iGEM 2024 — Team bgi-mammothedu-east

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}We officially formed our team in the spring of 2023 and participated in online courses from April to
June to gain a
solid understanding of synthetic biology and iGEM. During this period, we conducted brainstorming sessions to define our
project. In June, we completed training in laboratory safety, fundamental experimental techniques, and laboratory
equipment usage, which enabled us to begin over two months of intensive hands-on experimentation. Throughout this
journey, we encountered various challenges and made efforts to optimize experimental procedures to enhance our success
rate. Additionally, our project incorporated a diverse range of human practice activities, facilitating continuous
refinement and adjustment based on feedback and dialogue.{% endblock %}
{% block page\_content %}

Wet lab attendance record

![](https://static.igem.wiki/teams/5217/image/notebook/image1.png)

Online meeting attendance record

![](https://static.igem.wiki/teams/5217/image/notebook/image2.png)

# Lab Notebooks

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{%
endblock %}
{% block page\_left\_content %}

* [**Overview**](#1)
* [**Engineering Cycle 1:
    Validating Our System**](#2)
  + [Proof of Concept with Ocimene](#2.1)
  + [Design](#2.2)
  + [Build](#2.3)
  + [Test](#2.4)
  + [Learn](#2.5)
* [**Engineering Cycle 2:
    Expanding Our Terpene Library**](#3)
  + [Construction for producing Cineole..](#3.1)
  + [Design](#3.2)
  + [Build](#3.3)
  + [Test](#3.4)
  + [Learn](#3.5)
* [**Engineering Cycle 3:
    Conditional Release test**](#4)
  + [Regulated synthesis of aromatic...](#4.1)
  + [Design](#4.2)
  + [Build](#4.3)
  + [Test](#4.4)
  + [Learn](#4.5)
* [**Reference**](#5)

{% endblock %}
{% block page\_content %}

# Overview

As part of our project Scentinel, we aim to produce seven valuable terpenes: limonene, ocimene, cineole, borneol,
myrcene, nerolidol, and nerol. To achieve this goal, we successfully constructed 19 plasmids and transformed them into
Saccharomyces cerevisiae chassis strain. This strategic approach enabled us to engineer yeast for terpene
biosynthesis.
Ultimately, we achieved the successful production of four terpenoids: ocimene, cineole, nerolidol, and nerol.
Additionally, we explored the conditional release of these aromatic compounds in yeast.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image1.png)

Figure
1: Engineered pathway for the
production of terpenes in Saccharomyces cerevisiae

![](https://static.igem.wiki/teams/5217/image/engineering-success/image2.png)

Figure 2: Summary of experimental
results

Synthetic biology applies engineering design principles to modify or recreate organisms for specific functions,
typically following an iterative Design-Build-Test-Learn (DBTL) cycle. In accordance with this methodology, we
undertook
three cycles of genetic editing on Saccharomyces cerevisiae aimed at achieving heterologous expression and conditional
release of various terpenoids.

In the initial phase of our study, we investigated the mevalonate (MVA) pathway in Saccharomyces cerevisiae for the
production of terpene products, thereby confirming the viability of this metabolic route for biotechnological
applications. During this phase, we also identified the critical influence of promoter and terminator selection on
fermentation yield, which underscored the importance of regulatory elements in optimizing metabolic processes. This
foundational work established a robust basis for further research. In the subsequent phase, we directed our efforts
towards synthesizing a variety of functional terpenoid compounds to address product demands, with the objective of
enhancing both the yield and diversity of these compounds for industrial use. Finally, in the third phase, we explored
conditional expression strategies by employing the GAL1 promoter to tightly regulate the production of target
terpenes.
Through this iterative process, we aimed to refine our engineering strategies, ultimately leading to a more efficient
and controlled biosynthesis of terpenoids in S. cerevisiae.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image3.png)

Figure 3: Experimental operation
procedure

![](https://static.igem.wiki/teams/5217/image/engineering-success/image4.png)

Figure 4: Engineering cycle diagram
of our project (D=Design, B=Build, T=Test, L=Learn)

# Engineering Cycle 1: Validating Our System

#### Proof of Concept with Ocimene

#### Design

Our goal is to successfully produce terpenes in yeast and to verify the pathway feasibility. Due to the fast growth
rate
of E.coli and the ease of molecular cloning, we decide to construct and verify plasmids in E.coli. The gene CcOCS
encoding ocimene synthase from C.camphora were synthesized by Gene Synthesis Platform according to the codon bias
of
S. cerevisiae and ligated into the plasmid, we then assembled it into vector HcKan-O by golden gate cloning, and then
constructed with promoters (pTHD3, pGPM1, pINO1) and terminators (ADH1t, PGK1t, TEF1t) into shuttle plasmid vector.
Heterologous expression of CcOCS with different promoters and terminators in S. cerevisiae.

#### Snapgene diagrams of ocimene synthase gene: CcOCS.

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa1.jpg)

HcKan-O Vector

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa2.jpg)

HcKan-O-CcOCS

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa3.jpg)

pINO1

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa4.jpg)

pGPM1

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa5.jpg)

​
pTDH3

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa6.jpg)

ADH1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa7.jpg)

PGK1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa8.jpg)

TEF1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa9.jpg)

POT2\_pINO1 CcOCS\_TEF1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa10.jpg)

POT2\_pTDH3\_CcOCS\_ADH1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa11.jpg)

​
POT vector

![](https://static.igem.wiki/teams/5217/image/engineering-success/aaa12.jpg)

POT2\_pGPM1\_CcOCS\_PGK1t

### Build

We have completed the construction and validation of the final plasmid through three main processes.

* Plasmid Extraction.
* The TPS gene was cloned into the HcKan-O vector. （Colony PCR, Enzymatic Digestion, Gene
  sequencing）
* Assembled the TPS gene with different promoters and terminators into shuttle vectors. （Colony PCR, Enzymatic
  Digestion, gene sequencing）

![](https://static.igem.wiki/teams/5217/image/gel/hkccocs.png)

Figure 5: Colony PCR results
performed to screen strain HcKan-O-CcOCS

![](https://static.igem.wiki/teams/5217/image/gel/pot-cc.png)

Figure 6: Colony PCR results
performed to screen strain. A: POT2\_pGPM1\_CcOCS\_PGK1t, B: POT2\_pINO1\_CcOCS\_TEF1t, C:
POT2\_pTDH3\_CcOCS\_ADH1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image19.png)

Figure 7: Single colonies of
plasmid
transformants on LB Ampicillin plate

![](https://static.igem.wiki/teams/5217/image/engineering-success/image20.png)

Strain 2 and 16 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image21.png)

Strain 4 and 7 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image22.png)

Strain 6 and 8 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image23.png)

Strain 1 and 5 have been confirmed
positive by sequencing

### Test

#### The colony PCR Gel Electrophoresis Results of CcOCS

We introduced the constructed plasmids into Saccharomyces cerevisiae through transformation and verify successful
transformation by yeast colony PCR. The results showed that we successfully transformed plasmids
POT2\_pINO1\_CcOCS\_TEF1t
and POT2\_pTDH3\_CcOCS\_ADH1t into the yeast, while the verification for the POT2\_pGPM1\_CcOCS\_PGK1t plasmid failed.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image24.png)

Figure 8: Yeast colony PCR result
performed to screen strain A: BY4741-POT2\_pGPM1\_CcOCS\_PGK1t, B:
BY4741-POT2\_pINO1\_CcOCS\_TEF1t, C: BY4741-POT2\_pTDH3\_CcOCS\_ADH1t（M=DL5000 marker；P=Positive; N=Negative）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image25.png)

Figure 9: Yeast single colonies of
plasmid transformants on SC-URA plate

### GC-MS analysis of ocimene :

To evaluate ocimene production in S. cerevisiae cells, shake flask fermentation was performed. After 12 hours of
fermentation, a dodecane overlay culture was added, and the fermentation continued for a total of 120 hours. The
ocimene
titer reached 0.83 ± 0.06 mg/L when using the pINO1 promoter and TEF1t terminator, whereas it was 0.33 ± 0.09 mg/L
with
the pTDH3 promoter and ADH1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot-cc-gc.png)

Figure 10: Heterologous expression
of CcOCS in S. cerevisiae. A: GC-MS analyses of ocimene extracts of S. cerevisiae
expressing CcOCS with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of ocimene extracts of S. cerevisiae
expressing CcOCS with the pINO1 promoter and TEF1t terminator. C: the OD600 and ocimene yield by CcOCS under the
control
of different promoters and terminators. The data represent the mean ± standard deviation of three independent
experiments.

### Learn

The successful production of ocimene demonstrates the feasibility of the MVA pathway in Saccharomyces cerevisiae. The
production of ocimene varied significantly under the control of different promoters and terminators, despite no
notable
differences in OD600 values. In our human practice activities, we focused on synthesizing various terpenes to assist
visually impaired women by alerting them to the onset of menstruation and potential leakage. Based on our literature
research findings, we planed to utilize synthetic biology to produce a diverse range of terpenes.

# Engineering Cycle 2: Expanding Our Terpene Library

### Construction for producing Cineole, Nerol, Nerolidol etc.

### Design

Based on product characteristics and literature research, we synthesized terpene synthase genes for three compounds:
cineole, nerolidol, and nerolidol. The genes AcNES1 (nerolidol synthase from Actinidia chinensis), PgfB (nerol
synthase
from Penicillium griseofulvum), and HpyCins, StrCins, and SfCins (cineol synthases from Hypoxylon, Streptomyces
clavuligerus, and Salvia fruticosa) were synthesized by Gene Synthesis Platform to match the codon bias of S.
cerevisiae
and then ligated into the plasmid.

Building on the first cycle, we constructed shuttle plasmids with various combinations of promoters and terminators
using Golden Gate assembly. After transformed Saccharomyces cerevisiae and performed yeast colony PCR, we typically
employed two-phase fermentation system for terpenoid production. We then test terpenoid production using gas
chromatography–mass spectrometry.

### Snapgene diagrams of three Cineole synthase genes: HpyCins, StrCins, SfCins.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image27.png)

HcKan-O-HpyCins

![](https://static.igem.wiki/teams/5217/image/engineering-success/image28.png)

HcKan-O-StrCins

![](https://static.igem.wiki/teams/5217/image/engineering-success/image29.png)

HcKan-O-SfCins

![](https://static.igem.wiki/teams/5217/image/engineering-success/image30.png)

POT2\_pGPM1 StrCins\_PGK1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image31.png)

​
POT2\_pTDH3 HpyCins\_ADH1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image32.png)

POT2\_pINO1 HpyCins\_TEF1t

### Snapgene diagrams of nerol synthase gene: PgfB.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image33.png)

HcKan-O-PgfB

![](https://static.igem.wiki/teams/5217/image/engineering-success/image34.png)

POT2\_pGPM1\_PgfB\_PGK1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image35.png)

POT2\_pTDH3\_PgfB\_ADH1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image36.png)

POT2\_pINO1\_PgfB\_TEF1

### Snapgene diagrams of nerolidol synthase gene: AcNES1.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image37.png)

HcKan-O-AcNES1

![](https://static.igem.wiki/teams/5217/image/engineering-success/image38.png)

POT2\_pGPM1\_AcNES1\_PGK1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image39.png)

POT2\_pTDH3\_AcNES1\_ADH1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image40.png)

POT2\_pINO1\_AcNES1\_TEF1t

### Build

New primers were ordered, and then we have completed the construction and validation of the final plasmid through the
same method as cycle 1. We have successfully constructed 9 shuttle plasmids, which are

POT2\_pTDH3\_HpyCins\_ADH1t, POT2\_pINO1\_HpyCins\_TEF1t, POT2\_pGPM1\_StrCins\_PGK1t,

POT2\_pINO1\_PgfB\_TEF1t, POT2\_pGPM1\_PgfB\_PGK1t, POT2\_pTDH3\_PgfB\_ADH1t,

POT2\_pINO1\_AcNES1\_TEF1t, POT2\_pGPM1\_AcNES1\_PGK1t, POT2\_pTDH3\_AcNES1\_ADH1t.

#### The colony PCR Gel Electrophoresis Results of HpyCins, StrCins and SfCins:

![](https://static.igem.wiki/teams/5217/image/engineering-success/hk-cineole.png)

Figure 11: DH5α E.coli colony PCR
results of HpyCins, StrCins and SfCins A: HcKan-O-SfCins B: HcKan-O-StrCins C:
HcKan-O-HpyCins（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot-cineole.png)

Figure 12: DH5α E.coli colony PCR
results of HpyCins, StrCins and SfCins A: POT2\_pTDH3\_HpyCins\_ADH1t (M=DL5000 marker)
B: POT2\_pINO1\_HpyCins\_TEF1t (M=DL5000 marker) C: POT2\_pGPM1\_StrCins\_PGK1t（M=DL2000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image43.png)

Figure 13: Enzymatic digestion
result of StrCins

### The sequencing result:

![](https://static.igem.wiki/teams/5217/image/engineering-success/image44.png)

Strain 10 and 13 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image45.png)

Strain 2 and 3 have been confirmed
incorrect by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image46.png)

Strain 6 and 8 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image47.png)

Strain 3 and 4 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image48.png)

​
Strain 3 and 10 have been confirmed positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image49.png)

​
Strain 1 and 3 have been confirmed positive by sequencing

#### The colony PCR Gel Electrophoresis Results of PgfB

![](https://static.igem.wiki/teams/5217/image/gel/gel-pgfb-heng.png)

Figure 14: DH5α E.coli colony PCR
results of PgfB A: HcKan-O-PgfB, B: POT2\_pINO1\_PgfB\_TEF1t, C: POT2\_pGPM1\_PgfB\_PGK1t,
D: POT2\_pTDH3\_PgfB\_ADH1t（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image51.png)

Figure 15: Enzymatic digestion
result
of PgfB

### The sequencing result:

![](https://static.igem.wiki/teams/5217/image/engineering-success/image52.png)

Strain 2 and 3 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image53.png)

Strain 8 and 9 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image54.png)

Strain 1 and 2 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image55.png)

Strain 1 and 2 have been confirmed
positive by sequencing

#### The colony PCR Gel Electrophoresis Results of AcNES1

![](https://static.igem.wiki/teams/5217/image/gel/gel-ac.png)

Figure 16: DH5α E.coli colony PCR
results of AcNES1 A: HcKan-O-AcNES1. B: POT2\_pINO1\_AcNES1\_TEF1t,
POT2\_pGPM1\_AcNES1\_PGK1t, POT2\_pTDH3\_AcNES1\_ADH1t. C: Enzymatic digestion result of AcNES1. （M=DL5000 marker）

### The sequencing result:

![](https://static.igem.wiki/teams/5217/image/engineering-success/image57.png)

Strain 4 and 8 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot2-pgpm1-acnes1-pgk1t.png)

Strain 3 and 10 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image59.png)

Strain 2 and 4 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot2-ptdh3-acnes1-adh1t.png)

Strain 1 has been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image61.png)

Figure 17: Single colonies of
plasmid
transformants on LB agar plates containing ampicillin or kanamycin

### Test

#### The yeast colony PCR Gel Electrophoresis Results of HpyCins, PgfB and AcNES1:

Yeast colony PCR results confirmed that seven plasmids—POT2\_pTDH3\_HpyCins\_ADH1t,

POT2\_pINO1\_HpyCins\_TEF1t, POT2\_pINO1\_PgfB\_TEF1t, POT2\_pGPM1\_PgfB\_PGK1t,

POT2\_pTDH3\_PgfB\_ADH1t, POT2\_pGPM1\_AcNES1\_PGK1t, and POT2\_pTDH3\_AcNES1\_ADH1t—were positive in Saccharomyces cerevisiae.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image62.png)

Figure 18: Yeast colony PCR result
of
cineole performed to screen strain.（M=DL5000 marker；P=Positive; N=Negative）
HcKan-O-HpyCins（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image63.png)

Figure 19: Yeast colony PCR result
of
nerol performed to screen strain. A: BY4741-POT2\_pGPM1\_PgfB\_PGK1t. B:
BY4741-POT2\_pINO1\_PgfB\_TEF1t. C: BY4741-POT2\_pTDH3\_PgfB\_ADH1t.（M=DL5000 marker；P=Positive; N=Negative）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image64.png)

Figure 20: Yeast colony PCR result
of
nerolidol performed to screen strain. A: BY4741-POT2\_pGPM1\_AcNES1\_PGK1t. B:
BY4741-POT2\_pTDH3\_AcNES1\_ADH1t. C: BY4741-POT2\_pINO1\_AcNES1\_TEF1t.（M=DL5000 marker；P=Positive; N=Negative）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image65.png)

Figure 21: Yeast single colonies
of
plasmid transformants on SC-URA plate

### GC-MS analysis of cineole:

Flask fermentation of HpyCins yielded 11.88 ± 2.03 mg/l of ocimene with the pINO1 promoter and TEF1t terminator,
compared to 17.58 ± 3.20 mg/l with the pTDH3 promoter and ADH1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/gc-cineole.png)

Figure 22: Heterologous expression
of HpyCins in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae
expressing HpyCins with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of cineole extracts of S.
cerevisiae
expressing HpyCins with the pINO1 promoter and TEF1t terminator. C: the OD600 and cineole yield by HpyCins under
the
control of different promoters and terminators. The data represent the mean ± standard deviation of three
independent
experiments.

### GC-MS analysis of nerol:

Flask fermentation of PgfB yielded 6.07 ± 0.23 mg/l of nerol with the pTDH3 promoter and ADH1t terminator, but no
terpenes are produced with pINO1 and GPM1 promoter.

![](https://static.igem.wiki/teams/5217/image/engineering-success/gc-nerol.png)

Figure 23: Heterologous expression
of PgfB in S. cerevisiae. A: GC-MS analyses of nerol extracts of S. cerevisiae
expressing PgfB with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerol extracts of S. cerevisiae
expressing PgfB with the pINO1 promoter and TEF1t terminator. C: GC-MS analyses of nerol extracts of S. cerevisiae
expressing PgfB with the pGPM1 promoter and PGK1t terminator. D: The OD600 and nerol yield by PgfB under the
control of
different promoters and terminators. The data represent the mean ± standard deviation of three independent
experiments.

### GC-MS analysis of nerolidol:

Flask fermentation of AcNES1 yielded 45.06 ± 1.68 mg/l of nerolidol with the pTDH3 promoter and ADH1t terminator,
compared to 12.92 ± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image68.png)

Figure 24: Heterologous expression
of AcNES1 in S. cerevisiae. A: GC-MS analyses of nerolidol extracts of S. cerevisiae
expressing AcNES1 with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerolidol extracts of S.
cerevisiae
expressing AcNES1 with the pGPM1 promoter and PGK1t terminator. C: The OD600 and nerolidol yield by AcNES1 under
the
control of different promoters and terminators. The data represent the mean ± standard deviation of three
independent
experiments.

### Learn

The production of cineole varied significantly between different promoters and terminators. Flask fermentation using
the
pINO1 promoter and TEF1t terminator yielded 11.88 ± 2.03 mg/l of ocimene, while the pTDH3 promoter and ADH1t
terminator
produced 17.58 ± 3.20 mg/l, despite both constructs having similar OD600 values around 4.4. Similarly, flask
fermentation with the pTDH3 promoter and ADH1t terminator resulted in 45.06 ± 1.68 mg/l of nerolidol, compared to
12.92
± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator, even though OD600 values for both constructs were similar at
approximately 5. The results highlight the importance of the promoter in heterologous expression.

Although we are able to produce the scent molecules, we have not yet fully achieved the primary goal of our project:
to
provide reminders through the conditional release of sanitary napkins or laundry detergent and to make our product
accessible to visually impaired women. Part of this limitation arises from safety restrictions imposed by the
competition, but more significantly, we currently lack a viable implementation method. Our research confirms that
scented sanitary napkin products are available on the market; however, there are no studies on the conditional release
of fragrance in sanitary napkin products. Therefore, we have decided to focus on the conditional release of terpenes
to
explore the feasibility of this approach.

# Engineering Cycle 3: Conditional Release test

#### Regulated synthesis of aromatic compounds by inducible promoters

### Design

From our previous two experimental cycles, we have successfully engineered yeast to produce scents. Our next main
focus
will be to build a conditional release test for terpenes aimed at assisting visually impaired women. To achieve this,
we
will explore the use of regulatory promoters for the conditional release of terpenoids through synthetic biology. We
have preliminarily selected two genes for investigation: AcNES1 (nerolidol synthase from Actinidia chinensis) and PgfB
(nerol synthase from Penicillium griseofulvum). We will examine strategies for achieving conditional release from a
synthetic biology perspective. The pGAL1 promoter is known for its strength in driving high levels of enzyme
expression,
which facilitates efficient production of the desired product. Therefore, we plan to utilize the pGAL1 promoter for
plasmid construction, enabling the conditional release of terpenes from Saccharomyces cerevisiae.

#### Snapgene diagrams of three Cineole synthase genes: PgfB and AcNES1.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image69.png)

POT2\_pGAL1\_PgfB\_PGK1t

![](https://static.igem.wiki/teams/5217/image/engineering-success/image70.png)

POT2\_pGAL1\_AcNES1\_PGK1t

### Build

We have completed the construction and validation of the final plasmid through the same method as cycle 1.

We have successfully constructed two shuttle plasmids, which were POT2\_pGAL1\_PgfB\_PGK1t, POT2\_pGAL1\_AcNES1\_PGK1t.

#### The colony PCR Gel Electrophoresis Results of PgfB and AcNES1:

![](https://static.igem.wiki/teams/5217/image/engineering-success/gal-gel.png)

Figure 25: DH5α E.coli colony PCR
results of PgfB and AcNES1 A: POT2\_pGAL1\_PgfB\_PGK1t. B: POT2\_pGAL1\_AcNES1\_PGK1t. C:
Enzymatic digestion result of PgfB and AcNES1. （M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image72.png)

Figure 26: Single colonies of plasmid
transformants on LB ampicillin plate

### The sequencing result:

![](https://static.igem.wiki/teams/5217/image/engineering-success/image73.png)

Strain 2 and 6 have been confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/image74.png)

Strain 1 and 2 have been confirmed
positive by sequencing

### Test

#### The colony PCR gel electrophoresis Results of PgfB and AcNES1:

Yeast colony PCR results confirmed that two plasmids—BY4741-POT2\_pGAL1\_AcNES1\_PGK1t and
BY4741-POT2\_pGAL1\_PgfB\_PGK1t—were positive in Saccharomyces cerevisiae. We then selected single colonies for
fermentation. In contrast to the previous two cycles, we employed a galactose-inducible promoter and chose SC-URA
medium with both glucose and galactose as carbon sources for the control fermentation culture.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image75.png)

Figure 27: Yeast colony PCR results
of
nerolidol and nerol performed to screen strain. A:
BY4741-POT2\_pGAL1\_AcNES1\_PGK1t. B: BY4741-POT2\_pGAL1\_PgfB\_PGK1t. （M=DL5000 marker；P=Positive; N=Negative）

![](https://static.igem.wiki/teams/5217/image/engineering-success/image76.png)

Figure 28: Yeast single colonies of
plasmid transformants on SC-URA plate

#### GC-MS analysis of nerol and nerolidol:

Flask fermentation of AcNES1 with the pGAL1 promoter and PGK1t terminator yielded 4.78 ± 0.88 mg/l of nerolidol, while
no nerol production was detected.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image77.png)

Figure 29: Heterologous expression
of
AcNES1 in S. cerevisiae A: GC results of AcNES1 with glucose under the control of
the pGAL1 promoter and PGK1t terminator. B: GC results of AcNES1 with galactose under the control of the pGAL1
promoter
and PGK1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/image78.png)

Figure 30: Heterologous expression
of
PgfB in S. cerevisiae. A: GC results of PgfB with glucose under the control of the
pGAL1 promoter and PGK1t terminator. B: GC results of PgfB with galactose under the control of the pGAL1 promoter
and
PGK1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/gal-gc.png)

Figure 31: The OD600 and terpenes
production by AcNES1 and PgfB under the control of pGAL1 promoter and PGK1t terminator

### Learn

In detecting nerol from the PgfB gene with various combinations, only the pTDH3 and ADH1t combination yielded 6.07 ±
0.23 mg/l, while no terpenes were produced with the other combinations. GC-MS analysis revealed that the impurity peak
around 8.2 minutes was identified as a solvent peak (dodecane), whereas the retention time for nerol was 8.4 minutes.
Thus, the fermentation results from the three plasmids are likely influenced by the solvent peak. Future experiments
will consider using n-hexane or ethyl acetate as extraction agents for re-evaluation.

In our comparison of nerolidol yields from the AcNES1 gene using various promoter and terminator combinations, we
observed that galactose induction increased the yield 12-fold compared to glucose. However, this yield remained lower
than the yield achieved by the constitutive promoter (45.1 mg/l). These results indicate that our approach to
conditional release has shown initial success.

# Reference

Ignea, C.; Cvetkovic, I.; Loupassaki, S.; Kefalas, P.; Johnson, C. B.; Kampranis, S. C.; Makris, A. M. Improving Yeast Strains Using Recyclable Integration Cassettes, for the Production of Plant Terpenoids. Microb. Cell Factories 2011, 10 (1), 4. https://doi.org/10.1186/1475-2859-10-4.

Shaw, J. J.; Berbasova, T.; Sasaki, T.; Jefferson-George, K.; Spakowicz, D. J.; Dunican, B. F.; Portero, C. E.; Narváez-Trujillo, A.; Strobel, S. A. Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon Sp. with Specificity Determinants in Common with the Plant Synthases. J. Biol. Chem. 2015, 290 (13), 8511–8526. https://doi.org/10.1074/jbc.M114.636159.

Sun, J.; Wang, X.; Yu, K.; Zang, Y.; Qu, Z.; Wei, C.; Yuan, W. Expression of the Human Antiapoptotic Protein Bcl-2 Increases Nerolidol Production in Engineered Yeast. Process Biochem. 2022, 119, 90–95. https://doi.org/10.1016/j.procbio.2022.05.009.

Ma, R.; Su, P.; Guo, J.; Jin, B.; Ma, Q.; Haiyan, Z.; Chen, L.; Mao, L.; Tian, M.; Lai, C.; Tang, J.; Cui, G.; Huang, L. Bornyl Diphosphate Synthase From Cinnamomum Burmanni and Its Application for (+)-Borneol Biosynthesis in Yeast. Front. Bioeng. Biotechnol. 2021, 9, 631863. https://doi.org/10.3389/fbioe.2021.631863.

Cheng, S.; Liu, X.; Jiang, G.; Wu, J.; Zhang, J.; Lei, D.; Yuan, Y.-J.; Qiao, J.; Zhao, G.-R. Orthogonal Engineering of Biosynthetic Pathway for Efficient Production of Limonene in Saccharomyces Cerevisiae. ACS Synth. Biol. 2019, 8 (5), 968–975. https://doi.org/10.1021/acssynbio.9b00135

Zeng, W.; Jiang, Y.; Shan, X.; Zhou, J. Engineering Saccharomyces Cerevisiae for Synthesis of β-Myrcene and (E)-β-Ocimene. 3 Biotech 2023, 13 (12), 384. https://doi.org/10.1007/s13205-023-03818-2.

Guo, Y.; Dong, J.; Zhou, T.; Auxillos, J.; Li, T.; Zhang, W.; Wang, L.; Shen, Y.; Luo, Y.; Zheng, Y.; Lin, J.; Chen, G.-Q.; Wu, Q.; Cai, Y.; Dai, J. YeastFab: The Design and Construction of Standard Biological Parts for Metabolic Engineering in Saccharomyces Cerevisiae. Nucleic Acids Res. 2015, 43 (13), e88–e88. https://doi.org/10.1093/nar/gkv464.

Tang, H.; Wu, Y.; Deng, J.; Chen, N.; Zheng, Z.; Wei, Y.; Luo, X.; Keasling, J. D. Promoter Architecture and Promoter Engineering in Saccharomyces Cerevisiae. Metabolites 2020, 10 (8), 320. https://doi.org/10.3390/metabo10080320.

Tetali, S. D. Terpenes and Isoprenoids: A Wealth of Compounds for Global Use. Planta 2019, 249 (1), 1–8. https://doi.org/10.1007/s00425-018-3056-x.

McGinty, D.; Letizia, C. S.; Api, A. M. Addendum to Fragrance Material Review on Nerolidol (Isomer Unspecified). FOOD Chem. Toxicol. 2010, 48, S43–S45. https://doi.org/10.1016/j.fct.2009.11.008.

Mo, X.; Cai, X.; Hui, Q.; Sun, H.; Yu, R.; Bu, R.; Yan, B.; Ou, Q.; Li, Q.; He, S.; Jiang, C. Whole Genome Sequencing and Metabolomics Analyses Reveal the Biosynthesis of Nerol in a Multi-Stress-Tolerant Meyerozyma Guilliermondii GXDK6. Microb. CELL FACTORIES 2021, 20 (1), 4. https://doi.org/10.1186/s12934-020-01490-2.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_left\_content %}

* [Common laboratory ratio](#1)
* [Preparation of Agarose Gel and DNA Electrophoresis](#2)
* [Reagents and media configurations need to be sterilized](#3)
* [Plasmid Extraction method](#4)
* [Golden-gate assembly and bacterial transformation](#5)
* [Verfication Operation](#6)
* [Yeast Experiment Operation Procedure](#7)
* [Fermentation and detection](#8)

{% endblock %}
{% block page\_content %}

## 1. Common laboratory ratio

1.1 Solution inoculum volume (v / v): 1%

1.2 Antibiotic concentration (v / v): 1‰

1.3 Dye ratio: 0.1‰

## 2. Preparation of Agarose Gel and DNA Electrophoresis

2.1 Agarose gel electrophoresis, to prepare 1% gel.

2.1.1 Measure 1g of agarose, mix it with 100mL of TAE.

2.1.2 Microwave for 1-3 minutes until agarose dissolves.

2.1.3 Pour the mixture into a gel tray with well comb in place.

2.1.4 Let the mixture cool down for 10-15 minutes.

2.1.5 Once solidifyed place the agarose gel into a gel box.

2.1.6 Load samples and marker into the wells of the gel.

2.1.7 Put the gel into the electropheoric chamber.

2.1.8 Start the electropheoric chamber for 20 minutes, 140V.

2.1.9 Remove the gel afterwards and place it in an UV light source.

2.1.10 Compare the sample markings to the marker.

## 3. Reagents and media configurations need to be sterilized at 121℃ for 20 min

3.1 Commonly used reagent configuration

3.1.1 Reagents used for the yeast validation experiments

| Materials | Dosage |
| --- | --- |
| 1M Sodium Hydroxide | 4 g+100 mL Purified water |

3.1.2 Antibiotic preparation (Only filtration sterilization is employed in this process)

| Materials | Dosage |
| --- | --- |
| Carbenicillin（50 mg/ml） | 1 g+20 mL Purified water |
| Kanamycin（50 mg/ml） | 1 g+20 mL Purified water |

3.1.3 50% (V/V) Glycerol (suitable for making frozen stocks)

| Materials | Dosage |
| --- | --- |
| Glycerol | 50 ml |
| Purified water | 50 ml |

3.2 Culture medium preparation：

3.2.1 Preparing stock solutions of reagents

| Materials | Dosage | 200mL Purified water | Notes |
| --- | --- | --- | --- |
| Glucose（20x） | 400 g/L | 80 g | Add to medium at a final concentration of 1x |
| Ura（100x） | 2.24 g/L | 0.448 g | Add to medium at a final concentration of 1x |

3.2.2 YPD medium (Yeast extract peptone dextrose medium)

| Materials | Dosage | 800mL Purified water | Notes |
| --- | --- | --- | --- |
| Yeast extract | 10 g/L | 8 g |  |
| Peptone | 20 g/L | 16 g |  |
| Glucose（20×） | 20 g/L | 40 mL | Add it after sterilization |
| H2O | 1 L | 760 mL |  |
| Agar (solid plate) | 30 g/L |  |  |

3.2.3 Synthetic complete medium：

| Materials | Dosage | 800mL Purified water | Notes |
| --- | --- | --- | --- |
| Ammonium sulphate | 5 g/L | 4 g |  |
| YNB (Yeast Nitrogen Base) | 1.7 g/L | 1.36 g |  |
| DO Supplement-Ura | 0.78 g/L | 0.624 g |  |
| Glucose（20×） | 20 g/L | 40 mL | Add it after sterilization |
| Agar (solid plate) | 30 g/L | 760 mL |  |

3.2.4 LB medium （suitable for cultivating E.coli and other bacteria）

| Materials | Dosage | 800mL Purified water |
| --- | --- | --- |
| LB Broth | 30 g/L | 20 g |

## 4. Plasmid Extraction method

4.1.1 Place 1.5 ml of bacterial fluid into a 1.5 ml centrifuge tube and centrifuge at 12,000 rpm for one minute.
Repeat
step 1 until 3 ml of sample precipitate is collected.

4.1.2 Add 150ul of Buffer P1 and mix with a vortex mixer.

4.1.3 Add 150ul of Buffer P2 and softly invertate for eight times to mix well. (This step should be finished within 2
mins)

4.1.4 Add 350ul of Buffer P5 and softly invertate for eight times, until the color of mixture is even.

4.1.5 Centrifuge at 12000 rpm for 7 minutes.

4.1.6 Place the adsorbent column CP3 in the collection tube and pour the supernatant from the

4.1.7 Centrifuged tube (Do not mix in any sediment). After resting for 1 min, centrifuge at 12,000 rpm for 2 minutes
and
pour off the waste solution.

4.1.8 Add 300 ul of Buffer PWT to the adsorbent column CP3 and let it stand for two minutes.

4.1.9 Centrifuge at 12,000 rpm for one minute and pour off the waste solution.

4.1.10 Put adsorbent column CP3 with the waste solution removed into the collection tube and centrifuged at 12000 rpm
for 3 mins.

4.1.11 Remove the adsorbent column CP3 from the collection tube and place it in a 1.5ml.

4.1.12 Centrifuge tube. Place the combined centrifuge tube into a dry bath incubator at 50°C for 2 mins (with lid
opened).

4.1.13 Drop 50ul of ddH2O in the middle of the adsorption column CP3. Place in a dry bath. incubator at 50°C for 5
minutes (with lid closed). Then centrifuge at 12,000rpm for 2 mins.

4.1.14 The DNA plasmid solution is collected into the 1.5 ml centrifuge tube, then we could assay the concentration
with
qubit 4.

## 5. Golden-gate assembly and bacterial transformation

5.1 Connect the synthesized plasmid containing the target gene to HcKan-O (universal carrier) (enzymes: BSA1, T4 DNA
ligase)

5.1.1 Mix all the materials as the table mentioned below in the PCR tube.

| mixture | input |
| --- | --- |
| HCKAN\_O | 4 μL |
| Gene | 4 μL |
| 10×T4DNA ligase buffer | 1 μL |
| T4 DNA ligase | 0.5 μL |
| BsaI | 0.5 μL |
| ddH2O | 0 μL |

5.1.2 Put the tube with mixture into the thermal cycler and react according to the following procedure.

| Temperature | Time |
| --- | --- |
| 37℃ | 1 h |
| 25℃ | 1 h |
| 55℃ | 15 min |
| 80℃ | 15 min |
| 12℃ | - |

5.2 Connect the promoter+Gene+terminator to the POT plasmid (enzymes: ESP3I, T4 DNA ligase)

5.2.1 Mix all the materials as the table mentioned below in the PCR tube.

| mixture | input |
| --- | --- |
| POT | 1 μL |
| HCKAN\_P-X | 2 μL |
| HCKAN\_O-Gene | 3 μL |
| HCKAN\_T-X | 2 μL |
| 10×T4 DNA ligase buffer | 1 μL |
| T4 DNA ligase | 0.5 μL |
| ESP3I(BSMBI) | 0.5 μL |
| ddH2O | 0 μL |

5.2.2 Put the tube with mixture into the thermal cycler and react according to the following procedure.

| Temperature | Time |
| --- | --- |
| 37℃ | 1 h |
| 25℃ | 1 h |
| 55℃ | 15 min |
| 80℃ | 15 min |
| 12℃ | - |

5.3 DH5 α conversion operation

5.3.1 Thaw the competent cells DH5 α on ice 10 minutes in advance.

5.3.2 Take the PCR tube out of thermal cycler after the reaction is completed. Gently mix all the assembly system with
100 ul competent cell, and put it on ice bath for 20 minutes without shaking

5.3.3 After the ice bath is completed, water bath heat shock at 42 ℃ for 45 s without shaking.

5.3.4 Take a sample ice bath for 2 min.

5.3.5 Add 500 μL of non-resistant sterile LB liquid medium.

5.3.6 Place in a shaker at 37 ℃ and shake at 250 rpm for 45-60 minutes to revive and culture.

5.3.7 Take 100 μL of the bacterial solution and evenly spread it onto LB agar plates containing the appropriate
antibiotic. Incubate the plates at 37℃ overnight to observe the growth of bacterial colonies.

## 6. Verification Operation

6.1 Colony PCR

6.1.1 Pick and incubate single colony bacteria

* In the laminar flow hood, add 1 ml of LB liquid medium containing the appropriate antibiotics into a 1.5 ml EP
  tube,
  and use the pipette tip to pick (white) single colonies and discard them into the centrifuge tube. (Pick 10-15
  single
  colonies from each plate.)

6.1.2 Incubate at 37°C with 220 rpm shaking for 3 hours on a shaker. Proceed with single clony PCR validation.

6.1.3 Mix all the materials as the table mentioned below in the PCR tube.

| Materials | Dosage |
| --- | --- |
| 2×Taq PCR mix | 5 μL |
| Primer F | 0.4 μL |
| Primer R | 0.4 μL |
| ddH2O | 3.2 μL |
| Template DNA | 1 μL |

6.1.4 Mix the PCR system using a vortex mixer, and centrifuge shortly. Then place the PCR tube in the thermal cycler,
follow the steps below to set up the program, and close the cover to run the program.

| Temperature | Time |
| --- | --- |
| 94℃ | 3 min |
| 94℃ 20s | 30 cycle |
| 56℃ 30s |
| 72℃ 90s |
| 72℃ | 5 min |
| 12℃ | - |

6.1.5 After PCR, perform electrophoresis

6.2 Enzyme restriction validation operation.

6.2.1 Mix all the materials as the table mentioned below in the PCR tube.

| Materials | Dosage |
| --- | --- |
| Vector | 5 μL |
| 10x cut smart buffer | 1 μL |
| BSAI(POT)/ESP3I(HCKAN-O) | 1 μL |
| ddH2O | 3 μL |

6.2.2 Put the tube with mixture into the thermal cycler and react according to the following procedure.

| Temperature | Time |
| --- | --- |
|  |  |
| --- | --- |
| 37℃ | 30 min |
| 80℃ | 15 min |
| 12℃ | - |

6.2.3 After PCR, perform electrophoresis.

## 7. Yeast Experiment Operation Procedure

7.1 Yeast Competent Cell Preparation Protocol (Strain: BY4741)

7.1.1 Inoculate the BY4741 yeast strain into 5 mL of YPD medium and incubate overnight to prepare the seed culture.

7.1.2 The next day, dilute the seed culture 10-fold and measure its optical density at 600 nm (OD600).

7.1.3 Using the diluted culture, inoculate 20 mL of YPD medium to achieve an initial OD600 of 0.1. Incubate at 30°C in
a
shaker until the OD600 reaches 0.6 to 1 (growth rate approximately doubles every 1.5 hours).

7.1.4 Measure the OD600 again. Transfer 5 mL of this culture to a centrifuge tube and centrifuge at 3000 rpm for 5
minutes to collect the yeast cells.

7.1.5 Resuspend the yeast pellet in 4 mL of Solution A and wash the cells. Centrifuge again at 3000g for 5 minutes and
carefully remove the supernatant.

7.1.6 Resuspend the yeast pellet in 0.5 mL of Solution B to create competent cells.

7.1.7 Portion the competent cells into 50 μL aliquots in sterile 1.5 mL cryogenic vials. Slowly freeze these vials and
store at -80°C for future use.

7.1.8 When needed, remove the vials from the -80°C freezer, allow them to thaw at room temperature, and use the
competent
cells directly for transformation procedures.

7.2 Transformation

7.2.1 Prepare the premix for each plasmid transformation, requiring a total of 360 μL premix.

|  |  |
| --- | --- |
| Solution C | 350 μL |
| Plasmid (approximately 200 ng/μL) | 10 μL (Adjust the volume according to the plasmid concentration) |
| Total Volume | 360 μL (Top up any shortfall with ddH2O) |

7.2.2 Aspirate 360 μL of the premix and add it to resuspend the yeast competent cells. Use the pipette tip to
repeatedly
blow and draw the precipitate, thoroughly suspending the yeast cells in the premix at the bottom of the tube.

7.2.3 Place it in a 30°C water bath for heat shock for 45 min, shake to mix thoroughly every ten minutes. After the
water
bath, centrifuge at 3000g for 3 min.

7.2.4 Resuspend the precipitate in 1 mL of sterile water.

7.2.5 Spread 100 μL of the yeast suspension on SC-URA tablet.

7.2.6 Put the tablet after spreading into 30 ℃ celcius incubator for 2 days to gain successfully transformed
single-cloned yeast.

7.3 Colony PCR of S.cerevisiae

7.3.1 Plate streaking for bacterial enrichment

* mark and pick six single colonies to perform streaking for isolation. After incubating at 30°C for one day,
  colonies of
  an appropriate size (rice grain size) can be selected for clone PCR verification. Then the plate should be placed
  back
  into the incubator for further incubation for 2 days.

7.3.2 lyse yeast cells

* Dispense 50 μl of freshly prepared 20 mM Sodium Hydroxide (NaOH) solution into a PCR tube. Add yeast cells and
  thoroughly mix.
* Place the tube in the PCR machine and run the following program.

| Temperature | Time |
| --- | --- |
| 99℃ 5 min | 3 cycle |
| 12℃ 5 min |
| 12℃ | - |

* Upon completion, chill the tube on ice. Perform a quick spin, and use 1 μl of the supernatant as the template for
  colony
  PCR.

### 8. Fermentation and detection of Limonene/Ocimene/Myrcene/borneol/Cineole/Nerol/Nerolidol

8.1 Fermentation conditions

8.1.1 Inoculate A single colony of S.cerevisiae within YPD medium and cultivate overnight, then the seed solution
would
be obtained.

8.1.2 Measure the OD600.

8.1.3 The seed culture was inoculated to an initial OD600 of 0.1 into a fertilized 250 mL flask containing 50 mL
SC-URA
medium and cultivated at 30 ℃ and 250 rpm.

8.1.4 After fermentation for 12 h, 5 ml dodecane (10%) overlay culture was added.

8.1.5 The fermentation process was ended at 120 h.

8.1.6 Transfer the fermentation solution from the flask into a 5 mL centrifuge tube and centrifuge to obtain the
organic
phase.

8.1.7 Prepare a sample for GC-MS analysis by combining equal volumes (500 μL) of the dodecane extract and ethyl
acetate.

**8.2 GC-MS analysis**

8.2.1 1μL samples were injected into a GC-MS [7890A (GC) + 5975C (MS), Agilent] equipped with a HP5MS column (30 m ×
0.25 mm × 0.25 μm) and operated with He carrier gas at 1.0 mL/min.

8.2.2 Temperature and voltage were set as follows: ionization voltage 70 eV, split ratio of 30:1; transfer line
temperature 250°C; ion source temperature 230°C.

8.2.3 The oven program was as follows: 80°C (held for 1 min), 80–90°C (2°C min–1), 90–270°C (20°C min–1), and held for
5
min, with a total program of 21 min.

8.2.4 Analysis is carried out by SIM (Single Ion Monitoring) mode: m/z = 50–250.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}

{% block page\_left\_content %}

* [Overview](#1)
* [Verifying the Construction of plasmid in E.coli](#2)
* [Verifying the transformation of plasmid in yeast](#3)
* [Gas chromatography–mass spectrometry analysis](#4)
* [Reference](#5)

{% endblock %}
{% block page\_content %}

# Overview

We chose Saccharomyces cerevisiae (yeast) to express enzymes that catalyze the production of seven specific
terpenoids
which are pleasant-smelling, distinctive, and safe compounds that could serve as olfactory signals. The selected
terpenoids also have antibacterial properties, adding a functional benefit beyond their fragrance. Such as
Limonene(tLsCl), ocimene (CcOCS), cineole (HpyCins, StrCins, SfCins), nerol (PgfB), nerolidol (AcNES1), Myrcene
(AmMYS),
borneol (CbTPS1).In this section, we will present all the results that are important checkpoints in our experiments.
These include successful plasmid construction verification, successful transformation into yeast strain BY4741, and
successful protein expression in yeast strain BY4741.

![](https://static.igem.wiki/teams/5217/image/results/image1.jpeg)

Figure 1: Plates of microbial
medium used during the experiment

## Verifying the Construction of plasmid in E.coli

**Colony PCR of E.coli single colonies**

Below are the results of the gel electrophoresis of colony PCR of transformed single DH5α E. coli colonies with our
constructed plasmids. Two tubes of the sizes that are the same as the expected size are sequenced. The constructed
plasmids were initially assembled from the terpene synthase gene segments and the HcKan-O vector. Subsequently, we
combined these genes with our shuttle vector, along with the promoters and terminators.

**The colony PCR Gel Electrophoresis Results of AmMYS, CbTPS1 and tLsCl:**

![](https://static.igem.wiki/teams/5217/image/results/image2.png)

Figure 2: The growth of
POT-AmMYS, POT-CbTPS1 and POT-tLsCl colonies on petri dish.

![](https://static.igem.wiki/teams/5217/image/results/image3.png)

Figure 3: HcKan-AmMYS,
HcKan-CbTPS1, POT-AmMYS, POT-CbTPS1 and POT-tLsCl Colony PCR Gel Electrophoresis Results. A:
HcKan-AmMYS and HcKan-CbTPS1. B: Enzymatic digestion result of HcKan-AmMYS and HcKan-CbTPS1. C:
POT-AmMYS,
POT-CbTPS1 and
POT-tLsCl.

**The colony PCR Gel Electrophoresis Results of CcOCS:**

![](https://static.igem.wiki/teams/5217/image/results/image4.png)

Figure 4: Single colonies of
plasmid transformants on LB Ampicillin plate

![](https://static.igem.wiki/teams/5217/image/gel/hkccocs.png)

Figure 5: DH5α E.coli colony PCR
results of CcOCS A: HCKAN-O-CcOCS, B: POT2\_pGPM1\_CcOCS\_PGK1t, C:
POT2\_pINO1\_CcOCS\_TEF1t, D: POT2\_pTDH3\_CcOCS\_ADH1t（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/gel/pot-cc.png)

Figure 6: Enzymatic digestion
results of CcOCS

**The colony PCR Gel Electrophoresis Results of StrCins, HpyCins and SfCins:**

![](https://static.igem.wiki/teams/5217/image/results/image7.png)

Figure 7: The growth of
HcKan-HpyCins, HcKan-SfCins, POT-HpyCins, POT-StrCins and POT-SfCins colonies on petri dish.

![](https://static.igem.wiki/teams/5217/image/engineering-success/hk-cineole.png)

Figure 8: DH5α E.coli colony PCR
results of HpyCins, StrCins and SfCins A: HCKAN-O-SfCins, B: HCKAN-O-StrCins, C:
HCKAN-O-HpyCins（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot-cineole.png)

Figure 9: DH5α E.coli colony PCR
results of HpyCins, StrCins and SfCins A: POT2\_pTDH3\_HpyCins\_ADH1t, B:
POT2\_pINO1\_HpyCins\_TEF1t, C: POT2\_pGPM1\_StrCins\_PGK1t（M=DL2000 marker）

![](https://static.igem.wiki/teams/5217/image/results/image10.png)

Figure 10: Enzymatic digestion
result of StrCins

**The colony PCR Gel Electrophoresis Results of PgfB:**

![](https://static.igem.wiki/teams/5217/image/results/image11.png)

Figure 11: The growth of
HcKan-PgfB
and POT-PgfB colonies on petri dish.
HCKAN-O-HpyCins（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/gel/gel-pgfb-heng.png)

Figure 12: DH5α E.coli colony
PCR results of PgfB A: HCKAN-O-PgfB, B: POT2\_pINO1\_PgfB\_TEF1t, C: POT2\_pGPM1\_PgfB\_PGK1t,
D: POT2\_pTDH3\_PgfB\_ADH1t

![](https://static.igem.wiki/teams/5217/image/results/image13.png)

Figure 13: Enzymatic digestion
result of PgfB

**The colony PCR Gel Electrophoresis Results of AcNES1:**

![](https://static.igem.wiki/teams/5217/image/results/image14.png)

Figure 14: The growth of
HcKan-AcNES1 and POT-AcNES1 colonies on petri dish.
HCKAN-O-HpyCins（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/gel/gel-ac.png)

Figure 15: DH5α E.coli colony PCR
results of AcNES1 A: HCKAN-O-AcNES1. B: POT2\_pINO1\_AcNES1\_TEF1t,
POT2\_pGPM1\_AcNES1\_PGK1t, POT2\_pTDH3\_AcNES1\_ADH1t. C: Enzymatic digestion result of AcNES1. （M=DL5000 marker）
D: POT2\_pTDH3\_PgfB\_ADH1t（M=DL5000 marker）

**The colony PCR Gel Electrophoresis Results of PgfB and AcNES1 with pGAL1 promoter:**

![](https://static.igem.wiki/teams/5217/image/results/image16.png)

Figure 16: Single colonies of
plasmid transformants on LB ampicillin plate
HCKAN-O-HpyCins（M=DL5000 marker）

![](https://static.igem.wiki/teams/5217/image/engineering-success/gal-gel.png)

Figure 17: DH5α E.coli colony PCR
results of PgfB and AcNES1 A: POT2\_pGAL1\_PgfB\_PGK1t. B: POT2\_pGAL1\_AcNES1\_PGK1t. C:
Enzymatic digestion result of PgfB and AcNES1. （M=DL5000 marker）

**Sequencing results**

Sequencing of the plasmids can test whether our constructed plasmid is exactly the same as what we designed and
inserted. Below are the diagrams of the sequencing results. The solid red arrows present at the top of the diagrams mean
that the sequencing is correct.

**The sequencing result of AmMYS, CbTPS1 and tLsCl:**

![](https://static.igem.wiki/teams/5217/image/results/image18.png)

Strain 2 has been confirmed
positive
by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image19.png)

Strain 1 has been confirmed
positive
by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image20.png)

Strain 3 and 4 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image21.png)

Strain 2 and 7 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image22.png)

Strain 1 and 3 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image23.png)

Strain 9 has been confirmed
positive
by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image24.png)

Strain 1 and 7 have been
confirmed
positive by sequencing

**The sequencing result of CcOCS:**

![](https://static.igem.wiki/teams/5217/image/results/image25.png)

Strain 2 and 16 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image26.png)

Strain 4 and 7 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image27.png)

Strain 6 and 8 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image28.png)

Strain 1 and 5 have been
confirmed
positive by sequencing

**The sequencing result of HpyCins, StrCins and SfCins:**

![](https://static.igem.wiki/teams/5217/image/results/image29.png)

Strain 10 and 13 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image30.png)

Strain 2 and 3 have been
confirmed
incorrect by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image31.png)

Strain 6 and 8 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image32.png)

Strain 3 and 4 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image33.png)

​
Strain 3 and 10 have been confirmed positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image34.png)

Strain 1 and 3 have been
confirmed
positive by sequencing

**The sequencing result of PgfB:**

![](https://static.igem.wiki/teams/5217/image/results/image35.png)

Strain 2 and 3 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image36.png)

Strain 8 and 9 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image37.png)

Strain 1 and 2 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image38.png)

Strain 1 and 2 have been
confirmed
positive by sequencing

**The sequencing result of AcNES1:**

![](https://static.igem.wiki/teams/5217/image/results/image39.png)

Strain 4 and 8 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot2-pgpm1-acnes1-pgk1t.png)

Strain 3 and 10 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image41.png)

Strain 2 and 4 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot2-ptdh3-acnes1-adh1t.png)

Strain 1 has been
confirmed
positive by sequencing

**The sequencing result of PgfB and AcNES1 with pGAL1 promoter:**

![](https://static.igem.wiki/teams/5217/image/results/image43.png)

Strain 2 and 6 have been
confirmed
positive by sequencing

![](https://static.igem.wiki/teams/5217/image/results/image44.png)

Strain 1 and 2 have been
confirmed
positive by sequencing

**Primers used for monoclonal colony PCR**

| Terpene Synthase Gene | primer-F | primer-R | Expected Size (bp) | Results |
| --- | --- | --- | --- | --- |
| HcKan-O-CcOCS | CcOCS-seq-F | CcOCS-seq-R | 971 | Success |
| HcKan-O-StrCins | StrCins-seq-F | StrCins-seq-R | 925 | Success |
| HcKan-O-AmMYS | AmMYS-seq-F | AmMYS-seq-R | 914 | Success |
| HcKan-O-CbTPS1 | CbTPS1-seq-F | CbTPS1-seq-R | 1610 | Success |
| HcKan-O-pgfB | pgfB-seq-F | pgfB-seq-R | 986 | Success |
| HcKan-O-AcNES1 | AcNES1-seq-F | AcNES1-seq-R | 970 | Success |
| HcKan-O-HpyCins | HpyCins-seq-F | HpyCins-seq-R | 1039 | Success |
| HcKan-O-SfCins | SfCins-seq-F | SfCins-seq-R | 1574 | Failed |
| POT2-pINO1-tLsCl-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1936 | Failed |
| POT2-pGPM1-tLSCl-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1842 | Failed |
| POT2-pTDH3-tLSCl-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2073 | Success |
| POT2-pINO1-CcOCS-TEF1 | pINO1-seq-F | TEF1t-seq-R | 2052 | Success |
| POT2-pGPM1-CcOCS-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1965 | Failed |
| POT2-pTDH3-CcOCS-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2148 | Success |
| POT2-pGPM1-StrCins-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1167 | Failed |
| POT2-pINO1-AmMYS-TEF1t | pINO1-seq-F | TEF1t-seq-R | 2014 | Failed |
| POT2-pGPM1-CbTPS1 -PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1902 | Failed |
| POT2-pTDH3-pgfB-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1368 | Success |
| POT2-pGPM1-pgfB-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1185 | Success |
| POT2-pINO1-pgfB-TEF1t | pTDH3-seq-F | ADH1t-seq-R | 1270 | Success |
| POT2-pGAL1-pgfB-PGK1t | pgfB-seq-F | PGK1t-seq-R | 1069 | Success |
| POT2-pTDH3-AcNES1-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2079 | Success |
| POT2-pINO1-AcNES1-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1990 | Failed |
| POT2-pGPM1-AcNES1-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1896 | Success |
| POT2-pGAL1-AcNES1-PGK1t | AcNES1-seq-F | PGK1t-seq-R | 1432 | Success |
| POT2-pTDH3-HpyCins-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1476 | Success |
| POT2-pINO1-HpyCins-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1387 | Success |

# Verifying the transformation of plasmid in yeast

**Colony PCR of Yeast single colonies**

Below are the gel electrophoresis pictures of the colony PCR of transformed strain BY4741 with fully constructed
plasmids, which are detected positive by colony PCR. The single colonies are shown in figure 18. If we see that the
gene
segment is the correct size, then we know that the plasmid is inside the yeast, and we can begin fermenting them for
Gas
chromatography–mass spectrometry check.

![](https://static.igem.wiki/teams/5217/image/results/image45.png)

Figure 18: Streak monocultures
of transformed strain BY4741 with terpene synthase genes.

**The colony PCR Gel Electrophoresis Results of tLsCl:**

Yeast colony PCR results confirmed that the plasmid POT2\_pTDH3\_tLsCl\_ADH1t were positive in Saccharomyces
cerevisiae.

![](https://static.igem.wiki/teams/5217/image/results/image46.png)

Figure 19: Streak
monocultures
of
transformed stain BY4741 with terpene synthase genes tLsCl.

![](https://static.igem.wiki/teams/5217/image/results/image47.png)

Figure 20: Yeast colony PCR
result performed to screen strain

**The colony PCR Gel Electrophoresis Results of CcOCS:**

Yeast colony PCR results confirmed that two plasmids POT2\_pINO1\_CcOCS\_TEF1t and POT2\_pTDH3\_CcOCS\_ADH1t were positive
in
Saccharomyces cerevisiae.

![](https://static.igem.wiki/teams/5217/image/results/image48.png)

Figure 21: Streak
monocultures
of transformed stain BY4741 with terpene synthase genes CcOCS.

![](https://static.igem.wiki/teams/5217/image/results/image49.png)

Figure 22: Yeast colony PCR
result performed to screen strain A: BY4741-POT2\_pGPM1\_CcOCS\_PGK1t, B:
BY4741-POT2\_pINO1\_CcOCS\_TEF1t, C: BY4741-POT2\_pTDH3\_CcOCS\_ADH1t（M=DL5000 marker；P=Positive; N=Negative）

**The colony PCR Gel Electrophoresis Results of StrCins, HpyCins:**

Yeast colony PCR results confirmed that two plasmids POT2\_pTDH3\_HpyCins\_ADH1t and POT2\_pINO1\_HpyCins\_TEF1t were
positive
in Saccharomyces cerevisiae.

![](https://static.igem.wiki/teams/5217/image/results/image50.png)

Figure 23: Streak
monocultures
of transformed stain BY4741 with terpene synthase genes HpyCins, StrCins and SfCins.

![](https://static.igem.wiki/teams/5217/image/results/image51.png)

Figure 24: Yeast colony PCR
result of cineole performed to screen strain.（M=DL5000 marker；P=Positive; N=Negative）

**The colony PCR Gel Electrophoresis Results of PgfB:**

Yeast colony PCR results confirmed that three plasmids POT2\_pINO1\_PgfB\_TEF1t, POT2\_pGPM1\_PgfB\_PGK1t and
POT2\_pTDH3\_PgfB\_ADH1t, were positive in Saccharomyces cerevisiae.

![](https://static.igem.wiki/teams/5217/image/results/image52.png)

Figure 25: Streak
monocultures
of transformed stain BY4741 with terpene synthase genes PgfB.

![](https://static.igem.wiki/teams/5217/image/results/image53.png)

Figure 26: Yeast colony PCR
result of nerol performed to screen strain. A: BY4741-POT2\_pGPM1\_PgfB\_PGK1t. B:
BY4741-POT2\_pINO1\_PgfB\_TEF1t. C: BY4741-POT2\_pTDH3\_PgfB\_ADH1t.（M=DL5000 marker；P=Positive;
N=Negative）

**The colony PCR Gel Electrophoresis Results of AcNES1:**

Yeast colony PCR results confirmed that two plasmids POT2\_pGPM1\_AcNES1\_PGK1t and POT2\_pTDH3\_AcNES1\_ADH1t were
positive
in Saccharomyces cerevisiae.

![](https://static.igem.wiki/teams/5217/image/results/image54.png)

Figure 27: Streak
monocultures
of transformed stain BY4741 with terpene synthase genes AcNES1.

![](https://static.igem.wiki/teams/5217/image/results/image55.png)

Figure 28: Yeast colony PCR
result of nerolidol performed to screen strain. A: BY4741-POT2\_pGPM1\_AcNES1\_PGK1t. B:
BY4741-POT2\_pTDH3\_AcNES1\_ADH1t. C: BY4741-POT2\_pINO1\_AcNES1\_TEF1t.（M=DL5000 marker；P=Positive;
N=Negative）

**The colony PCR Gel Electrophoresis Results of PgfB and AcNES1 with pGAL1 promoter:**

Yeast colony PCR results confirmed that two plasmids BY4741-POT2\_pGAL1\_AcNES1\_PGK1t and BY4741-POT2\_pGAL1\_PgfB\_PGK1t
were positive in Saccharomyces cerevisiae.

![](https://static.igem.wiki/teams/5217/image/results/image56.png)

Figure 29: Yeast colony PCR
results of nerolidol and nerol performed to screen strain. A:
BY4741-POT2\_pGAL1\_AcNES1\_PGK1t. B: BY4741-POT2\_pGAL1\_PgfB\_PGK1t. （M=DL5000 marker；P=Positive;
N=Negative）

![](https://static.igem.wiki/teams/5217/image/results/image57.png)

Figure 30: Streak
monocultures
of transformed stain BY4741 with terpene synthase genes PgfB and AcNES1 with pGAL1
promoter.

**Primers used for yeast colony PCR**

| Terpene Synthase Gene | primer-F | primer-R | Expected Size (bp) | Results |
| --- | --- | --- | --- | --- |
| BY4741-POT2-pINO1-tLsCl-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1936 | Failed |
| BY4741-POT2-pGPM1-tLSCl-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1842 | Failed |
| BY4741-POT2-pTDH3-tLSCl-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2073 | Success |
| BY4741-POT2-pINO1-CcOCS-TEF1 | pINO1-seq-F | TEF1t-seq-R | 2052 | Success |
| BY4741-POT2-pGPM1-CcOCS-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1965 | Failed |
| BY4741-POT2-pTDH3-CcOCS-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2148 | Success |
| BY4741-POT2-pGPM1-StrCins-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1167 | Failed |
| BY4741-POT2-pINO1-AmMYS-TEF1t | pINO1-seq-F | TEF1t-seq-R | 2014 | Failed |
| BY4741-POT2-pGPM1-CbTPS1 -PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1902 | Failed |
| BY4741-POT2-pTDH3-pgfB-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1368 | Success |
| BY4741-POT2-pGPM1-pgfB-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1185 | Success |
| BY4741-POT2-pINO1-pgfB-TEF1t | pTDH3-seq-F | ADH1t-seq-R | 1270 | Success |
| BY4741-POT2-pGAL1-pgfB-PGK1t | pgfB-seq-F | PGK1t-seq-R | 1069 | Success |
| BY4741-POT2-pTDH3-AcNES1-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2079 | Success |
| BY4741-POT2-pINO1-AcNES1-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1990 | Failed |
| BY4741-POT2-pGPM1-AcNES1-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1896 | Success |
| BY4741-POT2-pGAL1-AcNES1-PGK1t | AcNES1-seq-F | PGK1t-seq-R | 1432 | Success |
| BY4741-POT2-pTDH3-HpyCins-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1476 | Success |
| BY4741-POT2-pINO1-HpyCins-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1387 | Success |

## Gas chromatography–mass spectrometry analysis

To evaluate the production of nerol in S.cerevisiae cells, a shake flask fermentation was conducted. After
fermentation
for 12 h, dodecane overlay culture was added. The fermentation process ended at 120 h.

**GC-MS analysis of Limonene :**

Flask fermentation of tLsCl yielded 3.17 ± 0.30 mg/l of Limonene with the pTDH3 promoter and ADH1t terminator. There
was
no significant difference in yield of empty shuttle vector strain.

![](https://static.igem.wiki/teams/5217/image/results/gc-ls.png)

Figure 31: The production of
limonene in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae with POT
vector. B: GC-MS analyses of cineole extracts of S. cerevisiae expressing tLsCl with the pTDH3 promoter and
ADH1t
terminator. C: the OD600 and Limonene yield by POT vector and gene tLsCl. The data represent the mean ±
standard
deviation of three independent experiments.

**GC-MS analysis of ocimene :**

The ocimene titer was 0.83 ± 0.06 mg/L with the pINO1 promoter and TEF1t terminator, while it was 0.33 ± 0.09 mg/L
with
the pTDH3 promoter and ADH1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/pot-cc-gc.png)

Figure 32: Heterologous
expression
of HpyCins in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae
expressing HpyCins with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of cineole extracts of S.
cerevisiae
expressing HpyCins with the pINO1 promoter and TEF1t terminator. C: the OD600 and cineole yield by HpyCins
under
the
control of different promoters and terminators. The data represent the mean ± standard deviation of three
independent
experiments.

**GC-MS analysis of cineole:**

Flask fermentation of HpyCins yielded 11.88 ± 2.03 mg/l of cineole with the pINO1 promoter and TEF1t terminator,
compared to 17.58 ± 3.20 mg/l with the pTDH3 promoter and ADH1t terminator.

![](https://static.igem.wiki/teams/5217/image/engineering-success/gc-cineole.png)

Figure 33: Heterologous
expression of HpyCins in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae
expressing HpyCins with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of cineole extracts of S.
cerevisiae
expressing HpyCins with the pINO1 promoter and TEF1t terminator. C: the OD600 and cineole yield by HpyCins
under the
control of different promoters and terminators. The data represent the mean ± standard deviation of three
independent
experiments.

**GC-MS analysis of nerol:**

Flask fermentation of PgfB yielded 6.07 ± 0.23 mg/l of nerol with the pTDH3 promoter and ADH1t terminator, but no
terpenes are produced with pINO1 and GPM1 promoter.

![](https://static.igem.wiki/teams/5217/image/engineering-success/gc-nerol.png)

Figure 34: Heterologous
expression of PgfB in S. cerevisiae. A: GC-MS analyses of nerol extracts of S. cerevisiae
expressing PgfB with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerol extracts of S.
cerevisiae
expressing PgfB with the pINO1 promoter and TEF1t terminator. C: GC-MS analyses of nerol extracts of S.
cerevisiae
expressing PgfB with the pGPM1 promoter and PGK1t terminator. D: The OD600 and nerol yield by PgfB under the
control of
different promoters and terminators. The data represent the mean ± standard deviation of three independent
experiments.

**GC-MS analysis of nerolidol:**

Flask fermentation of AcNES1 yielded 45.06 ± 1.68 mg/l of nerolidol with the pTDH3 promoter and ADH1t terminator,
compared to 12.92 ± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator.

![](https://static.igem.wiki/teams/5217/image/results/image61.png)

Figure 35: Heterologous
expression of AcNES1 in S. cerevisiae. A: GC-MS analyses of nerolidol extracts of S. cerevisiae
expressing AcNES1 with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerolidol extracts of S.
cerevisiae
expressing AcNES1 with the pGPM1 promoter and PGK1t terminator. C: The OD600 and nerolidol yield by AcNES1
under the
control of different promoters and terminators. The data represent the mean ± standard deviation of three
independent
experiments.

**GC-MS analysis of nerol and nerolidol:**

Flask fermentation of AcNES1 with the pGAL1 promoter and PGK1t terminator yielded 4.78 ± 0.88 mg/l of nerolidol,
while
no nerol production was detected.

![](https://static.igem.wiki/teams/5217/image/results/image62.png)

Figure 36: Heterologous
expression
of AcNES1 in S. cerevisiae A: GC results of AcNES1 with glucose under the control of
the pGAL1 promoter and PGK1t terminator. B: GC results of AcNES1 with galactose under the control
of
the
pGAL1
promoter
and PGK1t terminator.

![](https://static.igem.wiki/teams/5217/image/results/image63.png)

Figure 37: Heterologous
expression
of PgfB in S. cerevisiae. A: GC results of PgfB with glucose under the control of the
pGAL1 promoter  and PGK1t terminator.B: GC results of PgfB with galactose under the control of the
pGAL1
promoter and
PGK1t terminator.

![](https://static.igem.wiki/teams/5217/image/results/image64.png)

Figure 38: The OD600 and
terpenes
production by AcNES1 and PgfB under the control of pGAL1 promoter and PGK1t terminator

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