 times for 1 minute, cooling
for 2 minutes between stirring).

(6) Centrifuge the homogenate at 10000 g for 1 minute, and centrifuge the
supernatant at 100000 g for 1 hour. Designate 100000 g of supernatant as a
cell-free extract.

**Separate FPP:**

(1) Add a suspension of anion exchanger Macro Prep High Q (in chloride form)
to a disposable rotating column to obtain a column bed volume of 0.25ml.
Initial pressure is required to start flow, and subsequent flow is driven by
gravity. Firstly, wash the column with water (2 \* 0.5 ml), then wash with 50
mM Bis Tris propane/HCl, pH 7 (3 \* 0.5 ml).

(2) Slowly apply cell-free extract (up to 0.8ml) or buffer containing FPP to
the column at a rate of 0.5ml or less. The column was washed with 50 mM Bis
Tris propane/HCl+0.1M NaCl, pH 7 (2 \* 0.5 ml), methanol (4 \* 0.5 ml), and 50
mM glycine/NaOH+40% (v/v) methanol, pH 10.4 (0.5ml). Wash with 0.1M
glycine/NaOH+1 M NaCl+40% (v/v) methanol at pH 10.4 (2 \* 0.25 ml).

**Convert FPP to farnesol:**

Use a glass vial with a PTFE lined lid for hexane extraction. Add the
stock solutions of MaCl2 and ZnCl2 (both 0.1M) to the 0.5ml sample
obtained above, to their respective final concentrations of 1 mM, and
then add 200 units of alkaline phosphatase. Incubate the mixture at 37 °
C for 90 minutes and extract with 0.2ml hexane (vortex for 1 minute).
After centrifugation, analyze the hexane layer by GC-MS to determine E;
Use external standard E-farnesol. The recovered farnesol is expressed as
nmol/mg protein.

---

### Sclareol

**GC-MS analysis:**

Sclareol were analyzed on an SHIMADZU QP2010 Ultra GC-MS system
operating in electron ionization selected ionmonitoring mode.
Samples were analyzed on a RTX-5MS-silicacolumn (SHIMADZU
Corporation)(30 m long, 250 μm internal diame-ter, 0.25 μm film
thickness).

The injector was operated in pulsed splitless mode, with the injector
temperature maintained at 250°C. Scan range: m/z 40-400. The dwell
time was 50 msec. The oven program comprised 150°C for 5 min, ramps
of 10℃ min-1 to 250℃, for 10min.

**Preparation of standard curve:**

Take 5mg of Sclareol (provided by Innochem Co., Ltd.), dilute it
with methanol to a concentration of 50 mg/mL, and then further
dilute it to obtain gradient standard solutions of 20, 25, 30,
40, 50 mg/L. Quantitative analysis will be conducted using
GC-MS.

---

### Santalol

**GC-MS analysis:**

Santalol were analyzed on an SHIMADZU QP2010 Ultra GC-MS
system operating in electron ionization selected
ionmonitoring mode. Samples were analyzed on a
RTX-5MS-silicacolumn (SHIMADZU Corporation)(30 m long, 250
μm internal diame-ter, 0.25 μm film thickness).

The injector was operated in pulsedsplitless mode, with the
injector temperature maintained at 250°C. Scan range: m/z
40-400. The dwell time was 50 msec. The oven program
comprised 40°C for 3 min, ramps of 10℃ min-1 to 130℃, 3℃
min-1 to 180℃, for 1min.

Take 100μL of Sclareol (10mM in DMSO) (provided by Innochem
Co., Ltd.), dilute it with methanol to a concentration of 50
mg/mL, and then further dilute it to obtain gradient
standard solutions of 10, 20, 25, 30, 40, 50 mg/L.
Quantitative analysis will be conducted using GC-MS.

## Reference

1. [1] Xuan Cao, Wei Yu, Yu Chen, Shan Yang, Zongbao K. Zhao, Jens Nielsen, Hongwei Luan, Yongjin J. Zhou,Engineering yeast for high-level production of diterpenoid sclareol,Metabolic Engineering,Volume 75,2023,Pages 19-28,ISSN 1096-7176.
2. [2] Wang, Y., Gong, X., Li, F. et al. Optimized biosynthesis of santalenes and santalols in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 105, 8795–8804 (2021).

{% endblock %}