iGEM 2022 — Team gdsyzx

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* Project

# Project[¶](#project "Permalink to this heading")

* [Communication](#communication)

  + [Highschool Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
  + [Online
    Conferences](#online-conferences)
  + [Propaganda
    Speech](#propaganda-speech)
  + [Interview
    with Experts](#interview-with-experts)
  + [Interview
    with doctor](#interview-with-doctor)
* [Contribution](#contribution)

  + [1.Characterization of previous iGEM parts](#characterization-of-previous-igem-parts)
  + [2.Add New
    Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
  + [3.Add New
    Parts](#add-new-parts)
  + [4.Improvement of Existing Parts](#improvement-of-existing-parts)

    - [4.1 Optimized of farnesyl diphosphate synthase (ERG20,
      FPS) (BBa\_K517004)](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004)
    - [4.2
      Plasmid Construction](#plasmid-construction)
    - [4.3
      Results](#results)
  + [Reference](#reference)
* [Description](#description)

  + [Pogostemon cablin and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
  + [Patchoulol](#patchoulol)
  + [Plant
    extraction](#plant-extraction)
  + [Chemical
    synthesis](#chemical-synthesis)
  + [Microbial
    Fermentation](#microbial-fermentation)
  + [Patchoulol biosynthetic pathways](#patchoulol-biosynthetic-pathways)
  + [Vision](#vision)
  + [Reference](#id1)
* [Engineering](#engineering)

  + [Abstract](#abstract)
  + [Experiment
    design](#experiment-design)

    - [8 type of expression vector](#type-of-expression-vector)
  + [Experiment structure and testing](#experiment-structure-and-testing)

    - [PCR amplification and estriction enzyme cutting
      expression vector pSP-GM2](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2)
    - [Likage of expression vector and chemical transformation
      of E.coli DH5α](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5)
    - [Screening of transformants](#screening-of-transformants)
    - [Plasmids
      extraction and extract sequencing](#plasmids-extraction-and-extract-sequencing)
    - [Transformation
      and screening of yeast](#transformation-and-screening-of-yeast)
    - [Transform
      procedure](#transform-procedure)
    - [Fermentation of
      modified strains](#fermentation-of-modified-strains)
    - [Extraction
      and determining of metabolite](#extraction-and-determining-of-metabolite)
    - [Learning and design again:](#learning-and-design-again)
* [Experiments](#experiments)

  + [Protocol](#protocol)

    - [1.
      The enzyme digestion reaction of pSP-GM2 vector](#the-enzyme-digestion-reaction-of-psp-gm2-vector)
    - [2. PCR and electropherosis](#pcr-and-electropherosis)
    - [3. DNA gel electrophoresis](#dna-gel-electrophoresis)
    - [4.
      Transformation of plasmid to E.coli](#transformation-of-plasmid-to-e-coli)
    - [5.
      Selecting colonies](#selecting-colonies)
    - [6. DNA
      sequencing](#dna-sequencing)
    - [7. Yeast transformation procedure](#yeast-transformation-procedure)
* [Implementation](#implementation)

  + [Background](#background)
  + [Target
    customers](#target-customers)
  + [Possible
    challenges](#possible-challenges)
  + [Reference](#id2)
* [Notebook](#notebook)

  + [Week 1
    (6.20-6.26)](#week-1-6-20-6-26)
  + [Week 2
    (6.27-7.3)](#week-2-6-27-7-3)
  + [Week 3
    (7.4-7.10)](#week-3-7-4-7-10)
  + [Week 4
    (7.11-7.17)](#week-4-7-11-7-17)
  + [Week 5
    (7.18-7.24)](#week-5-7-18-7-24)
  + [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
  + [Week 7
    (8.15-8.27)](#week-7-8-15-8-27)
* [Proof of concept](#proof-of-concept)

  + [Method
    development](#method-development)
  + [Plasmid construction](#id3)
  + [Patchoulol
    detection](#patchoulol-detection)
  + [Reference](#id4)
* [Results](#id5)

  + [1. Codon
    Optimization](#codon-optimization)
  + [2. DNA
    Preparation](#dna-preparation)
  + [3.
    Construction of Plasmids](#construction-of-plasmids)
  + [4. Patchoulol
    detection](#id6)

## [Communication](#id12)[¶](#communication "Permalink to this heading")

### [Highschool Teams HP Experience Exchange](#id13)[¶](#highschool-teams-hp-experience-exchange "Permalink to this heading")

We were invited by SYSU-CHINA to visit their laboratory and communicate about our
projects. They showed us their advanced and practical scientific research, and provided
us with experience and advice on the use of the equipment, which made great
contributions to the direction and operation of our team’s experiment. At the same time,
we learned each other’s speech skills, which provided us with valuable experience in
speaking and writing manuscripts.

**Lab Visit**[¶](#id7 "Permalink to this table")

|  |  |  |  |
| --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication1.png](https://static.igem.wiki/teams/4270/wiki/communication1.png) | ![https://static.igem.wiki/teams/4270/wiki/communication2.png](https://static.igem.wiki/teams/4270/wiki/communication2.png) | ![https://static.igem.wiki/teams/4270/wiki/communication3.png](https://static.igem.wiki/teams/4270/wiki/communication3.png) | ![https://static.igem.wiki/teams/4270/wiki/communication4.png](https://static.igem.wiki/teams/4270/wiki/communication4.png) |

### [Online Conferences](#id14)[¶](#online-conferences "Permalink to this heading")

Later, we also had a video conference with SYSU-CHINA team. We presented and shared our
story of patchoulol, questionary experiences, and failures to SYSU-CHINA team members.
They gave us some valuable suggestions. SYSU-CHINA team also presented their research
content about salidroside. The communication with SYSU-CHINA team gave us extraordinary
inspiration for our project.

[![https://static.igem.wiki/teams/4270/wiki/communication5.png](https://static.igem.wiki/teams/4270/wiki/communication5.png)](https://static.igem.wiki/teams/4270/wiki/communication5.png)

**Online Metting**[¶](#id8 "Permalink to this table")

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication6.png](https://static.igem.wiki/teams/4270/wiki/communication6.png) | ![https://static.igem.wiki/teams/4270/wiki/communication7.png](https://static.igem.wiki/teams/4270/wiki/communication7.png) | ![https://static.igem.wiki/teams/4270/wiki/communication8.png](https://static.igem.wiki/teams/4270/wiki/communication8.png) | ![https://static.igem.wiki/teams/4270/wiki/communication9.png](https://static.igem.wiki/teams/4270/wiki/communication9.png) | ![https://static.igem.wiki/teams/4270/wiki/communication10.png](https://static.igem.wiki/teams/4270/wiki/communication10.png) | ![https://static.igem.wiki/teams/4270/wiki/communication11.png](https://static.igem.wiki/teams/4270/wiki/communication11.png) |

### [Propaganda Speech](#id15)[¶](#propaganda-speech "Permalink to this heading")

In order to bring our project closer to the masses, our team held public talks to
introduce ourselves and our results. Adhering to the principle of making synthetic
biology enjoyable, we selected three speakers from our team to describe our team’s
struggles and research results along the way. After returning to campus in September, we
also held another club presentation to show new students our latest progress and basic
information. At the club festival of our school, we publicized our project in the
biology club, so that all the high school freshmen and part of the high school seniors
who participated in the activity knew about our project and activity, and learned
something from it. In all the lectures we held, we received a lot of compliments and
suggestions, so we would adjust our presentation each time and do it better next time to
give everyone a better understanding of the project.

**Public Speaking**[¶](#id9 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication12.png](https://static.igem.wiki/teams/4270/wiki/communication12.png) | ![https://static.igem.wiki/teams/4270/wiki/communication13.png](https://static.igem.wiki/teams/4270/wiki/communication13.png) | ![https://static.igem.wiki/teams/4270/wiki/communication14.png](https://static.igem.wiki/teams/4270/wiki/communication14.png) |

### [Interview with Experts](#id16)[¶](#interview-with-experts "Permalink to this heading")

Considering that the light-induced promoter is used in our project, and the professor’s
research field is about the regulation of photosynthesis, we asked the professor to give
us some suggestions for improving the knowledge of photobiology used in our project.

Initially, the professor says, you need to learn to screen when doing experiments.
Photobiology is a very cutting-edge branch of life science. There have been previous
achievements in this field. When we make use of it, we should screen the suitable
conditions for our experiments according to the actual situation. For example, the
light-induced promoter of HCF173 used in this experiment should be screened before use
to screen out the light-induced promoter with the best-carrying effect with our target
gene to ensure that its activity matches each other with the gene.

In addition, promoter optimization should be carried out in combination with the actual
situation. For example, the light we are using to activate now in our experiments, white
light, means that daily light can activate the promoter. However, in the actual
production, because white light is too easy to appear, it is impossible to achieve a
real light environment. To solve this problem, the professor suggested that the light we
used to induce could be selected from specific bands of light, such as those rare in
nature, so as to avoid the interference of light in nature and make the experiment more
accurate.

What is more, the promoter’s light requirements need to be feasible. If we have a light
promoter that has very high light intensity requirements, and you have to meet these
ultra-high requirements to start, then most of the time you can’t produce. And if very
strong light is required, it will greatly increase the cost of our production.

Considering the problems that arise when moving from the laboratory to mass production,
we also discussed the whole synthetic biology industry with the professor. The professor
pointed out that the laboratory environment is very different from the actual production
environment. For example, in this project, we have a light-induced promoter, and in the
lab, the volume that we synthesize is actually very small, maybe a thin layer in yeast.
However, in mass production, it is still very difficult to ensure that each yeast light
promoter can feel the light in the face of the large volume of the factory. For these
problems, the professor pointed out a clear way for us: we should first form a
theoretical basis in the laboratory, and when we find problems in large-scale
production, we should timely feedback, optimize and solve them in the laboratory, and
then put them into large-scale production for trial. And so on and so on until the
desired effect is achieved.

Finally, we asked professors how they could connect the projects they were working on to
society and make a real contribution. First of all, the professor affirmed our research
project on patchouli and patchoulol, which are very distinctive ingredients derived from
Lingnan’s traditional Chinese medicine. It is a contribution to society to bring
characteristic plants into play. As for the role of patchoulol, the professor pointed
out the following points: one is that patchoulol can be directly used as a component in
medicine, and the other is that patchoulol can be used as a chassis for other products,
providing a prerequisite for other drugs.

According to the suggestions made by the professor during the interview, we plan to make
the following improvements in the future: First, for the optical promoter itself, reduce
its requirements on light and increase the practicability of the product. Second, if in
the future will be put into large-scale production, the problems encountered in the
production should be timely improved in the laboratory.

### [Interview with doctor](#id17)[¶](#interview-with-doctor "Permalink to this heading")

Considering the particularity of the research objects of our project (patchouli and
patchouli alcohol), the team decided to interview some practitioners of traditional
Chinese medicine working on the frontline in the community clinic, and further discuss
the current situation of the use of patchouli and patchouli alcohol and the social value
of our project.

First, we asked doctors what kind of concoctions they typically prescribe to patients,
such as sliced or ground medicine. Doctors say there are only one or two medicines that
usually contain patchouli or patchoulol in hospitals, such as Huoxiang Zhengqi capsule
and Huoxiang Zhengqi liquid.

Then, we briefly introduced our project and discussed the impact of the increased
production of patchoulol on doctors’ consultations and even society as a whole. Doctors
say that if the production of patchoulol can be increased and more patchoulol becomes
more available, the price of patchoulol will fall accordingly. Among them, pharmacists
in the pharmacy told us that the price of Chinese patent medicine has been soaring. If
the price of patchoulol can be reduced, doctors will have fewer concerns when
prescribing medicine to patients, and it will also be able to ensure that some poor
people have access to medicine, which is of great significance to society.

**Interviewing Doctors**[¶](#id10 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication15.png](https://static.igem.wiki/teams/4270/wiki/communication15.png) | ![https://static.igem.wiki/teams/4270/wiki/communication16.png](https://static.igem.wiki/teams/4270/wiki/communication16.png) | ![https://static.igem.wiki/teams/4270/wiki/communication17.png](https://static.igem.wiki/teams/4270/wiki/communication17.png) |

## [Contribution](#id18)[¶](#contribution "Permalink to this heading")

### [1.Characterization of previous iGEM parts](#id19)[¶](#characterization-of-previous-igem-parts "Permalink to this heading")

In this project, basic parts of previous projects are characterized, including pPGK1
promoter (BBa\_K122000), pTEF1 promoter (BBa\_K2765041), TADH1 terminator (BBa\_K122004),
and TCYC1 terminator (BBa\_K122003). Patchoulol synthase (PTS) gene and farnesyl
diphosphate synthase (ERG20, also named FPS) gene are expressed by this promoters and
terminators. The sequences were assembled into pSP-GM2 plasmids and constructed 4
recombinant vectors, namely pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS and
pSP-GM2-PTS-FPS (Figure 1-4). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 5**).

[![https://static.igem.wiki/teams/4270/wiki/contributions1.png](https://static.igem.wiki/teams/4270/wiki/contributions1.png)](https://static.igem.wiki/teams/4270/wiki/contributions1.png)

**Figure 1. The construction of pSP-GM2-FPS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions2.png](https://static.igem.wiki/teams/4270/wiki/contributions2.png)](https://static.igem.wiki/teams/4270/wiki/contributions2.png)

**Figure 2. The construction of pSP-GM2-PTS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions3.png](https://static.igem.wiki/teams/4270/wiki/contributions3.png)](https://static.igem.wiki/teams/4270/wiki/contributions3.png)

**Figure 3. The construction of pSP-GM2-PTS-linker-FPS plasmid with pPGK1 as
promoter and TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions4.png](https://static.igem.wiki/teams/4270/wiki/contributions4.png)](https://static.igem.wiki/teams/4270/wiki/contributions4.png)

**Figure 4. The construction of pSP-GM2-PTS-FPS plasmid with pPGK1 and pTEF1 as
promoter, TADH1 and TCYC1 as terminators, respectively.**

[![https://static.igem.wiki/teams/4270/wiki/contributions5.png](https://static.igem.wiki/teams/4270/wiki/contributions5.png)](https://static.igem.wiki/teams/4270/wiki/contributions5.png)

**Figure 5 A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

### [2.Add New Documentations To An Existing Part](#id20)[¶](#add-new-documentations-to-an-existing-part "Permalink to this heading")

We will artificially cloned 2 new genes including HCF173 promoter and PTS. The patchoulol
synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces
more than 20 valuable sesquiterpenes including patchoulol by conversion of the natural
substrate FPP.[1-3] HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173),
which is required for Arabidopsis thaliana PSII biogenesis. HCF173 transcript levels and
expression were induced by light. HCF173 promoter contains ACE motif and G-box
element.[4] In our project, the CDS sequence of PTS was optimized according to yeast
codon preference. PCR was conducted first for cloning PTS, FPS (ERG20) and HCF173
(**Figure 6**). Later a gene fusion of PTS and FPS namely PTS-linker-FPS
are acquired by removing the stop codon of PTS and replacing it by a short peptide
(Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs.

[![https://static.igem.wiki/teams/4270/wiki/contributions6.png](https://static.igem.wiki/teams/4270/wiki/contributions6.png)](https://static.igem.wiki/teams/4270/wiki/contributions6.png)

**Figure 6. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

Later, we assembled HCF173 promoters, PTS gene and ERG20 (FPS) and constructed 4
recombinant vectors, namely pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS and pSP-GM2-HCF173-PTS-FPS (**Figure 7-10**).
The colony PCR were conducted for further verification of this recombinant vectors
(**Figure 11**).
.. image:: <https://static.igem.wiki/teams/4270/wiki/contributions7.png>

> width
> :   1000px
>
> align
> :   center

**Figure 7. The construction of pSP-GM2-HCF173-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions8.png](https://static.igem.wiki/teams/4270/wiki/contributions8.png)](https://static.igem.wiki/teams/4270/wiki/contributions8.png)

**Figure 8. The construction of pSP-GM2-HCF173-PTS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions9.png](https://static.igem.wiki/teams/4270/wiki/contributions9.png)](https://static.igem.wiki/teams/4270/wiki/contributions9.png)

**Figure 9. The construction of pSP-GM2-HCF173-PTS-linker-FPS plasmid with HCF173
as promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions10.png](https://static.igem.wiki/teams/4270/wiki/contributions10.png)](https://static.igem.wiki/teams/4270/wiki/contributions10.png)

**Figure 10. The construction of pSP-GM2-HCF173-PTS-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions11.png](https://static.igem.wiki/teams/4270/wiki/contributions11.png)](https://static.igem.wiki/teams/4270/wiki/contributions11.png)

**Figure 11. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [3.Add New Parts](#id21)[¶](#add-new-parts "Permalink to this heading")

In our 2022 project, we provide 10 new parts:

> | part name | part number | website |
> | --- | --- | --- |
> | HCF173 promoter | BBa\_K4270001 | [http://parts.igem.org/Part:BBa\_K4270001](http://parts.igem.org/Part%3ABBa_K4270001) |
> | PTS (patchoulol synthase) | BBa\_K4270004 | [http://parts.igem.org/Part:BBa\_K4270004](http://parts.igem.org/Part%3ABBa_K4270004) |
> | erg20 K197P | BBa\_K4270007 | [http://parts.igem.org/Part:BBa\_K4270007](http://parts.igem.org/Part%3ABBa_K4270007) |
> | erg20 K197H | BBa\_K4270008 | [http://parts.igem.org/Part:BBa\_K4270008](http://parts.igem.org/Part%3ABBa_K4270008) |
> | FPS-linker-PTS | BBa\_K4270010 | [http://parts.igem.org/Part:BBa\_K4270010](http://parts.igem.org/Part%3ABBa_K4270010) |
> | pPGK1+PTS+TADH1 | BBa\_K4270011 | [http://parts.igem.org/Part:BBa\_K4270011](http://parts.igem.org/Part%3ABBa_K4270011) |
> | pPGK1+ERG20+TADH1 | BBa\_K4270012 | [http://parts.igem.org/Part:BBa\_K4270012](http://parts.igem.org/Part%3ABBa_K4270012) |
> | pTEF1+ERG20+TCYC1 | BBa\_K4270016 | [http://parts.igem.org/Part:BBa\_K4270016](http://parts.igem.org/Part%3ABBa_K4270016) |
> | HCF173+ ERG20+TADH1 | BBa\_K4270013 | [http://parts.igem.org/Part:BBa\_K4270013](http://parts.igem.org/Part%3ABBa_K4270013) |

### [4.Improvement of Existing Parts](#id22)[¶](#improvement-of-existing-parts "Permalink to this heading")

#### [4.1 Optimized of farnesyl diphosphate synthase (ERG20, FPS) (BBa\_K517004)](#id23)[¶](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004 "Permalink to this heading")

The ERG20 is a basic part comprised from a synthesized farnesyl diphosphate synthase
coding sequence. The basic part BBa\_K4270007 (erg20 K197P) and BBa\_K4270008 (erg20
K197H) are optimized versions of ERG20 (BBa\_K517004) that can be constructed into
pSP-GM2 plasmid. The resulted vectors are named pSP-GM2-FPS, pSP-GM2-FPS K197P and
pSP-GM2-FPS K197H respectively. Then this vectors were transformed into
Saccharomyces cerevisiae BY4741 to produce patchoulol.

#### [4.2 Plasmid Construction](#id24)[¶](#plasmid-construction "Permalink to this heading")

PCR was conducted for cloning FPS (ERG20), erg20 K197P and erg20 K197H. The purified
PCR products were assembled into pSP-GM2 plasmids and constructed 3 recombinant
vectors, namely pSP-GM2-FPS, pSP-GM2-FPS K197P and pSP-GM2-FPS K197H (**Figure
12-13**). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 14**).

[![https://static.igem.wiki/teams/4270/wiki/contributions12.png](https://static.igem.wiki/teams/4270/wiki/contributions12.png)](https://static.igem.wiki/teams/4270/wiki/contributions12.png)

**Figure 12. The construction of pSP-GM2-FPS K197P plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions13.png](https://static.igem.wiki/teams/4270/wiki/contributions13.png)](https://static.igem.wiki/teams/4270/wiki/contributions13.png)

**Figure 13. The construction of pSP-GM2-FPS K197H plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions14.png](https://static.igem.wiki/teams/4270/wiki/contributions14.png)](https://static.igem.wiki/teams/4270/wiki/contributions14.png)

**Figure 14. The colony PCR results of pSP-GM2-FPS K197P (left side) and
pSP-GM2-FPS K197H (right side).**

#### [4.3 Results](#id25)[¶](#results "Permalink to this heading")

Gas chromatography (GC) method to observe the patchoulol in Yeast fermentation broth.
Unfortunately, we did not detect patchoulol in the supernatant but detected
patchoulol in the standard substance containing patchoulol (Figure 15). The reason
we did not detect patchoulol in the supernatant may due to the low yield of
patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/contributions15.png](https://static.igem.wiki/teams/4270/wiki/contributions15.png)](https://static.igem.wiki/teams/4270/wiki/contributions15.png)

**Figure 15. The detection of patchoulol in the supernatant. A-C. The
detection of patchoulol in Yeast fermentation broth from Yeast transformed with
pSP-GM2-FPS, pSP-GM2- FPS K197P, pSP-GM2- FPS K197H plasmids, respectively. D.
The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id26)[¶](#reference "Permalink to this heading")

[1] Albertsen L, Chen Y,Bach L. S, Rattleff S, et al. Diversion of flux toward
sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous
enzymes. Appl. Environ. Microbiol. 2011, 77, 1033−1040.

[2] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid precursors elevates terpene production in plants. Nature Biotechnology, 2006,
24: 1441-47.

[3] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[4] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Description](#id27)[¶](#description "Permalink to this heading")

### [Pogostemon cablin and patchouli oil](#id28)[¶](#pogostemon-cablin-and-patchouli-oil "Permalink to this heading")

Pogostemon cablin is a kind of medicinal aromatic herb and one of the Top 10 drugs in
Guangdong province. P. cablin is mainly grown in Indonesia, Singapore, Vietnam,
Malaysia, China, and other areas.[1] Patchouli oil is made from the dried ground part of
P. cablin by distillation and is mainly used in two aspects.[2] First, it is used as a
drug because of its anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other
pharmacological effects.[3-5] On the other hand, its fragrance is unique and used as a
spices and food additive.[6] Benefiting from its good efficacy, patchouli oil is widely
used in the daily chemical, pharmaceutical, and food industries and has a broad market
prospect. The price of high-quality patchouli oil can be as high as ¥1300 per kilogram.
In terms of the retail price, it can rank 10th among the important essential oils in the
world and has obvious economic benefits. [7, 8]

**Pogostemon cablin**[¶](#id11 "Permalink to this table")

|  |  |
| --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/description1.png](https://static.igem.wiki/teams/4270/wiki/description1.png) | ![https://static.igem.wiki/teams/4270/wiki/description2.png](https://static.igem.wiki/teams/4270/wiki/description2.png) |

### [Patchoulol](#id29)[¶](#patchoulol "Permalink to this heading")

Patchoulol, a sesquiterpene, is one of the most abundant components in patchouli oil. [6,
9] As an important index for evaluating the quality of P. cablin and patchouli oil in
Chinese Pharmacopoeia, patchoulol is also considered the main component contributing to
the pharmacological effects and aroma of patchouli oil. [10] Nowadays, there is a
serious imbalance between the supply and demand of patchoulol and patchouli oil in the
world. The global annual demand for patchoulol and patchouli oil can reach over 2 000 t,
but the annual output is about 1300 t. Indonesia supplies 90 percent of all annual
output, while China produces less than 10 percent of that, which has plenty of room for
growth. [11, 12] Increasing the yield of patchoulol has become a key point in the study
of p. cablin.

At present, patchoulol is obtained in three main ways, namely, plant extraction, chemical
synthesis, and microbial fermentation.

[![https://static.igem.wiki/teams/4270/wiki/description3.png](https://static.igem.wiki/teams/4270/wiki/description3.png)](https://static.igem.wiki/teams/4270/wiki/description3.png)

### [Plant extraction](#id30)[¶](#plant-extraction "Permalink to this heading")

Typically, patchoulol is extracted from the leaves of P.cablin. However, the low content
of patchoulol in P. cablin is the bottleneck of its large-scale application. [2]

### [Chemical synthesis](#id31)[¶](#chemical-synthesis "Permalink to this heading")

Although the chemical synthesis method can support patchoulol with high purity, it needs
many steps, complex operation, high synthesis cost, and the yield are low, so it cannot
be applied to large-scale industrial production. [13]

### [Microbial Fermentation](#id32)[¶](#microbial-fermentation "Permalink to this heading")

Compared with plant extraction and chemical synthesis, microbial fermentation synthesis
exhibits several advantages in producing patchoulol, such as fast-growing, land-saving,
and controllable culture conditions. With the development of metabolic engineering and
synthetic biology, many valuable plant-derived terpenoids also have been produced in
microbial cell factories, such as artemisinic acid [14], ginsenosides [15], and
tanshinones [16]. Although naturally produced patchoulol can be obtained from plants,
patchoulol production using biobased microbial platforms is an economical and
sustainable alternative.

### [Patchoulol biosynthetic pathways](#id33)[¶](#patchoulol-biosynthetic-pathways "Permalink to this heading")

There are two biosynthetic pathways for patchoulol formation, namely the mevalonic acid
(MVA) pathway located in the cytoplasm and the methylerythritol 4-phosphate
(MEP) pathway located in plastids. [17, 18] In general, patchoulol is mainly synthesized
through the MVA pathway. There are 3 key enzymes in the synthesis of patchoulol, namely
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FPS),
and patchoulol synthase (PTS). HMGR catalyzes the irreversible formation of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into MVA. Because it can affect the
yield of IPP (isopentenyl diphosphate), the precursors of patchoulol, HMGR is the key to
the metabolism of patchoulol. FPS catalyzes IPP to FPP (farnesyl diphosphate). PTS is a
key rate-limiting enzyme in the formation of the patchoulol, which catalyzes the
formation of patchoulol skeleton from the FPP precursor, and the skeleton is modified by
secondary enzymes to form patchoulol.

[![https://static.igem.wiki/teams/4270/wiki/description4.png](https://static.igem.wiki/teams/4270/wiki/description4.png)](https://static.igem.wiki/teams/4270/wiki/description4.png)

### [Vision](#id34)[¶](#vision "Permalink to this heading")

Patchoulol production using biobased microbial platforms is an economical and sustainable
alternative. Recently, based on the endogenous mevalonate (MVA) pathway or
2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, heterologous production of patchoulol
has been accomplished by introducing the patchoulol synthase gene in Saccharomyces
cerevisiae [8] and Corynebacterium glutamicum [19], as well as in the moss
Physcomitrella patens[20] and eukaryotic microalga Chlamydomonas reinhardtii [21].
Nevertheless, the yield is still too low for industrial production. Therefore,
significant engineering is required to achieve sufficient yields for industrial
applications. Scientists see the use of environmental signals (light, temperature) and
endogenous signals (quorum sensing, pressure) to establish tightly controlled patchoulol
biological production systems as the future trend.

Promoters are crucial elements in controlling the expression of target genes, and the
emergence of inducible promoters provides the possibility to accurately and flexibly
regulate the expression of foreign genes, which has become one of the hot spots in the
research and development of genetic engineering. [22] Therefore, in the present study,
two metabolic engineering strategies were conducted to enhance the production of
patchoulol in yeast. First, ERG20 (encoding FPP synthase from yeast) and PTS (encoding
patchoulol synthase from P. cablin) were used to increase the utilization of the FPP
precursor. Second, the constituted promoter and light-inducible promoter are
respectively used to drive the overexpression of the critical gene FPS and PTS. In
addition, studies have reported that high-level patchoulol production in tobacco plants
was achieved by fusing FPS and PTS genes. [17] Hence, vectors consisting of a gene
fusion of PTS and FPS (PTS-FPS) inserted downstream of the constitutive and
light-inducible promoter were also constructed. Finally, we carried out fermentation in
yeast and envisioned our final product by meteorological chromatography.

[![https://static.igem.wiki/teams/4270/wiki/description5.png](https://static.igem.wiki/teams/4270/wiki/description5.png)](https://static.igem.wiki/teams/4270/wiki/description5.png)

### [Reference](#id35)[¶](#id1 "Permalink to this heading")

[1] Yao G, Drew B T, Yi T S, et al. Phylogenetic relationships, character evolution and
biogeographic diversification of Pogostemon s.l. (Lamiaceae) [J]. Mol Phylogenet Evol,
2016, 98(184-200.

[2] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[3] Zhang R, Yan P, Li Y, et al. A pharmacokinetic study of patchouli alcohol after a
single oral administration of patchouli alcohol or patchouli oil in rats [J]. European
Journal of Drug Metabolism and Pharmacokinetics, 2016, 41(4): 441-8.

[4] Wu Z, Zeng H, Zhang L, et al. Patchouli alcohol: a natural sesquiterpene against both
inflammation and intestinal barrier damage of ulcerative colitis [J]. Inflammation,
2020, 43(4): 1423-35.

[5] 闵琼, 曾海荣, 陆翠燕, et al. 广藿香醇通过调控上皮间质转化抑制人胃癌细胞HGC-27的侵袭和转移 [J]. 药学服务与研究, 2020, 20(01):
6-11.

[6] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[7] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Ind Crop Prod, 2016, 87(161-76.

[8] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[9] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[10] 徐雯, 吴艳清, 丁浩然, et al. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[11] Swamy M K, Mohanty S K, Sinniah U R, et al. Evaluation of patchouli (Pogostemon
cablin Benth.) cultivars for growth, yield and quality parameters [J]. Journal of
Essential Oil-Bearing Plants, 2015, 18(4): 826-32.

[12] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Industrial Crops & Products, 2016, 87(161-76.

[13] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of ()-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[14] Westfall P J, Pitera D J, Lenihan J R, et al. Production of amorphadiene in yeast,
and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent
artemisinin [J]. P Natl Acad Sci USA, 2012, 109(3): 655-6.

[15] Wei W, Wang P, Wei Y, et al. Characterization of Panax ginseng
UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive
ginsenosides F1 and Rh1 in metabolically engineered yeasts [J]. Molecular Plant,
2015,

[16] Guo J, Zhou Y J, Hillwig M L, et al. CYP76AH1 catalyzes turnover of miltiradiene in
tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts
[J]. Pnas, 2013, 110(29): 12108-13.

[17] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid
precursors elevates terpene production in plants. Nature Biotechnology, 2006, 24:
1441-47.

[18] Tang Y, Zhong L, Wang X, et al. Molecular identification and expression of
sesquiterpene pathway genes responsible for patchoulol biosynthesis and regulation in
Pogostemon cablin［J］.
Bot Stud, 2019, 60：11.

[19] Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum［J］. Genes (Basel), 2018, 9(4): E219.

[20] Zhan X, Zhang YH, Chen DF, et al. Metabolic engineering of the moss Physcomitrella
patens to produce the sesquiterpenoids patchoulol and α/β-santalene［J］. Front Plant Sci,
2014, 5 ：636.

[21] Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a
high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii［J］.
Metab Eng, 2016,
38: 331-343.

[22] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Engineering](#id36)[¶](#engineering "Permalink to this heading")

### [Abstract](#id37)[¶](#abstract "Permalink to this heading")

Patchoulol is one of the main active components of Pogostemon cablin, and it is also an
important indicator for evaluating the medicinal materials of P. cablin in the Chinese
Pharmacopoeia. Patchoulol can be obtained by natural extraction, chemical synthesis, and
synthetic biology engineering. Due to the low content of patchoulol in P. cablin and the
low yield of chemical synthesis, the study of combinatorial biosynthesis and molecular
regulation can provide the basis for obtaining high-content patchoulol. In this study,
on the basis of the biosynthetic pathway, molecular regulation mechanism, and synthetic
biology engineering of patchoulol, the key genes for its biosynthesis were combined and
expressed in heterologous host yeast to produce patchoulol, which provides the technical
foundations of the development and application of patchoulol.

### [Experiment design](#id38)[¶](#experiment-design "Permalink to this heading")

#### [8 type of expression vector](#id39)[¶](#type-of-expression-vector "Permalink to this heading")

We designed eight plasmids: The strong constitutive promoter (pGK1) was separately
used to connect FPS (BBa\_K517004), PTS (BBa\_K4270004), and FPS-linker-PTS
(BBa\_K4270010), and plasmids containing pGK1, FPS, terminator, pGK1, PTS terminator
were constructed .In the following four designs, all the above strong promoters were
replaced with light-induced promoters HCF173, and then the test was carried out to
verify (Figure 1).

[![https://static.igem.wiki/teams/4270/wiki/engineering1.png](https://static.igem.wiki/teams/4270/wiki/engineering1.png)](https://static.igem.wiki/teams/4270/wiki/engineering1.png)

### [Experiment structure and testing](#id40)[¶](#experiment-structure-and-testing "Permalink to this heading")

#### [PCR amplification and estriction enzyme cutting expression vector pSP-GM2](#id41)[¶](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2 "Permalink to this heading")

Amplified by PCR, the plasmids were double digestion by the restriction enzymes NotI
and SacI. Plenty of strands of DNA were obtained. Plasmid DNA, restriction enzyme,
reaction buffer, and ddH2O were added successively in a 1.5 mL sterilized centrifuge
tube according to the table below. Mix and mark well. The mixture reacted at 37℃ for
eight to ten hours. Agarose gel electrophoresis was used to detect the product size
and determine whether the enzyme was successfully cut.

**Table 1 Enzyme digestion reaction system**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | Not I | 0.5 |
> | Sac I | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

#### [Likage of expression vector and chemical transformation of E.coli DH5α](#id42)[¶](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5 "Permalink to this heading")

In vitro assembly gene, including promoter, control of the corresponding enzyme
synthesis gene, sequence of the terminator, DNA ligase access strip DNA, forming a
circular recombinant vector. Add 10 ng vector recombinant DNA solution to 50 μL
competent E.coli, mix well, and let stand in an ice bath for 30 min. By heat pulse
method, the mixture was shocked in a water bath at 42℃ for 30 s, then placed on ice
for 3-5 minutes. 1 mL fresh medium was added and cultured at 37℃ for 1 h for
amplification. Add 500 μL to sterilized LB medium into a centrifuge tube, mix
evenly, and culture for 1 h at 37℃ and 200 rpm. Adjust the speed to 5000rpm,
centrifuge at room temperature for 1min, take out the supernatant and pour it out
until there was 100 μL of thallus remaining.

#### [Screening of transformants](#id43)[¶](#screening-of-transformants "Permalink to this heading")

Pick a single bacteria placement from the conversion plate and place it on the new LB
solid plate containing antibiotics, culture it at 37℃ for 2-4 h and then pick up the
colonies. Put them into the centrifuge tube of the corresponding PCR reaction
solution. Take the colony as the template of PCR reaction and amplify the colony by
T5 Super PCR Mix.

**Table 2 Colony PCR reaction system**

> | Reagent | Dosage |
> | --- | --- |
> | 2×T5 Super PCR Mix | 5 μL |
> | M13F（10 μM） | 0.4 μL |
> | M13R（10 μM） | 0.4 μL |
> | ddH2O | 4.2 μL |

**Table 3 Colony PCR reaction procedure**

> | Temperature | Time | Cycle |
> | --- | --- | --- |
> | 98℃ | 10 min | 1 |
> | 98℃ | 10 s | 35 |
> | 50℃ | 10 s |  |
> | 72℃ | 30 s |  |
> | 72℃ | 2 min | 1 |

#### [Plasmids extraction and extract sequencing](#id44)[¶](#plasmids-extraction-and-extract-sequencing "Permalink to this heading")

The transformed E.coli was inoculated with 0.5% glycerin and ampicillin in sterilized
TB culture medium. Culture at 220 rpm for 12 h at 37℃ until saturated. Adjust the
speed to 10000 rpm, centrifuge at room temperature for 6 min, remove and pour off
the supernatant. The centrifugation process was repeated until the plasmid was
successfully extracted. After gene sequencing, verify whether the vector is
successfully transformed and consistent with the design sequence.

#### [Transformation and screening of yeast](#id45)[¶](#transformation-and-screening-of-yeast "Permalink to this heading")

About 16 colonies growing in LB solid medium containing ampicillin were selected and
verified by 2x Easy Taq enzyme. The PCR system is shown below.

**Table 4 PCR reaction (20 μL)**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | buffer | 8 |

The reaction time and temperature were set according to the table below, and
electrophoresis was performed after amplification.

**Table 5 experimental set**

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

The monoclones were selected into 5 mL of the corresponding defective medium (BY4741
into YPD containing glucose). The culture was incubated overnight at 28℃ at 200-220
rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial solution
into 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out the
supernatant. The monoclones were selected into 5mL of corresponding defective medium
(BY4741 into YPD containing glucose). The culture was incubated overnight at 28℃ at
200-220 rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial
solution into a 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out
the supernatant. Add 500 μL of EZ1 and resuspend thallus. Centrifuge 300-500 rpm for
3-5 min to remove the supernatant. Add 50μL of EZ2, flick, and resuspended the
thallus to obtain a competent state.

#### [Transform procedure](#id46)[¶](#transform-procedure "Permalink to this heading")

3-5 μL plasmid was added to 50 μL competent state, mixed evenly, then 500 μL EZ3 was
added, then evenly and lightly beaten with a blue gun since EZ3 is sticky. Incubated
in a water bath at 30℃ for 45 min-1 h. After centrifugation at 500 rpm for 5 min,
flocculent accumulation of thalli could be seen near the bottom of the tube. After
centrifugation, 400 μL centrifuged EZ3 was sucked off, leaving 100 μL resuspended
thallus and applied directly to the plate.

The vector was transformed into yeast and screened for nutritional deficiency.
Successfully transformed recipient cells can be grown on nutritionally deficient
media. The successfully transformed yeast cells were taken and examined again.

#### [Fermentation of modified strains](#id47)[¶](#fermentation-of-modified-strains "Permalink to this heading")

Provide suitable nutrients and environment for fermentation, so that the successful
transformation of yeast fermentation at room temperature.

#### [Extraction and determining of metabolite](#id48)[¶](#extraction-and-determining-of-metabolite "Permalink to this heading")

The content of patchoulol, a volatile sesquiterpene, was analyzed by gas
chromatography.

#### [Learning and design again:](#id49)[¶](#learning-and-design-again "Permalink to this heading")

We further optimized the original improved carrier. On this basis, the experimental
scheme was further optimized.

## [Experiments](#id50)[¶](#experiments "Permalink to this heading")

### [Protocol](#id51)[¶](#protocol "Permalink to this heading")

#### [1. The enzyme digestion reaction of pSP-GM2 vector](#id52)[¶](#the-enzyme-digestion-reaction-of-psp-gm2-vector "Permalink to this heading")

Material: pSP-GM2 vector, restriction enzyme, 10 x Cutsmart buffer, ddH2O.

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

**Procedure：**

1. Add ingredients in 1.5 mL test tubes at a time according to the above
   table.
2. Mix and mark well.
3. 37℃ for 8-10 h.

#### [2. PCR and electropherosis](#id53)[¶](#pcr-and-electropherosis "Permalink to this heading")

Material: DNA template、primer、dNTPs、5 × Q5 reaction buffer、Q5 DNA polymerase.

Procedure: (1) Mix up the recipe into 1.5 mL test tubes according to the following
table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

#### [3. DNA gel electrophoresis](#id54)[¶](#dna-gel-electrophoresis "Permalink to this heading")

(1) Preparation of 1% DNA gel
Weigh 1 g of agarose into a cone-shaped flask, and then add 100 mL of 10xTAE buffer
into this cone-shaped flask. Transfer the cone to the microwave and heat until
boiling (2-3 times). Cooling the cone-shaped flask to about 50℃, and then add the
nucleic acid dye into it. Mixing agarose and nucleic acid well and pour them into
the rubber rack. After 1 hour later, 1% DNA gel is ready.

(2) point sample
Mixing the DNA sample and the adding buffer (4:1). Then adding the mixture to the
sample tank with a micropipette gun. Each tank addes 20 μL mixture. Recording the
order of spot sampling and the amount of adding sample.

(3) Gel electrophoresis
After installing the electrode wire, connect one end of the sample hole to the
negative electrode and the other end to the positive electrode. Then switching on
the power supply and adjust the voltage to 120 V. After electrophoresis for 20-30
min, stop electrophoresis.

#### [4. Transformation of plasmid to E.coli](#id55)[¶](#transformation-of-plasmid-to-e-coli "Permalink to this heading")

1. Take 50 µL of E.coli cells and defrost E.coli with ice bath for 25 min.
   Mixing E.coli gently with 10 µL target recombinant plasmid.
2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for
   another 3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of
   supernatant and resuspend the bacteria. Coating on the LB broth containing
   ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

#### [5. Selecting colonies](#id56)[¶](#selecting-colonies "Permalink to this heading")

**Colony PCR**

1. Inoculate BL21 cells using a pipette tip，use 2x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

#### [6. DNA sequencing](#id57)[¶](#dna-sequencing "Permalink to this heading")

Expand and culture the positive colonies in LB liquid medium containing ampicillin at
37℃ for 10-12 h. Extract the plasmid by using the plasmid extraction kit. Sent 10 μL
plasmid to sequencing company for sequencing. The correctly sequenced plasmid was
stored at -20°C and the corresponding bacterial solution was stored at -80°C.

#### [7. Yeast transformation procedure](#id58)[¶](#yeast-transformation-procedure "Permalink to this heading")

1. selecte the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into 1.5mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into 1.5mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50μL yeast cells, mix evenly, then
   add 500μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1
   h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and
   coate them on a solid medium plate.

## [Implementation](#id59)[¶](#implementation "Permalink to this heading")

### [Background](#id60)[¶](#background "Permalink to this heading")

Patchoulol is one of the most abundant components in patchouli oil[1, 2] and is also
considered the main component contributing to the pharmacological effects and aroma of
patchouli oil[3], which is widely used in medicine, daily chemicals, food, and other
industries, with broad market prospects. However, there is a serious imbalance between
the supply and demand of patchoulol and patchouli oil in the world. There are three ways
to prepare patchouli so far. Typically, patchoulol is extracted from the leaves of
Pogostemon cablin. However, the low content of patchoulol in P. cablin is the bottleneck
of its large-scale application [4]. Although the chemical synthesis method can support
patchoulol with high purity, it has many drawbacks, such as too many steps, complex
operation, high synthesis cost, and low yield, so it is may not suitable for large-scale
industrial production [5]. Compared with plant extraction and chemical synthesis,
microbial fermentation synthesis exhibits several advantages in producing patchoulol,
such as fast-growing, land-saving, and controllable culture conditions. With the
development of synthetic biology, patchoulol has been produced in yeast under the
control of a constitutive promoter. [6] However, the production of patchoulol is
uncontrollable and yeast cells may produce too much patchoulol at a time, which is toxic
to yeast cells. Herein, our team has innovatively applied the light-induced promoter in
the biosynthesis of patchoulol in yeast, which makes the production of patchoulol
controllable. When the light is on, yeast cells produce patchoulol. Otherwise, it is not
produced patchoulol. We believe the increasing production of patchoulol will lower the
price of patchoulol. Furthermore, the lower price of patchoulol can effectively help
more people particularly those in outlying poverty-stricken areas.

### [Target customers](#id61)[¶](#target-customers "Permalink to this heading")

**1. Patchouli essential oil factory**

Patchouli essential oil is in great demand in the market because of its long history of
use, wide range of uses, and good fragrance fixing effect. Patchouli essential oil is
beneficial to the body and mind. For example, it can be mentally refreshing, relieve
anxiety, and make people feel excited. Physiologically, it has a good bactericidal
effect, promotes skin regeneration and reduces inflammation, and so on. These properties
have made patchouli popular. As we all know, patchoulol is one of the main ingredients
in patchouli oil. If our project can produce patchoulol on a large-scale industrial
production, it will generate a lot of business value.

**2. Pharmaceutical factory**

Patchoulol is also considered the main component contributing to the pharmacological
effects of patchouli oil. It is often used as raw materials for drugs because of its
anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other pharmacological
effects[7]. Nowadays, The market demand for patchoulol is increasing. For example,
Huoxiang Zhengqi Shui (KangShengTang), a kind of medicine that is always available in
every household in China, contains a huge amount of patchoulol. Therefore, when our
project achieves the efficient production of patchoulol, we can sell patchoulol to
pharmaceutical factories and profit from it.

**3. Customers who need environment-friendly pesticides**

Because of its insecticidal biological activity, patchoulol can be made into insecticides
to control some specific species of pests. And because it has the characteristics of low
toxicity, easy degradation, safety, and environmental friendliness, it is a good choice
to use as an insecticide. Therefore, we can also consider producing a large amount of
patchoulol by ourselves, preparing it into an environment-friendly insecticide, and
selling it to customers who need it in this regard.[8]

### [Possible challenges](#id62)[¶](#possible-challenges "Permalink to this heading")

Although our project has achieved initial results in the laboratory environment,
considering the difference between the laboratory environment and the actual large-scale
production environment, we are worried that there will be some problems during the
large-scale production. For example, the accumulation of too much yeast may lead to an
untimely and/or insensitive response of the light-induced prompter in yeast to light
conditions, and even cause the light-induced promoter to lose its original function,
making the whole biosynthesis process uncontrollable. These accidents that may occur in
the mass production process will greatly affect the mass production of patchoulol, which
may limit the availability of our product to the market. So for us, problems that arise
during mass production can be a challenge.

### [Reference](#id63)[¶](#id2 "Permalink to this heading")

[1] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[2] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[3] 徐雯, 吴艳清, 丁浩然, 等. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[4] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[5] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of (-)-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[6] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene
production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J].
Appl Environ Microbiol, 2011, 77(3): 1033-40.

[7] 魏晓露, 彭成, 万峰. 广藿香醇体外抗呼吸道病毒作用研究 [J]. 中药药理与临床, 2013, 29(1): 4.

[8] 陈义娟, 范能能, 蒋杰贤, et al. 百秋李醇的用途及杀虫剂 [M]. 2020.

## [Notebook](#id64)[¶](#notebook "Permalink to this heading")

### [Week 1 (6.20-6.26)](#id65)[¶](#week-1-6-20-6-26 "Permalink to this heading")

We inoculated and cultured Escherichia coli containing the target vector in a liquid LB
growth medium. Then we waited for the cultures to grow and develop. We extracted the
target vector and designed primers needed for the construction of our vectors and sent
them to the company for synthesis.

We purchased the reagents needed for our experiments.

### [Week 2 (6.27-7.3)](#id66)[¶](#week-2-6-27-7-3 "Permalink to this heading")

**Part one**
**First attempt to digest target vector.**

We used the target pSP-GM2 vector, restriction enzyme, reaction buffer, and ddH2O to
digest
the target vector. The reaction system is as follows:

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

Procedures to digest target vector:
(1) Add ingredients in 1.5 mL test tubes at a time according to the above table.

2. Mix and mark well.
3. 37℃ for 8-10 h.

**Part teo**
**PCR amplification of corresponding parts.**

PCR amplification for PST gene, FPS gene, and the HCF173 promoter. However, promoter
HCF173 first was not successfully amplified. After the second test, we successfully
amplified HCF173 promoter.

Procedure for PCR amplification:
(1) Mix up the recipe into 1.5 mL test tubes according to the following table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

### [Week 3 (7.4-7.10)](#id67)[¶](#week-3-7-4-7-10 "Permalink to this heading")

**Part one Construction of target vector.**

We construct the target vector by mixing pSP-GM2 vector with the genes we cloned. These
plasmids were later transformed into E.coli BL21.

**Part two Transformation of the plasmid to E.coli**

Procedure:
(1) Take 50 µL of E.coli cells and defrost E.coli with an ice bath for 25 min. Mixing
E.coli gently with 10 µL target recombinant plasmid.

2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for another
   3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix it upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of supernatant
   and resuspend the bacteria. The coating on the LB broth containing ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

### [Week 4 (7.11-7.17)](#id68)[¶](#week-4-7-11-7-17 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Colony PCR was used to select positive colonies, which was verified by sequencing and
then
in liquid LB.

General procedures:

1. Inoculate BL21 cells using a pipette tip, use 2 x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

### [Week 5 (7.18-7.24)](#id69)[¶](#week-5-7-18-7-24 "Permalink to this heading")

**Transformation of plasmid to Yeast**

**Procedure:**

1. select the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into a 1.5 mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into a 1.5 mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50 μL yeast cells, mix evenly, then add
   500 μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1 h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and coat
   them on a solid medium plate.

### [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#id70)[¶](#week-6-8-1-8-7-and-week-7-8-8-8-14 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Single yeast colonies were taken from stock plates and inoculated into 8 ml YPD. We carry
out PCR on these colonies and plan to test them again. Results showed that some plasmids
have been successfully transformed into Yeast.

### [Week 7 (8.15-8.27)](#id71)[¶](#week-7-8-15-8-27 "Permalink to this heading")

**Cultivation of recombinant yeasts in a Shaking Flask.**

The recombinant yeasts were precultured in 5 mL of YPD at 30℃, 220 rpm for 24 h.
Precultures were inoculated to 50 mL of YPGD or YPGL in 250 mL flasks at an initial
OD600 of 0.05 and were grown under the same condition. An overlay of 5 mL dodecane was
added to the flasks after 24 h and the light was opened.

**Analysis of Patchoulol**

Samples from the organic layer were centrifuged for 15 min at 6000 rpm to determine the
level of patchoulol during fermentation. The patchoulol was analyzed by gas
chromatography-mass spectrometry using the HP-5 column. The sample (1 μL) was injected
in splitless mode. The patchoulol standard was used to identify the substances.

## [Proof of concept](#id72)[¶](#proof-of-concept "Permalink to this heading")

### [Method development](#id73)[¶](#method-development "Permalink to this heading")

Two kinds of promoters were selected for gene expression, constitutive promoter PGK1 and
light-inducible promoter HCF173 (BBa\_K4270001). [1]

By fusing with FPS (farnesyl diphosphate synthase), PTS (patchoulol synthase), and
FPS-linker-PTS genes via PCR, they were linked into pSP-GM2 vector. After the
transformation of pSP-GM2 plasmids into E.coli, the DNA of each gene was amplified by
PCR and sequenced for verification. The recombinant vectors were then transformed into
yeast. Fermentation was then carried out and the target compounds were detected by
meteorological chromatography. A qualitative and quantitative analysis was carried out
for the production of patchoulol. The ones with the best performance were further
applied to real fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)

### [Plasmid construction](#id74)[¶](#id3 "Permalink to this heading")

The DNA sequence of PTS (encoding patchoulol synthase from P. cablin) was optimized
according to the preference of yeast for amino acid codons. Then FPS (BBa\_K517004), PTS
(BBa\_K4270004), and FPS-linker-PTS (BBa\_K4270010) were fused with promoter PGK1 or
light-inducible promoter HCF173 and constructed into pSP-GM2 plasmids separately, which
contains ampicillin resistance genes for colony selection.

The eight plasmids: pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS,
pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS,
pSP-GM2-HCF173-PTS-FPS were transformed into E.coli according to the protocol. The
colony PCR was conducted for verification of the correct plasmids (Figure 1 and Figure
2). Then the amplified DNAs were sequenced and compared with the designed DNA sequences
for further verification.

Then these vectors are all transformed into yeast. The colony PCR was conducted for
further verification of these recombinant vectors.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)

**Figure 1. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)

**Figure 2. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [Patchoulol detection](#id75)[¶](#patchoulol-detection "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 3). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)

**Figure 3 The detection of patchoulol in the supernatant. A-H. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS,
pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids,
respectively. I. The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id76)[¶](#id4 "Permalink to this heading")

[1] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Results](#id77)[¶](#id5 "Permalink to this heading")

**Overview**

Our results consist of our gene cloning, colony PCR results and Gas chromatography
results.

### [1. Codon Optimization](#id78)[¶](#codon-optimization "Permalink to this heading")

To improve the translation efficiency of PTS in yeast, we first optimized the Pogostemon
cablin PTS gene according to the codon preference of yeast. During optimization, the
amino acid sequence of the protein encoded by PTS remained unchanged (**Figure
1**) and the codon was adjusted.

[![https://static.igem.wiki/teams/4270/wiki/results1.png](https://static.igem.wiki/teams/4270/wiki/results1.png)](https://static.igem.wiki/teams/4270/wiki/results1.png)

**Figure 1. The original PTS protein sequence and optimized PTS protein
sequence.**

### [2. DNA Preparation](#id79)[¶](#dna-preparation "Permalink to this heading")

The FPS (ERG20) gene was PCR-amplified from the genome of S. cerevisiae 4741. The fusion
of FPS and PTS with a Gly-Ser-Gly (GSG) tag was constructed according to a previous
work,[1] which generated a fusion protein called PTS-linker-FPS. The FPS (ERG20) gene
was PCR-amplified from the genome of S. cerevisiae 4741. The light-inducible promoter
HCF173 (HIGH CHLOROPHYLL FLUORESCENCE173) was PCR-amplified from the genome of wild-type
Arabidopsis thaliana. [2] The results are shown in **Figure 2**.

[![https://static.igem.wiki/teams/4270/wiki/results2.png](https://static.igem.wiki/teams/4270/wiki/results2.png)](https://static.igem.wiki/teams/4270/wiki/results2.png)

**Figure 2A. The PCR results of FPS. B. The PCR results of PTS. C. The PCR results
of HCF173. D. The PCR results of PTS-linker-FPS.**

After HCF173 and FPS, PTS, PTS-linker-FPS were separately amplified, HCF173 and other
genes were overlapped and amplified using primers. The results are shown in **Figure
3**.

[![https://static.igem.wiki/teams/4270/wiki/results3.png](https://static.igem.wiki/teams/4270/wiki/results3.png)](https://static.igem.wiki/teams/4270/wiki/results3.png)

**Figure 3A. The PCR results of HCF173-FPS. B. The PCR results of HCF173-PTS. C.
The PCR results of HCF173- PTS-linker-FPS. D. The PCR results of PTS-FPS (namely
HCF173-FPS- terminator -HCF173- PTS- terminator)**

### [3. Construction of Plasmids](#id80)[¶](#construction-of-plasmids "Permalink to this heading")

We constructed 4 plasmids with the constitutive promoter (pGK1) separately connecting
FPS, PTS, FPS-linker-PTS. The plasmids containing promoter
pGK1-FPS-terminator-pGK1-PTS-terminator were also built. We also constructed 4 plasmids
with HCF173 as the promoter. The schematic diagram of these plasmids is shown in
**Figure 4**.

[![https://static.igem.wiki/teams/4270/wiki/results4.png](https://static.igem.wiki/teams/4270/wiki/results4.png)](https://static.igem.wiki/teams/4270/wiki/results4.png)

**Figure 4. The schematic diagram of plasmids. Pcp, constitutive promoter pGK1.
Plip, light-induced promoter HCF173. Tns, T ADH1 or T CYC1 terminator.**

The colony PCR was conducted for further verification of these recombinant vectors. The
results are shown in **Figure 5** and **Figure 6**.

[![https://static.igem.wiki/teams/4270/wiki/results5.png](https://static.igem.wiki/teams/4270/wiki/results5.png)](https://static.igem.wiki/teams/4270/wiki/results5.png)

**Figure 5A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/results6.png](https://static.igem.wiki/teams/4270/wiki/results6.png)](https://static.igem.wiki/teams/4270/wiki/results6.png)

**Figure 6A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [4. Patchoulol detection](#id81)[¶](#id6 "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 7). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/results7.png](https://static.igem.wiki/teams/4270/wiki/results7.png)](https://static.igem.wiki/teams/4270/wiki/results7.png)

**Figure 7. The detection of patchoulol in the supernatant. A-J. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2- FPS K197P, pSP -GM2- FPS K197H, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS,
pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids, respectively. I. The
detection of patchoulol in the standard substance containing patchoulol.**

### [Table of Contents](static/_static/index.html)

* [Team](team)
* Project
  + [Communication](#communication)
    - [Highschool
      Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
    - [Online
      Conferences](#online-conferences)
    - [Propaganda
      Speech](#propaganda-speech)
    - [Interview
      with Experts](#interview-with-experts)
    - [Interview
      with doctor](#interview-with-doctor)
  + [Contribution](#contribution)
    - [1.Characterization
      of previous iGEM parts](#characterization-of-previous-igem-parts)
    - [2.Add New
      Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
    - [3.Add New
      Parts](#add-new-parts)
    - [4.Improvement of
      Existing Parts](#improvement-of-existing-parts)
    - [Reference](#reference)
  + [Description](#description)
    - [Pogostemon cablin
      and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
    - [Patchoulol](#patchoulol)
    - [Plant
      extraction](#plant-extraction)
    - [Chemical
      synthesis](#chemical-synthesis)
    - [Microbial
      Fermentation](#microbial-fermentation)
    - [Patchoulol
      biosynthetic pathways](#patchoulol-biosynthetic-pathways)
    - [Vision](#vision)
    - [Reference](#id1)
  + [Engineering](#engineering)
    - [Abstract](#abstract)
    - [Experiment
      design](#experiment-design)
    - [Experiment structure
      and testing](#experiment-structure-and-testing)
  + [Experiments](#experiments)
    - [Protocol](#protocol)
  + [Implementation](#implementation)
    - [Background](#background)
    - [Target
      customers](#target-customers)
    - [Possible
      challenges](#possible-challenges)
    - [Reference](#id2)
  + [Notebook](#notebook)
    - [Week 1
      (6.20-6.26)](#week-1-6-20-6-26)
    - [Week 2
      (6.27-7.3)](#week-2-6-27-7-3)
    - [Week 3
      (7.4-7.10)](#week-3-7-4-7-10)
    - [Week 4
      (7.11-7.17)](#week-4-7-11-7-17)
    - [Week 5
      (7.18-7.24)](#week-5-7-18-7-24)
    - [Week 6 (8.1-8.7)
      and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
    - [Week 7
      (8.15-8.27)](#week-7-8-15-8-27)
  + [Proof of
    concept](#proof-of-concept)
    - [Method
      development](#method-development)
    - [Plasmid
      construction](#id3)
    - [Patchoulol
      detection](#patchoulol-detection)
    - [Reference](#id4)
  + [Results](#id5)
    - [1. Codon
      Optimization](#codon-optimization)
    - [2. DNA
      Preparation](#dna-preparation)
    - [3.
      Construction of Plasmids](#construction-of-plasmids)
    - [4. Patchoulol
      detection](#id6)
* [Parts](parts)
* [Safety](safety)
* [Human Practices](human-practices)
* [Model](model)
* [Education](education)

### Navigation

* [index](static/_static/genindex.html "General Index")
* [next](parts "Parts") |
* [previous](team "Team") |
* [GDSYZX v1.0 documentation](static/_static/index.html) »
* Project

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### Navigation

* [index](static/_static/genindex.html "General Index")
* [next](parts "Parts") |
* [previous](team "Team") |
* [GDSYZX v1.0 documentation](static/_static/index.html) »
* Project

# Project[¶](#project "Permalink to this heading")

* [Communication](#communication)

  + [Highschool Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
  + [Online
    Conferences](#online-conferences)
  + [Propaganda
    Speech](#propaganda-speech)
  + [Interview
    with Experts](#interview-with-experts)
  + [Interview
    with doctor](#interview-with-doctor)
* [Contribution](#contribution)

  + [1.Characterization of previous iGEM parts](#characterization-of-previous-igem-parts)
  + [2.Add New
    Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
  + [3.Add New
    Parts](#add-new-parts)
  + [4.Improvement of Existing Parts](#improvement-of-existing-parts)

    - [4.1 Optimized of farnesyl diphosphate synthase (ERG20,
      FPS) (BBa\_K517004)](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004)
    - [4.2
      Plasmid Construction](#plasmid-construction)
    - [4.3
      Results](#results)
  + [Reference](#reference)
* [Description](#description)

  + [Pogostemon cablin and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
  + [Patchoulol](#patchoulol)
  + [Plant
    extraction](#plant-extraction)
  + [Chemical
    synthesis](#chemical-synthesis)
  + [Microbial
    Fermentation](#microbial-fermentation)
  + [Patchoulol biosynthetic pathways](#patchoulol-biosynthetic-pathways)
  + [Vision](#vision)
  + [Reference](#id1)
* [Engineering](#engineering)

  + [Abstract](#abstract)
  + [Experiment
    design](#experiment-design)

    - [8 type of expression vector](#type-of-expression-vector)
  + [Experiment structure and testing](#experiment-structure-and-testing)

    - [PCR amplification and estriction enzyme cutting
      expression vector pSP-GM2](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2)
    - [Likage of expression vector and chemical transformation
      of E.coli DH5α](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5)
    - [Screening of transformants](#screening-of-transformants)
    - [Plasmids
      extraction and extract sequencing](#plasmids-extraction-and-extract-sequencing)
    - [Transformation
      and screening of yeast](#transformation-and-screening-of-yeast)
    - [Transform
      procedure](#transform-procedure)
    - [Fermentation of
      modified strains](#fermentation-of-modified-strains)
    - [Extraction
      and determining of metabolite](#extraction-and-determining-of-metabolite)
    - [Learning and design again:](#learning-and-design-again)
* [Experiments](#experiments)

  + [Protocol](#protocol)

    - [1.
      The enzyme digestion reaction of pSP-GM2 vector](#the-enzyme-digestion-reaction-of-psp-gm2-vector)
    - [2. PCR and electropherosis](#pcr-and-electropherosis)
    - [3. DNA gel electrophoresis](#dna-gel-electrophoresis)
    - [4.
      Transformation of plasmid to E.coli](#transformation-of-plasmid-to-e-coli)
    - [5.
      Selecting colonies](#selecting-colonies)
    - [6. DNA
      sequencing](#dna-sequencing)
    - [7. Yeast transformation procedure](#yeast-transformation-procedure)
* [Implementation](#implementation)

  + [Background](#background)
  + [Target
    customers](#target-customers)
  + [Possible
    challenges](#possible-challenges)
  + [Reference](#id2)
* [Notebook](#notebook)

  + [Week 1
    (6.20-6.26)](#week-1-6-20-6-26)
  + [Week 2
    (6.27-7.3)](#week-2-6-27-7-3)
  + [Week 3
    (7.4-7.10)](#week-3-7-4-7-10)
  + [Week 4
    (7.11-7.17)](#week-4-7-11-7-17)
  + [Week 5
    (7.18-7.24)](#week-5-7-18-7-24)
  + [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
  + [Week 7
    (8.15-8.27)](#week-7-8-15-8-27)
* [Proof of concept](#proof-of-concept)

  + [Method
    development](#method-development)
  + [Plasmid construction](#id3)
  + [Patchoulol
    detection](#patchoulol-detection)
  + [Reference](#id4)
* [Results](#id5)

  + [1. Codon
    Optimization](#codon-optimization)
  + [2. DNA
    Preparation](#dna-preparation)
  + [3.
    Construction of Plasmids](#construction-of-plasmids)
  + [4. Patchoulol
    detection](#id6)

## [Communication](#id12)[¶](#communication "Permalink to this heading")

### [Highschool Teams HP Experience Exchange](#id13)[¶](#highschool-teams-hp-experience-exchange "Permalink to this heading")

We were invited by SYSU-CHINA to visit their laboratory and communicate about our
projects. They showed us their advanced and practical scientific research, and provided
us with experience and advice on the use of the equipment, which made great
contributions to the direction and operation of our team’s experiment. At the same time,
we learned each other’s speech skills, which provided us with valuable experience in
speaking and writing manuscripts.

**Lab Visit**[¶](#id7 "Permalink to this table")

|  |  |  |  |
| --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication1.png](https://static.igem.wiki/teams/4270/wiki/communication1.png) | ![https://static.igem.wiki/teams/4270/wiki/communication2.png](https://static.igem.wiki/teams/4270/wiki/communication2.png) | ![https://static.igem.wiki/teams/4270/wiki/communication3.png](https://static.igem.wiki/teams/4270/wiki/communication3.png) | ![https://static.igem.wiki/teams/4270/wiki/communication4.png](https://static.igem.wiki/teams/4270/wiki/communication4.png) |

### [Online Conferences](#id14)[¶](#online-conferences "Permalink to this heading")

Later, we also had a video conference with SYSU-CHINA team. We presented and shared our
story of patchoulol, questionary experiences, and failures to SYSU-CHINA team members.
They gave us some valuable suggestions. SYSU-CHINA team also presented their research
content about salidroside. The communication with SYSU-CHINA team gave us extraordinary
inspiration for our project.

[![https://static.igem.wiki/teams/4270/wiki/communication5.png](https://static.igem.wiki/teams/4270/wiki/communication5.png)](https://static.igem.wiki/teams/4270/wiki/communication5.png)

**Online Metting**[¶](#id8 "Permalink to this table")

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication6.png](https://static.igem.wiki/teams/4270/wiki/communication6.png) | ![https://static.igem.wiki/teams/4270/wiki/communication7.png](https://static.igem.wiki/teams/4270/wiki/communication7.png) | ![https://static.igem.wiki/teams/4270/wiki/communication8.png](https://static.igem.wiki/teams/4270/wiki/communication8.png) | ![https://static.igem.wiki/teams/4270/wiki/communication9.png](https://static.igem.wiki/teams/4270/wiki/communication9.png) | ![https://static.igem.wiki/teams/4270/wiki/communication10.png](https://static.igem.wiki/teams/4270/wiki/communication10.png) | ![https://static.igem.wiki/teams/4270/wiki/communication11.png](https://static.igem.wiki/teams/4270/wiki/communication11.png) |

### [Propaganda Speech](#id15)[¶](#propaganda-speech "Permalink to this heading")

In order to bring our project closer to the masses, our team held public talks to
introduce ourselves and our results. Adhering to the principle of making synthetic
biology enjoyable, we selected three speakers from our team to describe our team’s
struggles and research results along the way. After returning to campus in September, we
also held another club presentation to show new students our latest progress and basic
information. At the club festival of our school, we publicized our project in the
biology club, so that all the high school freshmen and part of the high school seniors
who participated in the activity knew about our project and activity, and learned
something from it. In all the lectures we held, we received a lot of compliments and
suggestions, so we would adjust our presentation each time and do it better next time to
give everyone a better understanding of the project.

**Public Speaking**[¶](#id9 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication12.png](https://static.igem.wiki/teams/4270/wiki/communication12.png) | ![https://static.igem.wiki/teams/4270/wiki/communication13.png](https://static.igem.wiki/teams/4270/wiki/communication13.png) | ![https://static.igem.wiki/teams/4270/wiki/communication14.png](https://static.igem.wiki/teams/4270/wiki/communication14.png) |

### [Interview with Experts](#id16)[¶](#interview-with-experts "Permalink to this heading")

Considering that the light-induced promoter is used in our project, and the professor’s
research field is about the regulation of photosynthesis, we asked the professor to give
us some suggestions for improving the knowledge of photobiology used in our project.

Initially, the professor says, you need to learn to screen when doing experiments.
Photobiology is a very cutting-edge branch of life science. There have been previous
achievements in this field. When we make use of it, we should screen the suitable
conditions for our experiments according to the actual situation. For example, the
light-induced promoter of HCF173 used in this experiment should be screened before use
to screen out the light-induced promoter with the best-carrying effect with our target
gene to ensure that its activity matches each other with the gene.

In addition, promoter optimization should be carried out in combination with the actual
situation. For example, the light we are using to activate now in our experiments, white
light, means that daily light can activate the promoter. However, in the actual
production, because white light is too easy to appear, it is impossible to achieve a
real light environment. To solve this problem, the professor suggested that the light we
used to induce could be selected from specific bands of light, such as those rare in
nature, so as to avoid the interference of light in nature and make the experiment more
accurate.

What is more, the promoter’s light requirements need to be feasible. If we have a light
promoter that has very high light intensity requirements, and you have to meet these
ultra-high requirements to start, then most of the time you can’t produce. And if very
strong light is required, it will greatly increase the cost of our production.

Considering the problems that arise when moving from the laboratory to mass production,
we also discussed the whole synthetic biology industry with the professor. The professor
pointed out that the laboratory environment is very different from the actual production
environment. For example, in this project, we have a light-induced promoter, and in the
lab, the volume that we synthesize is actually very small, maybe a thin layer in yeast.
However, in mass production, it is still very difficult to ensure that each yeast light
promoter can feel the light in the face of the large volume of the factory. For these
problems, the professor pointed out a clear way for us: we should first form a
theoretical basis in the laboratory, and when we find problems in large-scale
production, we should timely feedback, optimize and solve them in the laboratory, and
then put them into large-scale production for trial. And so on and so on until the
desired effect is achieved.

Finally, we asked professors how they could connect the projects they were working on to
society and make a real contribution. First of all, the professor affirmed our research
project on patchouli and patchoulol, which are very distinctive ingredients derived from
Lingnan’s traditional Chinese medicine. It is a contribution to society to bring
characteristic plants into play. As for the role of patchoulol, the professor pointed
out the following points: one is that patchoulol can be directly used as a component in
medicine, and the other is that patchoulol can be used as a chassis for other products,
providing a prerequisite for other drugs.

According to the suggestions made by the professor during the interview, we plan to make
the following improvements in the future: First, for the optical promoter itself, reduce
its requirements on light and increase the practicability of the product. Second, if in
the future will be put into large-scale production, the problems encountered in the
production should be timely improved in the laboratory.

### [Interview with doctor](#id17)[¶](#interview-with-doctor "Permalink to this heading")

Considering the particularity of the research objects of our project (patchouli and
patchouli alcohol), the team decided to interview some practitioners of traditional
Chinese medicine working on the frontline in the community clinic, and further discuss
the current situation of the use of patchouli and patchouli alcohol and the social value
of our project.

First, we asked doctors what kind of concoctions they typically prescribe to patients,
such as sliced or ground medicine. Doctors say there are only one or two medicines that
usually contain patchouli or patchoulol in hospitals, such as Huoxiang Zhengqi capsule
and Huoxiang Zhengqi liquid.

Then, we briefly introduced our project and discussed the impact of the increased
production of patchoulol on doctors’ consultations and even society as a whole. Doctors
say that if the production of patchoulol can be increased and more patchoulol becomes
more available, the price of patchoulol will fall accordingly. Among them, pharmacists
in the pharmacy told us that the price of Chinese patent medicine has been soaring. If
the price of patchoulol can be reduced, doctors will have fewer concerns when
prescribing medicine to patients, and it will also be able to ensure that some poor
people have access to medicine, which is of great significance to society.

**Interviewing Doctors**[¶](#id10 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication15.png](https://static.igem.wiki/teams/4270/wiki/communication15.png) | ![https://static.igem.wiki/teams/4270/wiki/communication16.png](https://static.igem.wiki/teams/4270/wiki/communication16.png) | ![https://static.igem.wiki/teams/4270/wiki/communication17.png](https://static.igem.wiki/teams/4270/wiki/communication17.png) |

## [Contribution](#id18)[¶](#contribution "Permalink to this heading")

### [1.Characterization of previous iGEM parts](#id19)[¶](#characterization-of-previous-igem-parts "Permalink to this heading")

In this project, basic parts of previous projects are characterized, including pPGK1
promoter (BBa\_K122000), pTEF1 promoter (BBa\_K2765041), TADH1 terminator (BBa\_K122004),
and TCYC1 terminator (BBa\_K122003). Patchoulol synthase (PTS) gene and farnesyl
diphosphate synthase (ERG20, also named FPS) gene are expressed by this promoters and
terminators. The sequences were assembled into pSP-GM2 plasmids and constructed 4
recombinant vectors, namely pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS and
pSP-GM2-PTS-FPS (Figure 1-4). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 5**).

[![https://static.igem.wiki/teams/4270/wiki/contributions1.png](https://static.igem.wiki/teams/4270/wiki/contributions1.png)](https://static.igem.wiki/teams/4270/wiki/contributions1.png)

**Figure 1. The construction of pSP-GM2-FPS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions2.png](https://static.igem.wiki/teams/4270/wiki/contributions2.png)](https://static.igem.wiki/teams/4270/wiki/contributions2.png)

**Figure 2. The construction of pSP-GM2-PTS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions3.png](https://static.igem.wiki/teams/4270/wiki/contributions3.png)](https://static.igem.wiki/teams/4270/wiki/contributions3.png)

**Figure 3. The construction of pSP-GM2-PTS-linker-FPS plasmid with pPGK1 as
promoter and TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions4.png](https://static.igem.wiki/teams/4270/wiki/contributions4.png)](https://static.igem.wiki/teams/4270/wiki/contributions4.png)

**Figure 4. The construction of pSP-GM2-PTS-FPS plasmid with pPGK1 and pTEF1 as
promoter, TADH1 and TCYC1 as terminators, respectively.**

[![https://static.igem.wiki/teams/4270/wiki/contributions5.png](https://static.igem.wiki/teams/4270/wiki/contributions5.png)](https://static.igem.wiki/teams/4270/wiki/contributions5.png)

**Figure 5 A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

### [2.Add New Documentations To An Existing Part](#id20)[¶](#add-new-documentations-to-an-existing-part "Permalink to this heading")

We will artificially cloned 2 new genes including HCF173 promoter and PTS. The patchoulol
synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces
more than 20 valuable sesquiterpenes including patchoulol by conversion of the natural
substrate FPP.[1-3] HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173),
which is required for Arabidopsis thaliana PSII biogenesis. HCF173 transcript levels and
expression were induced by light. HCF173 promoter contains ACE motif and G-box
element.[4] In our project, the CDS sequence of PTS was optimized according to yeast
codon preference. PCR was conducted first for cloning PTS, FPS (ERG20) and HCF173
(**Figure 6**). Later a gene fusion of PTS and FPS namely PTS-linker-FPS
are acquired by removing the stop codon of PTS and replacing it by a short peptide
(Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs.

[![https://static.igem.wiki/teams/4270/wiki/contributions6.png](https://static.igem.wiki/teams/4270/wiki/contributions6.png)](https://static.igem.wiki/teams/4270/wiki/contributions6.png)

**Figure 6. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

Later, we assembled HCF173 promoters, PTS gene and ERG20 (FPS) and constructed 4
recombinant vectors, namely pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS and pSP-GM2-HCF173-PTS-FPS (**Figure 7-10**).
The colony PCR were conducted for further verification of this recombinant vectors
(**Figure 11**).
.. image:: <https://static.igem.wiki/teams/4270/wiki/contributions7.png>

> width
> :   1000px
>
> align
> :   center

**Figure 7. The construction of pSP-GM2-HCF173-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions8.png](https://static.igem.wiki/teams/4270/wiki/contributions8.png)](https://static.igem.wiki/teams/4270/wiki/contributions8.png)

**Figure 8. The construction of pSP-GM2-HCF173-PTS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions9.png](https://static.igem.wiki/teams/4270/wiki/contributions9.png)](https://static.igem.wiki/teams/4270/wiki/contributions9.png)

**Figure 9. The construction of pSP-GM2-HCF173-PTS-linker-FPS plasmid with HCF173
as promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions10.png](https://static.igem.wiki/teams/4270/wiki/contributions10.png)](https://static.igem.wiki/teams/4270/wiki/contributions10.png)

**Figure 10. The construction of pSP-GM2-HCF173-PTS-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions11.png](https://static.igem.wiki/teams/4270/wiki/contributions11.png)](https://static.igem.wiki/teams/4270/wiki/contributions11.png)

**Figure 11. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [3.Add New Parts](#id21)[¶](#add-new-parts "Permalink to this heading")

In our 2022 project, we provide 10 new parts:

> | part name | part number | website |
> | --- | --- | --- |
> | HCF173 promoter | BBa\_K4270001 | [http://parts.igem.org/Part:BBa\_K4270001](http://parts.igem.org/Part%3ABBa_K4270001) |
> | PTS (patchoulol synthase) | BBa\_K4270004 | [http://parts.igem.org/Part:BBa\_K4270004](http://parts.igem.org/Part%3ABBa_K4270004) |
> | erg20 K197P | BBa\_K4270007 | [http://parts.igem.org/Part:BBa\_K4270007](http://parts.igem.org/Part%3ABBa_K4270007) |
> | erg20 K197H | BBa\_K4270008 | [http://parts.igem.org/Part:BBa\_K4270008](http://parts.igem.org/Part%3ABBa_K4270008) |
> | FPS-linker-PTS | BBa\_K4270010 | [http://parts.igem.org/Part:BBa\_K4270010](http://parts.igem.org/Part%3ABBa_K4270010) |
> | pPGK1+PTS+TADH1 | BBa\_K4270011 | [http://parts.igem.org/Part:BBa\_K4270011](http://parts.igem.org/Part%3ABBa_K4270011) |
> | pPGK1+ERG20+TADH1 | BBa\_K4270012 | [http://parts.igem.org/Part:BBa\_K4270012](http://parts.igem.org/Part%3ABBa_K4270012) |
> | pTEF1+ERG20+TCYC1 | BBa\_K4270016 | [http://parts.igem.org/Part:BBa\_K4270016](http://parts.igem.org/Part%3ABBa_K4270016) |
> | HCF173+ ERG20+TADH1 | BBa\_K4270013 | [http://parts.igem.org/Part:BBa\_K4270013](http://parts.igem.org/Part%3ABBa_K4270013) |

### [4.Improvement of Existing Parts](#id22)[¶](#improvement-of-existing-parts "Permalink to this heading")

#### [4.1 Optimized of farnesyl diphosphate synthase (ERG20, FPS) (BBa\_K517004)](#id23)[¶](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004 "Permalink to this heading")

The ERG20 is a basic part comprised from a synthesized farnesyl diphosphate synthase
coding sequence. The basic part BBa\_K4270007 (erg20 K197P) and BBa\_K4270008 (erg20
K197H) are optimized versions of ERG20 (BBa\_K517004) that can be constructed into
pSP-GM2 plasmid. The resulted vectors are named pSP-GM2-FPS, pSP-GM2-FPS K197P and
pSP-GM2-FPS K197H respectively. Then this vectors were transformed into
Saccharomyces cerevisiae BY4741 to produce patchoulol.

#### [4.2 Plasmid Construction](#id24)[¶](#plasmid-construction "Permalink to this heading")

PCR was conducted for cloning FPS (ERG20), erg20 K197P and erg20 K197H. The purified
PCR products were assembled into pSP-GM2 plasmids and constructed 3 recombinant
vectors, namely pSP-GM2-FPS, pSP-GM2-FPS K197P and pSP-GM2-FPS K197H (**Figure
12-13**). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 14**).

[![https://static.igem.wiki/teams/4270/wiki/contributions12.png](https://static.igem.wiki/teams/4270/wiki/contributions12.png)](https://static.igem.wiki/teams/4270/wiki/contributions12.png)

**Figure 12. The construction of pSP-GM2-FPS K197P plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions13.png](https://static.igem.wiki/teams/4270/wiki/contributions13.png)](https://static.igem.wiki/teams/4270/wiki/contributions13.png)

**Figure 13. The construction of pSP-GM2-FPS K197H plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions14.png](https://static.igem.wiki/teams/4270/wiki/contributions14.png)](https://static.igem.wiki/teams/4270/wiki/contributions14.png)

**Figure 14. The colony PCR results of pSP-GM2-FPS K197P (left side) and
pSP-GM2-FPS K197H (right side).**

#### [4.3 Results](#id25)[¶](#results "Permalink to this heading")

Gas chromatography (GC) method to observe the patchoulol in Yeast fermentation broth.
Unfortunately, we did not detect patchoulol in the supernatant but detected
patchoulol in the standard substance containing patchoulol (Figure 15). The reason
we did not detect patchoulol in the supernatant may due to the low yield of
patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/contributions15.png](https://static.igem.wiki/teams/4270/wiki/contributions15.png)](https://static.igem.wiki/teams/4270/wiki/contributions15.png)

**Figure 15. The detection of patchoulol in the supernatant. A-C. The
detection of patchoulol in Yeast fermentation broth from Yeast transformed with
pSP-GM2-FPS, pSP-GM2- FPS K197P, pSP-GM2- FPS K197H plasmids, respectively. D.
The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id26)[¶](#reference "Permalink to this heading")

[1] Albertsen L, Chen Y,Bach L. S, Rattleff S, et al. Diversion of flux toward
sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous
enzymes. Appl. Environ. Microbiol. 2011, 77, 1033−1040.

[2] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid precursors elevates terpene production in plants. Nature Biotechnology, 2006,
24: 1441-47.

[3] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[4] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Description](#id27)[¶](#description "Permalink to this heading")

### [Pogostemon cablin and patchouli oil](#id28)[¶](#pogostemon-cablin-and-patchouli-oil "Permalink to this heading")

Pogostemon cablin is a kind of medicinal aromatic herb and one of the Top 10 drugs in
Guangdong province. P. cablin is mainly grown in Indonesia, Singapore, Vietnam,
Malaysia, China, and other areas.[1] Patchouli oil is made from the dried ground part of
P. cablin by distillation and is mainly used in two aspects.[2] First, it is used as a
drug because of its anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other
pharmacological effects.[3-5] On the other hand, its fragrance is unique and used as a
spices and food additive.[6] Benefiting from its good efficacy, patchouli oil is widely
used in the daily chemical, pharmaceutical, and food industries and has a broad market
prospect. The price of high-quality patchouli oil can be as high as ¥1300 per kilogram.
In terms of the retail price, it can rank 10th among the important essential oils in the
world and has obvious economic benefits. [7, 8]

**Pogostemon cablin**[¶](#id11 "Permalink to this table")

|  |  |
| --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/description1.png](https://static.igem.wiki/teams/4270/wiki/description1.png) | ![https://static.igem.wiki/teams/4270/wiki/description2.png](https://static.igem.wiki/teams/4270/wiki/description2.png) |

### [Patchoulol](#id29)[¶](#patchoulol "Permalink to this heading")

Patchoulol, a sesquiterpene, is one of the most abundant components in patchouli oil. [6,
9] As an important index for evaluating the quality of P. cablin and patchouli oil in
Chinese Pharmacopoeia, patchoulol is also considered the main component contributing to
the pharmacological effects and aroma of patchouli oil. [10] Nowadays, there is a
serious imbalance between the supply and demand of patchoulol and patchouli oil in the
world. The global annual demand for patchoulol and patchouli oil can reach over 2 000 t,
but the annual output is about 1300 t. Indonesia supplies 90 percent of all annual
output, while China produces less than 10 percent of that, which has plenty of room for
growth. [11, 12] Increasing the yield of patchoulol has become a key point in the study
of p. cablin.

At present, patchoulol is obtained in three main ways, namely, plant extraction, chemical
synthesis, and microbial fermentation.

[![https://static.igem.wiki/teams/4270/wiki/description3.png](https://static.igem.wiki/teams/4270/wiki/description3.png)](https://static.igem.wiki/teams/4270/wiki/description3.png)

### [Plant extraction](#id30)[¶](#plant-extraction "Permalink to this heading")

Typically, patchoulol is extracted from the leaves of P.cablin. However, the low content
of patchoulol in P. cablin is the bottleneck of its large-scale application. [2]

### [Chemical synthesis](#id31)[¶](#chemical-synthesis "Permalink to this heading")

Although the chemical synthesis method can support patchoulol with high purity, it needs
many steps, complex operation, high synthesis cost, and the yield are low, so it cannot
be applied to large-scale industrial production. [13]

### [Microbial Fermentation](#id32)[¶](#microbial-fermentation "Permalink to this heading")

Compared with plant extraction and chemical synthesis, microbial fermentation synthesis
exhibits several advantages in producing patchoulol, such as fast-growing, land-saving,
and controllable culture conditions. With the development of metabolic engineering and
synthetic biology, many valuable plant-derived terpenoids also have been produced in
microbial cell factories, such as artemisinic acid [14], ginsenosides [15], and
tanshinones [16]. Although naturally produced patchoulol can be obtained from plants,
patchoulol production using biobased microbial platforms is an economical and
sustainable alternative.

### [Patchoulol biosynthetic pathways](#id33)[¶](#patchoulol-biosynthetic-pathways "Permalink to this heading")

There are two biosynthetic pathways for patchoulol formation, namely the mevalonic acid
(MVA) pathway located in the cytoplasm and the methylerythritol 4-phosphate
(MEP) pathway located in plastids. [17, 18] In general, patchoulol is mainly synthesized
through the MVA pathway. There are 3 key enzymes in the synthesis of patchoulol, namely
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FPS),
and patchoulol synthase (PTS). HMGR catalyzes the irreversible formation of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into MVA. Because it can affect the
yield of IPP (isopentenyl diphosphate), the precursors of patchoulol, HMGR is the key to
the metabolism of patchoulol. FPS catalyzes IPP to FPP (farnesyl diphosphate). PTS is a
key rate-limiting enzyme in the formation of the patchoulol, which catalyzes the
formation of patchoulol skeleton from the FPP precursor, and the skeleton is modified by
secondary enzymes to form patchoulol.

[![https://static.igem.wiki/teams/4270/wiki/description4.png](https://static.igem.wiki/teams/4270/wiki/description4.png)](https://static.igem.wiki/teams/4270/wiki/description4.png)

### [Vision](#id34)[¶](#vision "Permalink to this heading")

Patchoulol production using biobased microbial platforms is an economical and sustainable
alternative. Recently, based on the endogenous mevalonate (MVA) pathway or
2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, heterologous production of patchoulol
has been accomplished by introducing the patchoulol synthase gene in Saccharomyces
cerevisiae [8] and Corynebacterium glutamicum [19], as well as in the moss
Physcomitrella patens[20] and eukaryotic microalga Chlamydomonas reinhardtii [21].
Nevertheless, the yield is still too low for industrial production. Therefore,
significant engineering is required to achieve sufficient yields for industrial
applications. Scientists see the use of environmental signals (light, temperature) and
endogenous signals (quorum sensing, pressure) to establish tightly controlled patchoulol
biological production systems as the future trend.

Promoters are crucial elements in controlling the expression of target genes, and the
emergence of inducible promoters provides the possibility to accurately and flexibly
regulate the expression of foreign genes, which has become one of the hot spots in the
research and development of genetic engineering. [22] Therefore, in the present study,
two metabolic engineering strategies were conducted to enhance the production of
patchoulol in yeast. First, ERG20 (encoding FPP synthase from yeast) and PTS (encoding
patchoulol synthase from P. cablin) were used to increase the utilization of the FPP
precursor. Second, the constituted promoter and light-inducible promoter are
respectively used to drive the overexpression of the critical gene FPS and PTS. In
addition, studies have reported that high-level patchoulol production in tobacco plants
was achieved by fusing FPS and PTS genes. [17] Hence, vectors consisting of a gene
fusion of PTS and FPS (PTS-FPS) inserted downstream of the constitutive and
light-inducible promoter were also constructed. Finally, we carried out fermentation in
yeast and envisioned our final product by meteorological chromatography.

[![https://static.igem.wiki/teams/4270/wiki/description5.png](https://static.igem.wiki/teams/4270/wiki/description5.png)](https://static.igem.wiki/teams/4270/wiki/description5.png)

### [Reference](#id35)[¶](#id1 "Permalink to this heading")

[1] Yao G, Drew B T, Yi T S, et al. Phylogenetic relationships, character evolution and
biogeographic diversification of Pogostemon s.l. (Lamiaceae) [J]. Mol Phylogenet Evol,
2016, 98(184-200.

[2] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[3] Zhang R, Yan P, Li Y, et al. A pharmacokinetic study of patchouli alcohol after a
single oral administration of patchouli alcohol or patchouli oil in rats [J]. European
Journal of Drug Metabolism and Pharmacokinetics, 2016, 41(4): 441-8.

[4] Wu Z, Zeng H, Zhang L, et al. Patchouli alcohol: a natural sesquiterpene against both
inflammation and intestinal barrier damage of ulcerative colitis [J]. Inflammation,
2020, 43(4): 1423-35.

[5] 闵琼, 曾海荣, 陆翠燕, et al. 广藿香醇通过调控上皮间质转化抑制人胃癌细胞HGC-27的侵袭和转移 [J]. 药学服务与研究, 2020, 20(01):
6-11.

[6] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[7] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Ind Crop Prod, 2016, 87(161-76.

[8] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[9] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[10] 徐雯, 吴艳清, 丁浩然, et al. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[11] Swamy M K, Mohanty S K, Sinniah U R, et al. Evaluation of patchouli (Pogostemon
cablin Benth.) cultivars for growth, yield and quality parameters [J]. Journal of
Essential Oil-Bearing Plants, 2015, 18(4): 826-32.

[12] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Industrial Crops & Products, 2016, 87(161-76.

[13] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of ()-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[14] Westfall P J, Pitera D J, Lenihan J R, et al. Production of amorphadiene in yeast,
and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent
artemisinin [J]. P Natl Acad Sci USA, 2012, 109(3): 655-6.

[15] Wei W, Wang P, Wei Y, et al. Characterization of Panax ginseng
UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive
ginsenosides F1 and Rh1 in metabolically engineered yeasts [J]. Molecular Plant,
2015,

[16] Guo J, Zhou Y J, Hillwig M L, et al. CYP76AH1 catalyzes turnover of miltiradiene in
tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts
[J]. Pnas, 2013, 110(29): 12108-13.

[17] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid
precursors elevates terpene production in plants. Nature Biotechnology, 2006, 24:
1441-47.

[18] Tang Y, Zhong L, Wang X, et al. Molecular identification and expression of
sesquiterpene pathway genes responsible for patchoulol biosynthesis and regulation in
Pogostemon cablin［J］.
Bot Stud, 2019, 60：11.

[19] Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum［J］. Genes (Basel), 2018, 9(4): E219.

[20] Zhan X, Zhang YH, Chen DF, et al. Metabolic engineering of the moss Physcomitrella
patens to produce the sesquiterpenoids patchoulol and α/β-santalene［J］. Front Plant Sci,
2014, 5 ：636.

[21] Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a
high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii［J］.
Metab Eng, 2016,
38: 331-343.

[22] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Engineering](#id36)[¶](#engineering "Permalink to this heading")

### [Abstract](#id37)[¶](#abstract "Permalink to this heading")

Patchoulol is one of the main active components of Pogostemon cablin, and it is also an
important indicator for evaluating the medicinal materials of P. cablin in the Chinese
Pharmacopoeia. Patchoulol can be obtained by natural extraction, chemical synthesis, and
synthetic biology engineering. Due to the low content of patchoulol in P. cablin and the
low yield of chemical synthesis, the study of combinatorial biosynthesis and molecular
regulation can provide the basis for obtaining high-content patchoulol. In this study,
on the basis of the biosynthetic pathway, molecular regulation mechanism, and synthetic
biology engineering of patchoulol, the key genes for its biosynthesis were combined and
expressed in heterologous host yeast to produce patchoulol, which provides the technical
foundations of the development and application of patchoulol.

### [Experiment design](#id38)[¶](#experiment-design "Permalink to this heading")

#### [8 type of expression vector](#id39)[¶](#type-of-expression-vector "Permalink to this heading")

We designed eight plasmids: The strong constitutive promoter (pGK1) was separately
used to connect FPS (BBa\_K517004), PTS (BBa\_K4270004), and FPS-linker-PTS
(BBa\_K4270010), and plasmids containing pGK1, FPS, terminator, pGK1, PTS terminator
were constructed .In the following four designs, all the above strong promoters were
replaced with light-induced promoters HCF173, and then the test was carried out to
verify (Figure 1).

[![https://static.igem.wiki/teams/4270/wiki/engineering1.png](https://static.igem.wiki/teams/4270/wiki/engineering1.png)](https://static.igem.wiki/teams/4270/wiki/engineering1.png)

### [Experiment structure and testing](#id40)[¶](#experiment-structure-and-testing "Permalink to this heading")

#### [PCR amplification and estriction enzyme cutting expression vector pSP-GM2](#id41)[¶](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2 "Permalink to this heading")

Amplified by PCR, the plasmids were double digestion by the restriction enzymes NotI
and SacI. Plenty of strands of DNA were obtained. Plasmid DNA, restriction enzyme,
reaction buffer, and ddH2O were added successively in a 1.5 mL sterilized centrifuge
tube according to the table below. Mix and mark well. The mixture reacted at 37℃ for
eight to ten hours. Agarose gel electrophoresis was used to detect the product size
and determine whether the enzyme was successfully cut.

**Table 1 Enzyme digestion reaction system**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | Not I | 0.5 |
> | Sac I | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

#### [Likage of expression vector and chemical transformation of E.coli DH5α](#id42)[¶](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5 "Permalink to this heading")

In vitro assembly gene, including promoter, control of the corresponding enzyme
synthesis gene, sequence of the terminator, DNA ligase access strip DNA, forming a
circular recombinant vector. Add 10 ng vector recombinant DNA solution to 50 μL
competent E.coli, mix well, and let stand in an ice bath for 30 min. By heat pulse
method, the mixture was shocked in a water bath at 42℃ for 30 s, then placed on ice
for 3-5 minutes. 1 mL fresh medium was added and cultured at 37℃ for 1 h for
amplification. Add 500 μL to sterilized LB medium into a centrifuge tube, mix
evenly, and culture for 1 h at 37℃ and 200 rpm. Adjust the speed to 5000rpm,
centrifuge at room temperature for 1min, take out the supernatant and pour it out
until there was 100 μL of thallus remaining.

#### [Screening of transformants](#id43)[¶](#screening-of-transformants "Permalink to this heading")

Pick a single bacteria placement from the conversion plate and place it on the new LB
solid plate containing antibiotics, culture it at 37℃ for 2-4 h and then pick up the
colonies. Put them into the centrifuge tube of the corresponding PCR reaction
solution. Take the colony as the template of PCR reaction and amplify the colony by
T5 Super PCR Mix.

**Table 2 Colony PCR reaction system**

> | Reagent | Dosage |
> | --- | --- |
> | 2×T5 Super PCR Mix | 5 μL |
> | M13F（10 μM） | 0.4 μL |
> | M13R（10 μM） | 0.4 μL |
> | ddH2O | 4.2 μL |

**Table 3 Colony PCR reaction procedure**

> | Temperature | Time | Cycle |
> | --- | --- | --- |
> | 98℃ | 10 min | 1 |
> | 98℃ | 10 s | 35 |
> | 50℃ | 10 s |  |
> | 72℃ | 30 s |  |
> | 72℃ | 2 min | 1 |

#### [Plasmids extraction and extract sequencing](#id44)[¶](#plasmids-extraction-and-extract-sequencing "Permalink to this heading")

The transformed E.coli was inoculated with 0.5% glycerin and ampicillin in sterilized
TB culture medium. Culture at 220 rpm for 12 h at 37℃ until saturated. Adjust the
speed to 10000 rpm, centrifuge at room temperature for 6 min, remove and pour off
the supernatant. The centrifugation process was repeated until the plasmid was
successfully extracted. After gene sequencing, verify whether the vector is
successfully transformed and consistent with the design sequence.

#### [Transformation and screening of yeast](#id45)[¶](#transformation-and-screening-of-yeast "Permalink to this heading")

About 16 colonies growing in LB solid medium containing ampicillin were selected and
verified by 2x Easy Taq enzyme. The PCR system is shown below.

**Table 4 PCR reaction (20 μL)**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | buffer | 8 |

The reaction time and temperature were set according to the table below, and
electrophoresis was performed after amplification.

**Table 5 experimental set**

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

The monoclones were selected into 5 mL of the corresponding defective medium (BY4741
into YPD containing glucose). The culture was incubated overnight at 28℃ at 200-220
rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial solution
into 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out the
supernatant. The monoclones were selected into 5mL of corresponding defective medium
(BY4741 into YPD containing glucose). The culture was incubated overnight at 28℃ at
200-220 rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial
solution into a 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out
the supernatant. Add 500 μL of EZ1 and resuspend thallus. Centrifuge 300-500 rpm for
3-5 min to remove the supernatant. Add 50μL of EZ2, flick, and resuspended the
thallus to obtain a competent state.

#### [Transform procedure](#id46)[¶](#transform-procedure "Permalink to this heading")

3-5 μL plasmid was added to 50 μL competent state, mixed evenly, then 500 μL EZ3 was
added, then evenly and lightly beaten with a blue gun since EZ3 is sticky. Incubated
in a water bath at 30℃ for 45 min-1 h. After centrifugation at 500 rpm for 5 min,
flocculent accumulation of thalli could be seen near the bottom of the tube. After
centrifugation, 400 μL centrifuged EZ3 was sucked off, leaving 100 μL resuspended
thallus and applied directly to the plate.

The vector was transformed into yeast and screened for nutritional deficiency.
Successfully transformed recipient cells can be grown on nutritionally deficient
media. The successfully transformed yeast cells were taken and examined again.

#### [Fermentation of modified strains](#id47)[¶](#fermentation-of-modified-strains "Permalink to this heading")

Provide suitable nutrients and environment for fermentation, so that the successful
transformation of yeast fermentation at room temperature.

#### [Extraction and determining of metabolite](#id48)[¶](#extraction-and-determining-of-metabolite "Permalink to this heading")

The content of patchoulol, a volatile sesquiterpene, was analyzed by gas
chromatography.

#### [Learning and design again:](#id49)[¶](#learning-and-design-again "Permalink to this heading")

We further optimized the original improved carrier. On this basis, the experimental
scheme was further optimized.

## [Experiments](#id50)[¶](#experiments "Permalink to this heading")

### [Protocol](#id51)[¶](#protocol "Permalink to this heading")

#### [1. The enzyme digestion reaction of pSP-GM2 vector](#id52)[¶](#the-enzyme-digestion-reaction-of-psp-gm2-vector "Permalink to this heading")

Material: pSP-GM2 vector, restriction enzyme, 10 x Cutsmart buffer, ddH2O.

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

**Procedure：**

1. Add ingredients in 1.5 mL test tubes at a time according to the above
   table.
2. Mix and mark well.
3. 37℃ for 8-10 h.

#### [2. PCR and electropherosis](#id53)[¶](#pcr-and-electropherosis "Permalink to this heading")

Material: DNA template、primer、dNTPs、5 × Q5 reaction buffer、Q5 DNA polymerase.

Procedure: (1) Mix up the recipe into 1.5 mL test tubes according to the following
table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

#### [3. DNA gel electrophoresis](#id54)[¶](#dna-gel-electrophoresis "Permalink to this heading")

(1) Preparation of 1% DNA gel
Weigh 1 g of agarose into a cone-shaped flask, and then add 100 mL of 10xTAE buffer
into this cone-shaped flask. Transfer the cone to the microwave and heat until
boiling (2-3 times). Cooling the cone-shaped flask to about 50℃, and then add the
nucleic acid dye into it. Mixing agarose and nucleic acid well and pour them into
the rubber rack. After 1 hour later, 1% DNA gel is ready.

(2) point sample
Mixing the DNA sample and the adding buffer (4:1). Then adding the mixture to the
sample tank with a micropipette gun. Each tank addes 20 μL mixture. Recording the
order of spot sampling and the amount of adding sample.

(3) Gel electrophoresis
After installing the electrode wire, connect one end of the sample hole to the
negative electrode and the other end to the positive electrode. Then switching on
the power supply and adjust the voltage to 120 V. After electrophoresis for 20-30
min, stop electrophoresis.

#### [4. Transformation of plasmid to E.coli](#id55)[¶](#transformation-of-plasmid-to-e-coli "Permalink to this heading")

1. Take 50 µL of E.coli cells and defrost E.coli with ice bath for 25 min.
   Mixing E.coli gently with 10 µL target recombinant plasmid.
2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for
   another 3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of
   supernatant and resuspend the bacteria. Coating on the LB broth containing
   ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

#### [5. Selecting colonies](#id56)[¶](#selecting-colonies "Permalink to this heading")

**Colony PCR**

1. Inoculate BL21 cells using a pipette tip，use 2x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

#### [6. DNA sequencing](#id57)[¶](#dna-sequencing "Permalink to this heading")

Expand and culture the positive colonies in LB liquid medium containing ampicillin at
37℃ for 10-12 h. Extract the plasmid by using the plasmid extraction kit. Sent 10 μL
plasmid to sequencing company for sequencing. The correctly sequenced plasmid was
stored at -20°C and the corresponding bacterial solution was stored at -80°C.

#### [7. Yeast transformation procedure](#id58)[¶](#yeast-transformation-procedure "Permalink to this heading")

1. selecte the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into 1.5mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into 1.5mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50μL yeast cells, mix evenly, then
   add 500μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1
   h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and
   coate them on a solid medium plate.

## [Implementation](#id59)[¶](#implementation "Permalink to this heading")

### [Background](#id60)[¶](#background "Permalink to this heading")

Patchoulol is one of the most abundant components in patchouli oil[1, 2] and is also
considered the main component contributing to the pharmacological effects and aroma of
patchouli oil[3], which is widely used in medicine, daily chemicals, food, and other
industries, with broad market prospects. However, there is a serious imbalance between
the supply and demand of patchoulol and patchouli oil in the world. There are three ways
to prepare patchouli so far. Typically, patchoulol is extracted from the leaves of
Pogostemon cablin. However, the low content of patchoulol in P. cablin is the bottleneck
of its large-scale application [4]. Although the chemical synthesis method can support
patchoulol with high purity, it has many drawbacks, such as too many steps, complex
operation, high synthesis cost, and low yield, so it is may not suitable for large-scale
industrial production [5]. Compared with plant extraction and chemical synthesis,
microbial fermentation synthesis exhibits several advantages in producing patchoulol,
such as fast-growing, land-saving, and controllable culture conditions. With the
development of synthetic biology, patchoulol has been produced in yeast under the
control of a constitutive promoter. [6] However, the production of patchoulol is
uncontrollable and yeast cells may produce too much patchoulol at a time, which is toxic
to yeast cells. Herein, our team has innovatively applied the light-induced promoter in
the biosynthesis of patchoulol in yeast, which makes the production of patchoulol
controllable. When the light is on, yeast cells produce patchoulol. Otherwise, it is not
produced patchoulol. We believe the increasing production of patchoulol will lower the
price of patchoulol. Furthermore, the lower price of patchoulol can effectively help
more people particularly those in outlying poverty-stricken areas.

### [Target customers](#id61)[¶](#target-customers "Permalink to this heading")

**1. Patchouli essential oil factory**

Patchouli essential oil is in great demand in the market because of its long history of
use, wide range of uses, and good fragrance fixing effect. Patchouli essential oil is
beneficial to the body and mind. For example, it can be mentally refreshing, relieve
anxiety, and make people feel excited. Physiologically, it has a good bactericidal
effect, promotes skin regeneration and reduces inflammation, and so on. These properties
have made patchouli popular. As we all know, patchoulol is one of the main ingredients
in patchouli oil. If our project can produce patchoulol on a large-scale industrial
production, it will generate a lot of business value.

**2. Pharmaceutical factory**

Patchoulol is also considered the main component contributing to the pharmacological
effects of patchouli oil. It is often used as raw materials for drugs because of its
anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other pharmacological
effects[7]. Nowadays, The market demand for patchoulol is increasing. For example,
Huoxiang Zhengqi Shui (KangShengTang), a kind of medicine that is always available in
every household in China, contains a huge amount of patchoulol. Therefore, when our
project achieves the efficient production of patchoulol, we can sell patchoulol to
pharmaceutical factories and profit from it.

**3. Customers who need environment-friendly pesticides**

Because of its insecticidal biological activity, patchoulol can be made into insecticides
to control some specific species of pests. And because it has the characteristics of low
toxicity, easy degradation, safety, and environmental friendliness, it is a good choice
to use as an insecticide. Therefore, we can also consider producing a large amount of
patchoulol by ourselves, preparing it into an environment-friendly insecticide, and
selling it to customers who need it in this regard.[8]

### [Possible challenges](#id62)[¶](#possible-challenges "Permalink to this heading")

Although our project has achieved initial results in the laboratory environment,
considering the difference between the laboratory environment and the actual large-scale
production environment, we are worried that there will be some problems during the
large-scale production. For example, the accumulation of too much yeast may lead to an
untimely and/or insensitive response of the light-induced prompter in yeast to light
conditions, and even cause the light-induced promoter to lose its original function,
making the whole biosynthesis process uncontrollable. These accidents that may occur in
the mass production process will greatly affect the mass production of patchoulol, which
may limit the availability of our product to the market. So for us, problems that arise
during mass production can be a challenge.

### [Reference](#id63)[¶](#id2 "Permalink to this heading")

[1] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[2] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[3] 徐雯, 吴艳清, 丁浩然, 等. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[4] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[5] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of (-)-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[6] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene
production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J].
Appl Environ Microbiol, 2011, 77(3): 1033-40.

[7] 魏晓露, 彭成, 万峰. 广藿香醇体外抗呼吸道病毒作用研究 [J]. 中药药理与临床, 2013, 29(1): 4.

[8] 陈义娟, 范能能, 蒋杰贤, et al. 百秋李醇的用途及杀虫剂 [M]. 2020.

## [Notebook](#id64)[¶](#notebook "Permalink to this heading")

### [Week 1 (6.20-6.26)](#id65)[¶](#week-1-6-20-6-26 "Permalink to this heading")

We inoculated and cultured Escherichia coli containing the target vector in a liquid LB
growth medium. Then we waited for the cultures to grow and develop. We extracted the
target vector and designed primers needed for the construction of our vectors and sent
them to the company for synthesis.

We purchased the reagents needed for our experiments.

### [Week 2 (6.27-7.3)](#id66)[¶](#week-2-6-27-7-3 "Permalink to this heading")

**Part one**
**First attempt to digest target vector.**

We used the target pSP-GM2 vector, restriction enzyme, reaction buffer, and ddH2O to
digest
the target vector. The reaction system is as follows:

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

Procedures to digest target vector:
(1) Add ingredients in 1.5 mL test tubes at a time according to the above table.

2. Mix and mark well.
3. 37℃ for 8-10 h.

**Part teo**
**PCR amplification of corresponding parts.**

PCR amplification for PST gene, FPS gene, and the HCF173 promoter. However, promoter
HCF173 first was not successfully amplified. After the second test, we successfully
amplified HCF173 promoter.

Procedure for PCR amplification:
(1) Mix up the recipe into 1.5 mL test tubes according to the following table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

### [Week 3 (7.4-7.10)](#id67)[¶](#week-3-7-4-7-10 "Permalink to this heading")

**Part one Construction of target vector.**

We construct the target vector by mixing pSP-GM2 vector with the genes we cloned. These
plasmids were later transformed into E.coli BL21.

**Part two Transformation of the plasmid to E.coli**

Procedure:
(1) Take 50 µL of E.coli cells and defrost E.coli with an ice bath for 25 min. Mixing
E.coli gently with 10 µL target recombinant plasmid.

2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for another
   3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix it upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of supernatant
   and resuspend the bacteria. The coating on the LB broth containing ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

### [Week 4 (7.11-7.17)](#id68)[¶](#week-4-7-11-7-17 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Colony PCR was used to select positive colonies, which was verified by sequencing and
then
in liquid LB.

General procedures:

1. Inoculate BL21 cells using a pipette tip, use 2 x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

### [Week 5 (7.18-7.24)](#id69)[¶](#week-5-7-18-7-24 "Permalink to this heading")

**Transformation of plasmid to Yeast**

**Procedure:**

1. select the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into a 1.5 mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into a 1.5 mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50 μL yeast cells, mix evenly, then add
   500 μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1 h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and coat
   them on a solid medium plate.

### [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#id70)[¶](#week-6-8-1-8-7-and-week-7-8-8-8-14 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Single yeast colonies were taken from stock plates and inoculated into 8 ml YPD. We carry
out PCR on these colonies and plan to test them again. Results showed that some plasmids
have been successfully transformed into Yeast.

### [Week 7 (8.15-8.27)](#id71)[¶](#week-7-8-15-8-27 "Permalink to this heading")

**Cultivation of recombinant yeasts in a Shaking Flask.**

The recombinant yeasts were precultured in 5 mL of YPD at 30℃, 220 rpm for 24 h.
Precultures were inoculated to 50 mL of YPGD or YPGL in 250 mL flasks at an initial
OD600 of 0.05 and were grown under the same condition. An overlay of 5 mL dodecane was
added to the flasks after 24 h and the light was opened.

**Analysis of Patchoulol**

Samples from the organic layer were centrifuged for 15 min at 6000 rpm to determine the
level of patchoulol during fermentation. The patchoulol was analyzed by gas
chromatography-mass spectrometry using the HP-5 column. The sample (1 μL) was injected
in splitless mode. The patchoulol standard was used to identify the substances.

## [Proof of concept](#id72)[¶](#proof-of-concept "Permalink to this heading")

### [Method development](#id73)[¶](#method-development "Permalink to this heading")

Two kinds of promoters were selected for gene expression, constitutive promoter PGK1 and
light-inducible promoter HCF173 (BBa\_K4270001). [1]

By fusing with FPS (farnesyl diphosphate synthase), PTS (patchoulol synthase), and
FPS-linker-PTS genes via PCR, they were linked into pSP-GM2 vector. After the
transformation of pSP-GM2 plasmids into E.coli, the DNA of each gene was amplified by
PCR and sequenced for verification. The recombinant vectors were then transformed into
yeast. Fermentation was then carried out and the target compounds were detected by
meteorological chromatography. A qualitative and quantitative analysis was carried out
for the production of patchoulol. The ones with the best performance were further
applied to real fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)

### [Plasmid construction](#id74)[¶](#id3 "Permalink to this heading")

The DNA sequence of PTS (encoding patchoulol synthase from P. cablin) was optimized
according to the preference of yeast for amino acid codons. Then FPS (BBa\_K517004), PTS
(BBa\_K4270004), and FPS-linker-PTS (BBa\_K4270010) were fused with promoter PGK1 or
light-inducible promoter HCF173 and constructed into pSP-GM2 plasmids separately, which
contains ampicillin resistance genes for colony selection.

The eight plasmids: pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS,
pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS,
pSP-GM2-HCF173-PTS-FPS were transformed into E.coli according to the protocol. The
colony PCR was conducted for verification of the correct plasmids (Figure 1 and Figure
2). Then the amplified DNAs were sequenced and compared with the designed DNA sequences
for further verification.

Then these vectors are all transformed into yeast. The colony PCR was conducted for
further verification of these recombinant vectors.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)

**Figure 1. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)

**Figure 2. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [Patchoulol detection](#id75)[¶](#patchoulol-detection "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 3). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)

**Figure 3 The detection of patchoulol in the supernatant. A-H. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS,
pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids,
respectively. I. The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id76)[¶](#id4 "Permalink to this heading")

[1] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Results](#id77)[¶](#id5 "Permalink to this heading")

**Overview**

Our results consist of our gene cloning, colony PCR results and Gas chromatography
results.

### [1. Codon Optimization](#id78)[¶](#codon-optimization "Permalink to this heading")

To improve the translation efficiency of PTS in yeast, we first optimized the Pogostemon
cablin PTS gene according to the codon preference of yeast. During optimization, the
amino acid sequence of the protein encoded by PTS remained unchanged (**Figure
1**) and the codon was adjusted.

[![https://static.igem.wiki/teams/4270/wiki/results1.png](https://static.igem.wiki/teams/4270/wiki/results1.png)](https://static.igem.wiki/teams/4270/wiki/results1.png)

**Figure 1. The original PTS protein sequence and optimized PTS protein
sequence.**

### [2. DNA Preparation](#id79)[¶](#dna-preparation "Permalink to this heading")

The FPS (ERG20) gene was PCR-amplified from the genome of S. cerevisiae 4741. The fusion
of FPS and PTS with a Gly-Ser-Gly (GSG) tag was constructed according to a previous
work,[1] which generated a fusion protein called PTS-linker-FPS. The FPS (ERG20) gene
was PCR-amplified from the genome of S. cerevisiae 4741. The light-inducible promoter
HCF173 (HIGH CHLOROPHYLL FLUORESCENCE173) was PCR-amplified from the genome of wild-type
Arabidopsis thaliana. [2] The results are shown in **Figure 2**.

[![https://static.igem.wiki/teams/4270/wiki/results2.png](https://static.igem.wiki/teams/4270/wiki/results2.png)](https://static.igem.wiki/teams/4270/wiki/results2.png)

**Figure 2A. The PCR results of FPS. B. The PCR results of PTS. C. The PCR results
of HCF173. D. The PCR results of PTS-linker-FPS.**

After HCF173 and FPS, PTS, PTS-linker-FPS were separately amplified, HCF173 and other
genes were overlapped and amplified using primers. The results are shown in **Figure
3**.

[![https://static.igem.wiki/teams/4270/wiki/results3.png](https://static.igem.wiki/teams/4270/wiki/results3.png)](https://static.igem.wiki/teams/4270/wiki/results3.png)

**Figure 3A. The PCR results of HCF173-FPS. B. The PCR results of HCF173-PTS. C.
The PCR results of HCF173- PTS-linker-FPS. D. The PCR results of PTS-FPS (namely
HCF173-FPS- terminator -HCF173- PTS- terminator)**

### [3. Construction of Plasmids](#id80)[¶](#construction-of-plasmids "Permalink to this heading")

We constructed 4 plasmids with the constitutive promoter (pGK1) separately connecting
FPS, PTS, FPS-linker-PTS. The plasmids containing promoter
pGK1-FPS-terminator-pGK1-PTS-terminator were also built. We also constructed 4 plasmids
with HCF173 as the promoter. The schematic diagram of these plasmids is shown in
**Figure 4**.

[![https://static.igem.wiki/teams/4270/wiki/results4.png](https://static.igem.wiki/teams/4270/wiki/results4.png)](https://static.igem.wiki/teams/4270/wiki/results4.png)

**Figure 4. The schematic diagram of plasmids. Pcp, constitutive promoter pGK1.
Plip, light-induced promoter HCF173. Tns, T ADH1 or T CYC1 terminator.**

The colony PCR was conducted for further verification of these recombinant vectors. The
results are shown in **Figure 5** and **Figure 6**.

[![https://static.igem.wiki/teams/4270/wiki/results5.png](https://static.igem.wiki/teams/4270/wiki/results5.png)](https://static.igem.wiki/teams/4270/wiki/results5.png)

**Figure 5A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/results6.png](https://static.igem.wiki/teams/4270/wiki/results6.png)](https://static.igem.wiki/teams/4270/wiki/results6.png)

**Figure 6A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [4. Patchoulol detection](#id81)[¶](#id6 "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 7). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/results7.png](https://static.igem.wiki/teams/4270/wiki/results7.png)](https://static.igem.wiki/teams/4270/wiki/results7.png)

**Figure 7. The detection of patchoulol in the supernatant. A-J. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2- FPS K197P, pSP -GM2- FPS K197H, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS,
pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids, respectively. I. The
detection of patchoulol in the standard substance containing patchoulol.**

### [Table of Contents](static/_static/index.html)

* [Team](team)
* Project
  + [Communication](#communication)
    - [Highschool
      Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
    - [Online
      Conferences](#online-conferences)
    - [Propaganda
      Speech](#propaganda-speech)
    - [Interview
      with Experts](#interview-with-experts)
    - [Interview
      with doctor](#interview-with-doctor)
  + [Contribution](#contribution)
    - [1.Characterization
      of previous iGEM parts](#characterization-of-previous-igem-parts)
    - [2.Add New
      Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
    - [3.Add New
      Parts](#add-new-parts)
    - [4.Improvement of
      Existing Parts](#improvement-of-existing-parts)
    - [Reference](#reference)
  + [Description](#description)
    - [Pogostemon cablin
      and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
    - [Patchoulol](#patchoulol)
    - [Plant
      extraction](#plant-extraction)
    - [Chemical
      synthesis](#chemical-synthesis)
    - [Microbial
      Fermentation](#microbial-fermentation)
    - [Patchoulol
      biosynthetic pathways](#patchoulol-biosynthetic-pathways)
    - [Vision](#vision)
    - [Reference](#id1)
  + [Engineering](#engineering)
    - [Abstract](#abstract)
    - [Experiment
      design](#experiment-design)
    - [Experiment structure
      and testing](#experiment-structure-and-testing)
  + [Experiments](#experiments)
    - [Protocol](#protocol)
  + [Implementation](#implementation)
    - [Background](#background)
    - [Target
      customers](#target-customers)
    - [Possible
      challenges](#possible-challenges)
    - [Reference](#id2)
  + [Notebook](#notebook)
    - [Week 1
      (6.20-6.26)](#week-1-6-20-6-26)
    - [Week 2
      (6.27-7.3)](#week-2-6-27-7-3)
    - [Week 3
      (7.4-7.10)](#week-3-7-4-7-10)
    - [Week 4
      (7.11-7.17)](#week-4-7-11-7-17)
    - [Week 5
      (7.18-7.24)](#week-5-7-18-7-24)
    - [Week 6 (8.1-8.7)
      and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
    - [Week 7
      (8.15-8.27)](#week-7-8-15-8-27)
  + [Proof of
    concept](#proof-of-concept)
    - [Method
      development](#method-development)
    - [Plasmid
      construction](#id3)
    - [Patchoulol
      detection](#patchoulol-detection)
    - [Reference](#id4)
  + [Results](#id5)
    - [1. Codon
      Optimization](#codon-optimization)
    - [2. DNA
      Preparation](#dna-preparation)
    - [3.
      Construction of Plasmids](#construction-of-plasmids)
    - [4. Patchoulol
      detection](#id6)
* [Parts](parts)
* [Safety](safety)
* [Human Practices](human-practices)
* [Model](model)
* [Education](education)

### Navigation

* [index](static/_static/genindex.html "General Index")
* [next](parts "Parts") |
* [previous](team "Team") |
* [GDSYZX v1.0 documentation](static/_static/index.html) »
* Project

© Copyright 2022, chenzhr23.
Created using [Sphinx](https://www.sphinx-doc.org/) 5.1.1.

### Navigation

* [index](static/_static/genindex.html "General Index")
* [next](parts "Parts") |
* [previous](team "Team") |
* [GDSYZX v1.0 documentation](static/_static/index.html) »
* Project

# Project[¶](#project "Permalink to this heading")

* [Communication](#communication)

  + [Highschool Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
  + [Online
    Conferences](#online-conferences)
  + [Propaganda
    Speech](#propaganda-speech)
  + [Interview
    with Experts](#interview-with-experts)
  + [Interview
    with doctor](#interview-with-doctor)
* [Contribution](#contribution)

  + [1.Characterization of previous iGEM parts](#characterization-of-previous-igem-parts)
  + [2.Add New
    Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
  + [3.Add New
    Parts](#add-new-parts)
  + [4.Improvement of Existing Parts](#improvement-of-existing-parts)

    - [4.1 Optimized of farnesyl diphosphate synthase (ERG20,
      FPS) (BBa\_K517004)](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004)
    - [4.2
      Plasmid Construction](#plasmid-construction)
    - [4.3
      Results](#results)
  + [Reference](#reference)
* [Description](#description)

  + [Pogostemon cablin and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
  + [Patchoulol](#patchoulol)
  + [Plant
    extraction](#plant-extraction)
  + [Chemical
    synthesis](#chemical-synthesis)
  + [Microbial
    Fermentation](#microbial-fermentation)
  + [Patchoulol biosynthetic pathways](#patchoulol-biosynthetic-pathways)
  + [Vision](#vision)
  + [Reference](#id1)
* [Engineering](#engineering)

  + [Abstract](#abstract)
  + [Experiment
    design](#experiment-design)

    - [8 type of expression vector](#type-of-expression-vector)
  + [Experiment structure and testing](#experiment-structure-and-testing)

    - [PCR amplification and estriction enzyme cutting
      expression vector pSP-GM2](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2)
    - [Likage of expression vector and chemical transformation
      of E.coli DH5α](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5)
    - [Screening of transformants](#screening-of-transformants)
    - [Plasmids
      extraction and extract sequencing](#plasmids-extraction-and-extract-sequencing)
    - [Transformation
      and screening of yeast](#transformation-and-screening-of-yeast)
    - [Transform
      procedure](#transform-procedure)
    - [Fermentation of
      modified strains](#fermentation-of-modified-strains)
    - [Extraction
      and determining of metabolite](#extraction-and-determining-of-metabolite)
    - [Learning and design again:](#learning-and-design-again)
* [Experiments](#experiments)

  + [Protocol](#protocol)

    - [1.
      The enzyme digestion reaction of pSP-GM2 vector](#the-enzyme-digestion-reaction-of-psp-gm2-vector)
    - [2. PCR and electropherosis](#pcr-and-electropherosis)
    - [3. DNA gel electrophoresis](#dna-gel-electrophoresis)
    - [4.
      Transformation of plasmid to E.coli](#transformation-of-plasmid-to-e-coli)
    - [5.
      Selecting colonies](#selecting-colonies)
    - [6. DNA
      sequencing](#dna-sequencing)
    - [7. Yeast transformation procedure](#yeast-transformation-procedure)
* [Implementation](#implementation)

  + [Background](#background)
  + [Target
    customers](#target-customers)
  + [Possible
    challenges](#possible-challenges)
  + [Reference](#id2)
* [Notebook](#notebook)

  + [Week 1
    (6.20-6.26)](#week-1-6-20-6-26)
  + [Week 2
    (6.27-7.3)](#week-2-6-27-7-3)
  + [Week 3
    (7.4-7.10)](#week-3-7-4-7-10)
  + [Week 4
    (7.11-7.17)](#week-4-7-11-7-17)
  + [Week 5
    (7.18-7.24)](#week-5-7-18-7-24)
  + [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
  + [Week 7
    (8.15-8.27)](#week-7-8-15-8-27)
* [Proof of concept](#proof-of-concept)

  + [Method
    development](#method-development)
  + [Plasmid construction](#id3)
  + [Patchoulol
    detection](#patchoulol-detection)
  + [Reference](#id4)
* [Results](#id5)

  + [1. Codon
    Optimization](#codon-optimization)
  + [2. DNA
    Preparation](#dna-preparation)
  + [3.
    Construction of Plasmids](#construction-of-plasmids)
  + [4. Patchoulol
    detection](#id6)

## [Communication](#id12)[¶](#communication "Permalink to this heading")

### [Highschool Teams HP Experience Exchange](#id13)[¶](#highschool-teams-hp-experience-exchange "Permalink to this heading")

We were invited by SYSU-CHINA to visit their laboratory and communicate about our
projects. They showed us their advanced and practical scientific research, and provided
us with experience and advice on the use of the equipment, which made great
contributions to the direction and operation of our team’s experiment. At the same time,
we learned each other’s speech skills, which provided us with valuable experience in
speaking and writing manuscripts.

**Lab Visit**[¶](#id7 "Permalink to this table")

|  |  |  |  |
| --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication1.png](https://static.igem.wiki/teams/4270/wiki/communication1.png) | ![https://static.igem.wiki/teams/4270/wiki/communication2.png](https://static.igem.wiki/teams/4270/wiki/communication2.png) | ![https://static.igem.wiki/teams/4270/wiki/communication3.png](https://static.igem.wiki/teams/4270/wiki/communication3.png) | ![https://static.igem.wiki/teams/4270/wiki/communication4.png](https://static.igem.wiki/teams/4270/wiki/communication4.png) |

### [Online Conferences](#id14)[¶](#online-conferences "Permalink to this heading")

Later, we also had a video conference with SYSU-CHINA team. We presented and shared our
story of patchoulol, questionary experiences, and failures to SYSU-CHINA team members.
They gave us some valuable suggestions. SYSU-CHINA team also presented their research
content about salidroside. The communication with SYSU-CHINA team gave us extraordinary
inspiration for our project.

[![https://static.igem.wiki/teams/4270/wiki/communication5.png](https://static.igem.wiki/teams/4270/wiki/communication5.png)](https://static.igem.wiki/teams/4270/wiki/communication5.png)

**Online Metting**[¶](#id8 "Permalink to this table")

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication6.png](https://static.igem.wiki/teams/4270/wiki/communication6.png) | ![https://static.igem.wiki/teams/4270/wiki/communication7.png](https://static.igem.wiki/teams/4270/wiki/communication7.png) | ![https://static.igem.wiki/teams/4270/wiki/communication8.png](https://static.igem.wiki/teams/4270/wiki/communication8.png) | ![https://static.igem.wiki/teams/4270/wiki/communication9.png](https://static.igem.wiki/teams/4270/wiki/communication9.png) | ![https://static.igem.wiki/teams/4270/wiki/communication10.png](https://static.igem.wiki/teams/4270/wiki/communication10.png) | ![https://static.igem.wiki/teams/4270/wiki/communication11.png](https://static.igem.wiki/teams/4270/wiki/communication11.png) |

### [Propaganda Speech](#id15)[¶](#propaganda-speech "Permalink to this heading")

In order to bring our project closer to the masses, our team held public talks to
introduce ourselves and our results. Adhering to the principle of making synthetic
biology enjoyable, we selected three speakers from our team to describe our team’s
struggles and research results along the way. After returning to campus in September, we
also held another club presentation to show new students our latest progress and basic
information. At the club festival of our school, we publicized our project in the
biology club, so that all the high school freshmen and part of the high school seniors
who participated in the activity knew about our project and activity, and learned
something from it. In all the lectures we held, we received a lot of compliments and
suggestions, so we would adjust our presentation each time and do it better next time to
give everyone a better understanding of the project.

**Public Speaking**[¶](#id9 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication12.png](https://static.igem.wiki/teams/4270/wiki/communication12.png) | ![https://static.igem.wiki/teams/4270/wiki/communication13.png](https://static.igem.wiki/teams/4270/wiki/communication13.png) | ![https://static.igem.wiki/teams/4270/wiki/communication14.png](https://static.igem.wiki/teams/4270/wiki/communication14.png) |

### [Interview with Experts](#id16)[¶](#interview-with-experts "Permalink to this heading")

Considering that the light-induced promoter is used in our project, and the professor’s
research field is about the regulation of photosynthesis, we asked the professor to give
us some suggestions for improving the knowledge of photobiology used in our project.

Initially, the professor says, you need to learn to screen when doing experiments.
Photobiology is a very cutting-edge branch of life science. There have been previous
achievements in this field. When we make use of it, we should screen the suitable
conditions for our experiments according to the actual situation. For example, the
light-induced promoter of HCF173 used in this experiment should be screened before use
to screen out the light-induced promoter with the best-carrying effect with our target
gene to ensure that its activity matches each other with the gene.

In addition, promoter optimization should be carried out in combination with the actual
situation. For example, the light we are using to activate now in our experiments, white
light, means that daily light can activate the promoter. However, in the actual
production, because white light is too easy to appear, it is impossible to achieve a
real light environment. To solve this problem, the professor suggested that the light we
used to induce could be selected from specific bands of light, such as those rare in
nature, so as to avoid the interference of light in nature and make the experiment more
accurate.

What is more, the promoter’s light requirements need to be feasible. If we have a light
promoter that has very high light intensity requirements, and you have to meet these
ultra-high requirements to start, then most of the time you can’t produce. And if very
strong light is required, it will greatly increase the cost of our production.

Considering the problems that arise when moving from the laboratory to mass production,
we also discussed the whole synthetic biology industry with the professor. The professor
pointed out that the laboratory environment is very different from the actual production
environment. For example, in this project, we have a light-induced promoter, and in the
lab, the volume that we synthesize is actually very small, maybe a thin layer in yeast.
However, in mass production, it is still very difficult to ensure that each yeast light
promoter can feel the light in the face of the large volume of the factory. For these
problems, the professor pointed out a clear way for us: we should first form a
theoretical basis in the laboratory, and when we find problems in large-scale
production, we should timely feedback, optimize and solve them in the laboratory, and
then put them into large-scale production for trial. And so on and so on until the
desired effect is achieved.

Finally, we asked professors how they could connect the projects they were working on to
society and make a real contribution. First of all, the professor affirmed our research
project on patchouli and patchoulol, which are very distinctive ingredients derived from
Lingnan’s traditional Chinese medicine. It is a contribution to society to bring
characteristic plants into play. As for the role of patchoulol, the professor pointed
out the following points: one is that patchoulol can be directly used as a component in
medicine, and the other is that patchoulol can be used as a chassis for other products,
providing a prerequisite for other drugs.

According to the suggestions made by the professor during the interview, we plan to make
the following improvements in the future: First, for the optical promoter itself, reduce
its requirements on light and increase the practicability of the product. Second, if in
the future will be put into large-scale production, the problems encountered in the
production should be timely improved in the laboratory.

### [Interview with doctor](#id17)[¶](#interview-with-doctor "Permalink to this heading")

Considering the particularity of the research objects of our project (patchouli and
patchouli alcohol), the team decided to interview some practitioners of traditional
Chinese medicine working on the frontline in the community clinic, and further discuss
the current situation of the use of patchouli and patchouli alcohol and the social value
of our project.

First, we asked doctors what kind of concoctions they typically prescribe to patients,
such as sliced or ground medicine. Doctors say there are only one or two medicines that
usually contain patchouli or patchoulol in hospitals, such as Huoxiang Zhengqi capsule
and Huoxiang Zhengqi liquid.

Then, we briefly introduced our project and discussed the impact of the increased
production of patchoulol on doctors’ consultations and even society as a whole. Doctors
say that if the production of patchoulol can be increased and more patchoulol becomes
more available, the price of patchoulol will fall accordingly. Among them, pharmacists
in the pharmacy told us that the price of Chinese patent medicine has been soaring. If
the price of patchoulol can be reduced, doctors will have fewer concerns when
prescribing medicine to patients, and it will also be able to ensure that some poor
people have access to medicine, which is of great significance to society.

**Interviewing Doctors**[¶](#id10 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication15.png](https://static.igem.wiki/teams/4270/wiki/communication15.png) | ![https://static.igem.wiki/teams/4270/wiki/communication16.png](https://static.igem.wiki/teams/4270/wiki/communication16.png) | ![https://static.igem.wiki/teams/4270/wiki/communication17.png](https://static.igem.wiki/teams/4270/wiki/communication17.png) |

## [Contribution](#id18)[¶](#contribution "Permalink to this heading")

### [1.Characterization of previous iGEM parts](#id19)[¶](#characterization-of-previous-igem-parts "Permalink to this heading")

In this project, basic parts of previous projects are characterized, including pPGK1
promoter (BBa\_K122000), pTEF1 promoter (BBa\_K2765041), TADH1 terminator (BBa\_K122004),
and TCYC1 terminator (BBa\_K122003). Patchoulol synthase (PTS) gene and farnesyl
diphosphate synthase (ERG20, also named FPS) gene are expressed by this promoters and
terminators. The sequences were assembled into pSP-GM2 plasmids and constructed 4
recombinant vectors, namely pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS and
pSP-GM2-PTS-FPS (Figure 1-4). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 5**).

[![https://static.igem.wiki/teams/4270/wiki/contributions1.png](https://static.igem.wiki/teams/4270/wiki/contributions1.png)](https://static.igem.wiki/teams/4270/wiki/contributions1.png)

**Figure 1. The construction of pSP-GM2-FPS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions2.png](https://static.igem.wiki/teams/4270/wiki/contributions2.png)](https://static.igem.wiki/teams/4270/wiki/contributions2.png)

**Figure 2. The construction of pSP-GM2-PTS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions3.png](https://static.igem.wiki/teams/4270/wiki/contributions3.png)](https://static.igem.wiki/teams/4270/wiki/contributions3.png)

**Figure 3. The construction of pSP-GM2-PTS-linker-FPS plasmid with pPGK1 as
promoter and TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions4.png](https://static.igem.wiki/teams/4270/wiki/contributions4.png)](https://static.igem.wiki/teams/4270/wiki/contributions4.png)

**Figure 4. The construction of pSP-GM2-PTS-FPS plasmid with pPGK1 and pTEF1 as
promoter, TADH1 and TCYC1 as terminators, respectively.**

[![https://static.igem.wiki/teams/4270/wiki/contributions5.png](https://static.igem.wiki/teams/4270/wiki/contributions5.png)](https://static.igem.wiki/teams/4270/wiki/contributions5.png)

**Figure 5 A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

### [2.Add New Documentations To An Existing Part](#id20)[¶](#add-new-documentations-to-an-existing-part "Permalink to this heading")

We will artificially cloned 2 new genes including HCF173 promoter and PTS. The patchoulol
synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces
more than 20 valuable sesquiterpenes including patchoulol by conversion of the natural
substrate FPP.[1-3] HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173),
which is required for Arabidopsis thaliana PSII biogenesis. HCF173 transcript levels and
expression were induced by light. HCF173 promoter contains ACE motif and G-box
element.[4] In our project, the CDS sequence of PTS was optimized according to yeast
codon preference. PCR was conducted first for cloning PTS, FPS (ERG20) and HCF173
(**Figure 6**). Later a gene fusion of PTS and FPS namely PTS-linker-FPS
are acquired by removing the stop codon of PTS and replacing it by a short peptide
(Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs.

[![https://static.igem.wiki/teams/4270/wiki/contributions6.png](https://static.igem.wiki/teams/4270/wiki/contributions6.png)](https://static.igem.wiki/teams/4270/wiki/contributions6.png)

**Figure 6. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

Later, we assembled HCF173 promoters, PTS gene and ERG20 (FPS) and constructed 4
recombinant vectors, namely pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS and pSP-GM2-HCF173-PTS-FPS (**Figure 7-10**).
The colony PCR were conducted for further verification of this recombinant vectors
(**Figure 11**).
.. image:: <https://static.igem.wiki/teams/4270/wiki/contributions7.png>

> width
> :   1000px
>
> align
> :   center

**Figure 7. The construction of pSP-GM2-HCF173-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions8.png](https://static.igem.wiki/teams/4270/wiki/contributions8.png)](https://static.igem.wiki/teams/4270/wiki/contributions8.png)

**Figure 8. The construction of pSP-GM2-HCF173-PTS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions9.png](https://static.igem.wiki/teams/4270/wiki/contributions9.png)](https://static.igem.wiki/teams/4270/wiki/contributions9.png)

**Figure 9. The construction of pSP-GM2-HCF173-PTS-linker-FPS plasmid with HCF173
as promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions10.png](https://static.igem.wiki/teams/4270/wiki/contributions10.png)](https://static.igem.wiki/teams/4270/wiki/contributions10.png)

**Figure 10. The construction of pSP-GM2-HCF173-PTS-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions11.png](https://static.igem.wiki/teams/4270/wiki/contributions11.png)](https://static.igem.wiki/teams/4270/wiki/contributions11.png)

**Figure 11. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [3.Add New Parts](#id21)[¶](#add-new-parts "Permalink to this heading")

In our 2022 project, we provide 10 new parts:

> | part name | part number | website |
> | --- | --- | --- |
> | HCF173 promoter | BBa\_K4270001 | [http://parts.igem.org/Part:BBa\_K4270001](http://parts.igem.org/Part%3ABBa_K4270001) |
> | PTS (patchoulol synthase) | BBa\_K4270004 | [http://parts.igem.org/Part:BBa\_K4270004](http://parts.igem.org/Part%3ABBa_K4270004) |
> | erg20 K197P | BBa\_K4270007 | [http://parts.igem.org/Part:BBa\_K4270007](http://parts.igem.org/Part%3ABBa_K4270007) |
> | erg20 K197H | BBa\_K4270008 | [http://parts.igem.org/Part:BBa\_K4270008](http://parts.igem.org/Part%3ABBa_K4270008) |
> | FPS-linker-PTS | BBa\_K4270010 | [http://parts.igem.org/Part:BBa\_K4270010](http://parts.igem.org/Part%3ABBa_K4270010) |
> | pPGK1+PTS+TADH1 | BBa\_K4270011 | [http://parts.igem.org/Part:BBa\_K4270011](http://parts.igem.org/Part%3ABBa_K4270011) |
> | pPGK1+ERG20+TADH1 | BBa\_K4270012 | [http://parts.igem.org/Part:BBa\_K4270012](http://parts.igem.org/Part%3ABBa_K4270012) |
> | pTEF1+ERG20+TCYC1 | BBa\_K4270016 | [http://parts.igem.org/Part:BBa\_K4270016](http://parts.igem.org/Part%3ABBa_K4270016) |
> | HCF173+ ERG20+TADH1 | BBa\_K4270013 | [http://parts.igem.org/Part:BBa\_K4270013](http://parts.igem.org/Part%3ABBa_K4270013) |

### [4.Improvement of Existing Parts](#id22)[¶](#improvement-of-existing-parts "Permalink to this heading")

#### [4.1 Optimized of farnesyl diphosphate synthase (ERG20, FPS) (BBa\_K517004)](#id23)[¶](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004 "Permalink to this heading")

The ERG20 is a basic part comprised from a synthesized farnesyl diphosphate synthase
coding sequence. The basic part BBa\_K4270007 (erg20 K197P) and BBa\_K4270008 (erg20
K197H) are optimized versions of ERG20 (BBa\_K517004) that can be constructed into
pSP-GM2 plasmid. The resulted vectors are named pSP-GM2-FPS, pSP-GM2-FPS K197P and
pSP-GM2-FPS K197H respectively. Then this vectors were transformed into
Saccharomyces cerevisiae BY4741 to produce patchoulol.

#### [4.2 Plasmid Construction](#id24)[¶](#plasmid-construction "Permalink to this heading")

PCR was conducted for cloning FPS (ERG20), erg20 K197P and erg20 K197H. The purified
PCR products were assembled into pSP-GM2 plasmids and constructed 3 recombinant
vectors, namely pSP-GM2-FPS, pSP-GM2-FPS K197P and pSP-GM2-FPS K197H (**Figure
12-13**). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 14**).

[![https://static.igem.wiki/teams/4270/wiki/contributions12.png](https://static.igem.wiki/teams/4270/wiki/contributions12.png)](https://static.igem.wiki/teams/4270/wiki/contributions12.png)

**Figure 12. The construction of pSP-GM2-FPS K197P plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions13.png](https://static.igem.wiki/teams/4270/wiki/contributions13.png)](https://static.igem.wiki/teams/4270/wiki/contributions13.png)

**Figure 13. The construction of pSP-GM2-FPS K197H plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions14.png](https://static.igem.wiki/teams/4270/wiki/contributions14.png)](https://static.igem.wiki/teams/4270/wiki/contributions14.png)

**Figure 14. The colony PCR results of pSP-GM2-FPS K197P (left side) and
pSP-GM2-FPS K197H (right side).**

#### [4.3 Results](#id25)[¶](#results "Permalink to this heading")

Gas chromatography (GC) method to observe the patchoulol in Yeast fermentation broth.
Unfortunately, we did not detect patchoulol in the supernatant but detected
patchoulol in the standard substance containing patchoulol (Figure 15). The reason
we did not detect patchoulol in the supernatant may due to the low yield of
patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/contributions15.png](https://static.igem.wiki/teams/4270/wiki/contributions15.png)](https://static.igem.wiki/teams/4270/wiki/contributions15.png)

**Figure 15. The detection of patchoulol in the supernatant. A-C. The
detection of patchoulol in Yeast fermentation broth from Yeast transformed with
pSP-GM2-FPS, pSP-GM2- FPS K197P, pSP-GM2- FPS K197H plasmids, respectively. D.
The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id26)[¶](#reference "Permalink to this heading")

[1] Albertsen L, Chen Y,Bach L. S, Rattleff S, et al. Diversion of flux toward
sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous
enzymes. Appl. Environ. Microbiol. 2011, 77, 1033−1040.

[2] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid precursors elevates terpene production in plants. Nature Biotechnology, 2006,
24: 1441-47.

[3] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[4] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Description](#id27)[¶](#description "Permalink to this heading")

### [Pogostemon cablin and patchouli oil](#id28)[¶](#pogostemon-cablin-and-patchouli-oil "Permalink to this heading")

Pogostemon cablin is a kind of medicinal aromatic herb and one of the Top 10 drugs in
Guangdong province. P. cablin is mainly grown in Indonesia, Singapore, Vietnam,
Malaysia, China, and other areas.[1] Patchouli oil is made from the dried ground part of
P. cablin by distillation and is mainly used in two aspects.[2] First, it is used as a
drug because of its anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other
pharmacological effects.[3-5] On the other hand, its fragrance is unique and used as a
spices and food additive.[6] Benefiting from its good efficacy, patchouli oil is widely
used in the daily chemical, pharmaceutical, and food industries and has a broad market
prospect. The price of high-quality patchouli oil can be as high as ¥1300 per kilogram.
In terms of the retail price, it can rank 10th among the important essential oils in the
world and has obvious economic benefits. [7, 8]

**Pogostemon cablin**[¶](#id11 "Permalink to this table")

|  |  |
| --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/description1.png](https://static.igem.wiki/teams/4270/wiki/description1.png) | ![https://static.igem.wiki/teams/4270/wiki/description2.png](https://static.igem.wiki/teams/4270/wiki/description2.png) |

### [Patchoulol](#id29)[¶](#patchoulol "Permalink to this heading")

Patchoulol, a sesquiterpene, is one of the most abundant components in patchouli oil. [6,
9] As an important index for evaluating the quality of P. cablin and patchouli oil in
Chinese Pharmacopoeia, patchoulol is also considered the main component contributing to
the pharmacological effects and aroma of patchouli oil. [10] Nowadays, there is a
serious imbalance between the supply and demand of patchoulol and patchouli oil in the
world. The global annual demand for patchoulol and patchouli oil can reach over 2 000 t,
but the annual output is about 1300 t. Indonesia supplies 90 percent of all annual
output, while China produces less than 10 percent of that, which has plenty of room for
growth. [11, 12] Increasing the yield of patchoulol has become a key point in the study
of p. cablin.

At present, patchoulol is obtained in three main ways, namely, plant extraction, chemical
synthesis, and microbial fermentation.

[![https://static.igem.wiki/teams/4270/wiki/description3.png](https://static.igem.wiki/teams/4270/wiki/description3.png)](https://static.igem.wiki/teams/4270/wiki/description3.png)

### [Plant extraction](#id30)[¶](#plant-extraction "Permalink to this heading")

Typically, patchoulol is extracted from the leaves of P.cablin. However, the low content
of patchoulol in P. cablin is the bottleneck of its large-scale application. [2]

### [Chemical synthesis](#id31)[¶](#chemical-synthesis "Permalink to this heading")

Although the chemical synthesis method can support patchoulol with high purity, it needs
many steps, complex operation, high synthesis cost, and the yield are low, so it cannot
be applied to large-scale industrial production. [13]

### [Microbial Fermentation](#id32)[¶](#microbial-fermentation "Permalink to this heading")

Compared with plant extraction and chemical synthesis, microbial fermentation synthesis
exhibits several advantages in producing patchoulol, such as fast-growing, land-saving,
and controllable culture conditions. With the development of metabolic engineering and
synthetic biology, many valuable plant-derived terpenoids also have been produced in
microbial cell factories, such as artemisinic acid [14], ginsenosides [15], and
tanshinones [16]. Although naturally produced patchoulol can be obtained from plants,
patchoulol production using biobased microbial platforms is an economical and
sustainable alternative.

### [Patchoulol biosynthetic pathways](#id33)[¶](#patchoulol-biosynthetic-pathways "Permalink to this heading")

There are two biosynthetic pathways for patchoulol formation, namely the mevalonic acid
(MVA) pathway located in the cytoplasm and the methylerythritol 4-phosphate
(MEP) pathway located in plastids. [17, 18] In general, patchoulol is mainly synthesized
through the MVA pathway. There are 3 key enzymes in the synthesis of patchoulol, namely
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FPS),
and patchoulol synthase (PTS). HMGR catalyzes the irreversible formation of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into MVA. Because it can affect the
yield of IPP (isopentenyl diphosphate), the precursors of patchoulol, HMGR is the key to
the metabolism of patchoulol. FPS catalyzes IPP to FPP (farnesyl diphosphate). PTS is a
key rate-limiting enzyme in the formation of the patchoulol, which catalyzes the
formation of patchoulol skeleton from the FPP precursor, and the skeleton is modified by
secondary enzymes to form patchoulol.

[![https://static.igem.wiki/teams/4270/wiki/description4.png](https://static.igem.wiki/teams/4270/wiki/description4.png)](https://static.igem.wiki/teams/4270/wiki/description4.png)

### [Vision](#id34)[¶](#vision "Permalink to this heading")

Patchoulol production using biobased microbial platforms is an economical and sustainable
alternative. Recently, based on the endogenous mevalonate (MVA) pathway or
2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, heterologous production of patchoulol
has been accomplished by introducing the patchoulol synthase gene in Saccharomyces
cerevisiae [8] and Corynebacterium glutamicum [19], as well as in the moss
Physcomitrella patens[20] and eukaryotic microalga Chlamydomonas reinhardtii [21].
Nevertheless, the yield is still too low for industrial production. Therefore,
significant engineering is required to achieve sufficient yields for industrial
applications. Scientists see the use of environmental signals (light, temperature) and
endogenous signals (quorum sensing, pressure) to establish tightly controlled patchoulol
biological production systems as the future trend.

Promoters are crucial elements in controlling the expression of target genes, and the
emergence of inducible promoters provides the possibility to accurately and flexibly
regulate the expression of foreign genes, which has become one of the hot spots in the
research and development of genetic engineering. [22] Therefore, in the present study,
two metabolic engineering strategies were conducted to enhance the production of
patchoulol in yeast. First, ERG20 (encoding FPP synthase from yeast) and PTS (encoding
patchoulol synthase from P. cablin) were used to increase the utilization of the FPP
precursor. Second, the constituted promoter and light-inducible promoter are
respectively used to drive the overexpression of the critical gene FPS and PTS. In
addition, studies have reported that high-level patchoulol production in tobacco plants
was achieved by fusing FPS and PTS genes. [17] Hence, vectors consisting of a gene
fusion of PTS and FPS (PTS-FPS) inserted downstream of the constitutive and
light-inducible promoter were also constructed. Finally, we carried out fermentation in
yeast and envisioned our final product by meteorological chromatography.

[![https://static.igem.wiki/teams/4270/wiki/description5.png](https://static.igem.wiki/teams/4270/wiki/description5.png)](https://static.igem.wiki/teams/4270/wiki/description5.png)

### [Reference](#id35)[¶](#id1 "Permalink to this heading")

[1] Yao G, Drew B T, Yi T S, et al. Phylogenetic relationships, character evolution and
biogeographic diversification of Pogostemon s.l. (Lamiaceae) [J]. Mol Phylogenet Evol,
2016, 98(184-200.

[2] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[3] Zhang R, Yan P, Li Y, et al. A pharmacokinetic study of patchouli alcohol after a
single oral administration of patchouli alcohol or patchouli oil in rats [J]. European
Journal of Drug Metabolism and Pharmacokinetics, 2016, 41(4): 441-8.

[4] Wu Z, Zeng H, Zhang L, et al. Patchouli alcohol: a natural sesquiterpene against both
inflammation and intestinal barrier damage of ulcerative colitis [J]. Inflammation,
2020, 43(4): 1423-35.

[5] 闵琼, 曾海荣, 陆翠燕, et al. 广藿香醇通过调控上皮间质转化抑制人胃癌细胞HGC-27的侵袭和转移 [J]. 药学服务与研究, 2020, 20(01):
6-11.

[6] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[7] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Ind Crop Prod, 2016, 87(161-76.

[8] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[9] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[10] 徐雯, 吴艳清, 丁浩然, et al. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[11] Swamy M K, Mohanty S K, Sinniah U R, et al. Evaluation of patchouli (Pogostemon
cablin Benth.) cultivars for growth, yield and quality parameters [J]. Journal of
Essential Oil-Bearing Plants, 2015, 18(4): 826-32.

[12] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Industrial Crops & Products, 2016, 87(161-76.

[13] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of ()-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[14] Westfall P J, Pitera D J, Lenihan J R, et al. Production of amorphadiene in yeast,
and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent
artemisinin [J]. P Natl Acad Sci USA, 2012, 109(3): 655-6.

[15] Wei W, Wang P, Wei Y, et al. Characterization of Panax ginseng
UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive
ginsenosides F1 and Rh1 in metabolically engineered yeasts [J]. Molecular Plant,
2015,

[16] Guo J, Zhou Y J, Hillwig M L, et al. CYP76AH1 catalyzes turnover of miltiradiene in
tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts
[J]. Pnas, 2013, 110(29): 12108-13.

[17] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid
precursors elevates terpene production in plants. Nature Biotechnology, 2006, 24:
1441-47.

[18] Tang Y, Zhong L, Wang X, et al. Molecular identification and expression of
sesquiterpene pathway genes responsible for patchoulol biosynthesis and regulation in
Pogostemon cablin［J］.
Bot Stud, 2019, 60：11.

[19] Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum［J］. Genes (Basel), 2018, 9(4): E219.

[20] Zhan X, Zhang YH, Chen DF, et al. Metabolic engineering of the moss Physcomitrella
patens to produce the sesquiterpenoids patchoulol and α/β-santalene［J］. Front Plant Sci,
2014, 5 ：636.

[21] Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a
high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii［J］.
Metab Eng, 2016,
38: 331-343.

[22] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Engineering](#id36)[¶](#engineering "Permalink to this heading")

### [Abstract](#id37)[¶](#abstract "Permalink to this heading")

Patchoulol is one of the main active components of Pogostemon cablin, and it is also an
important indicator for evaluating the medicinal materials of P. cablin in the Chinese
Pharmacopoeia. Patchoulol can be obtained by natural extraction, chemical synthesis, and
synthetic biology engineering. Due to the low content of patchoulol in P. cablin and the
low yield of chemical synthesis, the study of combinatorial biosynthesis and molecular
regulation can provide the basis for obtaining high-content patchoulol. In this study,
on the basis of the biosynthetic pathway, molecular regulation mechanism, and synthetic
biology engineering of patchoulol, the key genes for its biosynthesis were combined and
expressed in heterologous host yeast to produce patchoulol, which provides the technical
foundations of the development and application of patchoulol.

### [Experiment design](#id38)[¶](#experiment-design "Permalink to this heading")

#### [8 type of expression vector](#id39)[¶](#type-of-expression-vector "Permalink to this heading")

We designed eight plasmids: The strong constitutive promoter (pGK1) was separately
used to connect FPS (BBa\_K517004), PTS (BBa\_K4270004), and FPS-linker-PTS
(BBa\_K4270010), and plasmids containing pGK1, FPS, terminator, pGK1, PTS terminator
were constructed .In the following four designs, all the above strong promoters were
replaced with light-induced promoters HCF173, and then the test was carried out to
verify (Figure 1).

[![https://static.igem.wiki/teams/4270/wiki/engineering1.png](https://static.igem.wiki/teams/4270/wiki/engineering1.png)](https://static.igem.wiki/teams/4270/wiki/engineering1.png)

### [Experiment structure and testing](#id40)[¶](#experiment-structure-and-testing "Permalink to this heading")

#### [PCR amplification and estriction enzyme cutting expression vector pSP-GM2](#id41)[¶](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2 "Permalink to this heading")

Amplified by PCR, the plasmids were double digestion by the restriction enzymes NotI
and SacI. Plenty of strands of DNA were obtained. Plasmid DNA, restriction enzyme,
reaction buffer, and ddH2O were added successively in a 1.5 mL sterilized centrifuge
tube according to the table below. Mix and mark well. The mixture reacted at 37℃ for
eight to ten hours. Agarose gel electrophoresis was used to detect the product size
and determine whether the enzyme was successfully cut.

**Table 1 Enzyme digestion reaction system**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | Not I | 0.5 |
> | Sac I | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

#### [Likage of expression vector and chemical transformation of E.coli DH5α](#id42)[¶](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5 "Permalink to this heading")

In vitro assembly gene, including promoter, control of the corresponding enzyme
synthesis gene, sequence of the terminator, DNA ligase access strip DNA, forming a
circular recombinant vector. Add 10 ng vector recombinant DNA solution to 50 μL
competent E.coli, mix well, and let stand in an ice bath for 30 min. By heat pulse
method, the mixture was shocked in a water bath at 42℃ for 30 s, then placed on ice
for 3-5 minutes. 1 mL fresh medium was added and cultured at 37℃ for 1 h for
amplification. Add 500 μL to sterilized LB medium into a centrifuge tube, mix
evenly, and culture for 1 h at 37℃ and 200 rpm. Adjust the speed to 5000rpm,
centrifuge at room temperature for 1min, take out the supernatant and pour it out
until there was 100 μL of thallus remaining.

#### [Screening of transformants](#id43)[¶](#screening-of-transformants "Permalink to this heading")

Pick a single bacteria placement from the conversion plate and place it on the new LB
solid plate containing antibiotics, culture it at 37℃ for 2-4 h and then pick up the
colonies. Put them into the centrifuge tube of the corresponding PCR reaction
solution. Take the colony as the template of PCR reaction and amplify the colony by
T5 Super PCR Mix.

**Table 2 Colony PCR reaction system**

> | Reagent | Dosage |
> | --- | --- |
> | 2×T5 Super PCR Mix | 5 μL |
> | M13F（10 μM） | 0.4 μL |
> | M13R（10 μM） | 0.4 μL |
> | ddH2O | 4.2 μL |

**Table 3 Colony PCR reaction procedure**

> | Temperature | Time | Cycle |
> | --- | --- | --- |
> | 98℃ | 10 min | 1 |
> | 98℃ | 10 s | 35 |
> | 50℃ | 10 s |  |
> | 72℃ | 30 s |  |
> | 72℃ | 2 min | 1 |

#### [Plasmids extraction and extract sequencing](#id44)[¶](#plasmids-extraction-and-extract-sequencing "Permalink to this heading")

The transformed E.coli was inoculated with 0.5% glycerin and ampicillin in sterilized
TB culture medium. Culture at 220 rpm for 12 h at 37℃ until saturated. Adjust the
speed to 10000 rpm, centrifuge at room temperature for 6 min, remove and pour off
the supernatant. The centrifugation process was repeated until the plasmid was
successfully extracted. After gene sequencing, verify whether the vector is
successfully transformed and consistent with the design sequence.

#### [Transformation and screening of yeast](#id45)[¶](#transformation-and-screening-of-yeast "Permalink to this heading")

About 16 colonies growing in LB solid medium containing ampicillin were selected and
verified by 2x Easy Taq enzyme. The PCR system is shown below.

**Table 4 PCR reaction (20 μL)**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | buffer | 8 |

The reaction time and temperature were set according to the table below, and
electrophoresis was performed after amplification.

**Table 5 experimental set**

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

The monoclones were selected into 5 mL of the corresponding defective medium (BY4741
into YPD containing glucose). The culture was incubated overnight at 28℃ at 200-220
rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial solution
into 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out the
supernatant. The monoclones were selected into 5mL of corresponding defective medium
(BY4741 into YPD containing glucose). The culture was incubated overnight at 28℃ at
200-220 rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial
solution into a 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out
the supernatant. Add 500 μL of EZ1 and resuspend thallus. Centrifuge 300-500 rpm for
3-5 min to remove the supernatant. Add 50μL of EZ2, flick, and resuspended the
thallus to obtain a competent state.

#### [Transform procedure](#id46)[¶](#transform-procedure "Permalink to this heading")

3-5 μL plasmid was added to 50 μL competent state, mixed evenly, then 500 μL EZ3 was
added, then evenly and lightly beaten with a blue gun since EZ3 is sticky. Incubated
in a water bath at 30℃ for 45 min-1 h. After centrifugation at 500 rpm for 5 min,
flocculent accumulation of thalli could be seen near the bottom of the tube. After
centrifugation, 400 μL centrifuged EZ3 was sucked off, leaving 100 μL resuspended
thallus and applied directly to the plate.

The vector was transformed into yeast and screened for nutritional deficiency.
Successfully transformed recipient cells can be grown on nutritionally deficient
media. The successfully transformed yeast cells were taken and examined again.

#### [Fermentation of modified strains](#id47)[¶](#fermentation-of-modified-strains "Permalink to this heading")

Provide suitable nutrients and environment for fermentation, so that the successful
transformation of yeast fermentation at room temperature.

#### [Extraction and determining of metabolite](#id48)[¶](#extraction-and-determining-of-metabolite "Permalink to this heading")

The content of patchoulol, a volatile sesquiterpene, was analyzed by gas
chromatography.

#### [Learning and design again:](#id49)[¶](#learning-and-design-again "Permalink to this heading")

We further optimized the original improved carrier. On this basis, the experimental
scheme was further optimized.

## [Experiments](#id50)[¶](#experiments "Permalink to this heading")

### [Protocol](#id51)[¶](#protocol "Permalink to this heading")

#### [1. The enzyme digestion reaction of pSP-GM2 vector](#id52)[¶](#the-enzyme-digestion-reaction-of-psp-gm2-vector "Permalink to this heading")

Material: pSP-GM2 vector, restriction enzyme, 10 x Cutsmart buffer, ddH2O.

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

**Procedure：**

1. Add ingredients in 1.5 mL test tubes at a time according to the above
   table.
2. Mix and mark well.
3. 37℃ for 8-10 h.

#### [2. PCR and electropherosis](#id53)[¶](#pcr-and-electropherosis "Permalink to this heading")

Material: DNA template、primer、dNTPs、5 × Q5 reaction buffer、Q5 DNA polymerase.

Procedure: (1) Mix up the recipe into 1.5 mL test tubes according to the following
table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

#### [3. DNA gel electrophoresis](#id54)[¶](#dna-gel-electrophoresis "Permalink to this heading")

(1) Preparation of 1% DNA gel
Weigh 1 g of agarose into a cone-shaped flask, and then add 100 mL of 10xTAE buffer
into this cone-shaped flask. Transfer the cone to the microwave and heat until
boiling (2-3 times). Cooling the cone-shaped flask to about 50℃, and then add the
nucleic acid dye into it. Mixing agarose and nucleic acid well and pour them into
the rubber rack. After 1 hour later, 1% DNA gel is ready.

(2) point sample
Mixing the DNA sample and the adding buffer (4:1). Then adding the mixture to the
sample tank with a micropipette gun. Each tank addes 20 μL mixture. Recording the
order of spot sampling and the amount of adding sample.

(3) Gel electrophoresis
After installing the electrode wire, connect one end of the sample hole to the
negative electrode and the other end to the positive electrode. Then switching on
the power supply and adjust the voltage to 120 V. After electrophoresis for 20-30
min, stop electrophoresis.

#### [4. Transformation of plasmid to E.coli](#id55)[¶](#transformation-of-plasmid-to-e-coli "Permalink to this heading")

1. Take 50 µL of E.coli cells and defrost E.coli with ice bath for 25 min.
   Mixing E.coli gently with 10 µL target recombinant plasmid.
2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for
   another 3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of
   supernatant and resuspend the bacteria. Coating on the LB broth containing
   ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

#### [5. Selecting colonies](#id56)[¶](#selecting-colonies "Permalink to this heading")

**Colony PCR**

1. Inoculate BL21 cells using a pipette tip，use 2x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

#### [6. DNA sequencing](#id57)[¶](#dna-sequencing "Permalink to this heading")

Expand and culture the positive colonies in LB liquid medium containing ampicillin at
37℃ for 10-12 h. Extract the plasmid by using the plasmid extraction kit. Sent 10 μL
plasmid to sequencing company for sequencing. The correctly sequenced plasmid was
stored at -20°C and the corresponding bacterial solution was stored at -80°C.

#### [7. Yeast transformation procedure](#id58)[¶](#yeast-transformation-procedure "Permalink to this heading")

1. selecte the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into 1.5mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into 1.5mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50μL yeast cells, mix evenly, then
   add 500μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1
   h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and
   coate them on a solid medium plate.

## [Implementation](#id59)[¶](#implementation "Permalink to this heading")

### [Background](#id60)[¶](#background "Permalink to this heading")

Patchoulol is one of the most abundant components in patchouli oil[1, 2] and is also
considered the main component contributing to the pharmacological effects and aroma of
patchouli oil[3], which is widely used in medicine, daily chemicals, food, and other
industries, with broad market prospects. However, there is a serious imbalance between
the supply and demand of patchoulol and patchouli oil in the world. There are three ways
to prepare patchouli so far. Typically, patchoulol is extracted from the leaves of
Pogostemon cablin. However, the low content of patchoulol in P. cablin is the bottleneck
of its large-scale application [4]. Although the chemical synthesis method can support
patchoulol with high purity, it has many drawbacks, such as too many steps, complex
operation, high synthesis cost, and low yield, so it is may not suitable for large-scale
industrial production [5]. Compared with plant extraction and chemical synthesis,
microbial fermentation synthesis exhibits several advantages in producing patchoulol,
such as fast-growing, land-saving, and controllable culture conditions. With the
development of synthetic biology, patchoulol has been produced in yeast under the
control of a constitutive promoter. [6] However, the production of patchoulol is
uncontrollable and yeast cells may produce too much patchoulol at a time, which is toxic
to yeast cells. Herein, our team has innovatively applied the light-induced promoter in
the biosynthesis of patchoulol in yeast, which makes the production of patchoulol
controllable. When the light is on, yeast cells produce patchoulol. Otherwise, it is not
produced patchoulol. We believe the increasing production of patchoulol will lower the
price of patchoulol. Furthermore, the lower price of patchoulol can effectively help
more people particularly those in outlying poverty-stricken areas.

### [Target customers](#id61)[¶](#target-customers "Permalink to this heading")

**1. Patchouli essential oil factory**

Patchouli essential oil is in great demand in the market because of its long history of
use, wide range of uses, and good fragrance fixing effect. Patchouli essential oil is
beneficial to the body and mind. For example, it can be mentally refreshing, relieve
anxiety, and make people feel excited. Physiologically, it has a good bactericidal
effect, promotes skin regeneration and reduces inflammation, and so on. These properties
have made patchouli popular. As we all know, patchoulol is one of the main ingredients
in patchouli oil. If our project can produce patchoulol on a large-scale industrial
production, it will generate a lot of business value.

**2. Pharmaceutical factory**

Patchoulol is also considered the main component contributing to the pharmacological
effects of patchouli oil. It is often used as raw materials for drugs because of its
anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other pharmacological
effects[7]. Nowadays, The market demand for patchoulol is increasing. For example,
Huoxiang Zhengqi Shui (KangShengTang), a kind of medicine that is always available in
every household in China, contains a huge amount of patchoulol. Therefore, when our
project achieves the efficient production of patchoulol, we can sell patchoulol to
pharmaceutical factories and profit from it.

**3. Customers who need environment-friendly pesticides**

Because of its insecticidal biological activity, patchoulol can be made into insecticides
to control some specific species of pests. And because it has the characteristics of low
toxicity, easy degradation, safety, and environmental friendliness, it is a good choice
to use as an insecticide. Therefore, we can also consider producing a large amount of
patchoulol by ourselves, preparing it into an environment-friendly insecticide, and
selling it to customers who need it in this regard.[8]

### [Possible challenges](#id62)[¶](#possible-challenges "Permalink to this heading")

Although our project has achieved initial results in the laboratory environment,
considering the difference between the laboratory environment and the actual large-scale
production environment, we are worried that there will be some problems during the
large-scale production. For example, the accumulation of too much yeast may lead to an
untimely and/or insensitive response of the light-induced prompter in yeast to light
conditions, and even cause the light-induced promoter to lose its original function,
making the whole biosynthesis process uncontrollable. These accidents that may occur in
the mass production process will greatly affect the mass production of patchoulol, which
may limit the availability of our product to the market. So for us, problems that arise
during mass production can be a challenge.

### [Reference](#id63)[¶](#id2 "Permalink to this heading")

[1] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[2] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[3] 徐雯, 吴艳清, 丁浩然, 等. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[4] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[5] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of (-)-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[6] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene
production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J].
Appl Environ Microbiol, 2011, 77(3): 1033-40.

[7] 魏晓露, 彭成, 万峰. 广藿香醇体外抗呼吸道病毒作用研究 [J]. 中药药理与临床, 2013, 29(1): 4.

[8] 陈义娟, 范能能, 蒋杰贤, et al. 百秋李醇的用途及杀虫剂 [M]. 2020.

## [Notebook](#id64)[¶](#notebook "Permalink to this heading")

### [Week 1 (6.20-6.26)](#id65)[¶](#week-1-6-20-6-26 "Permalink to this heading")

We inoculated and cultured Escherichia coli containing the target vector in a liquid LB
growth medium. Then we waited for the cultures to grow and develop. We extracted the
target vector and designed primers needed for the construction of our vectors and sent
them to the company for synthesis.

We purchased the reagents needed for our experiments.

### [Week 2 (6.27-7.3)](#id66)[¶](#week-2-6-27-7-3 "Permalink to this heading")

**Part one**
**First attempt to digest target vector.**

We used the target pSP-GM2 vector, restriction enzyme, reaction buffer, and ddH2O to
digest
the target vector. The reaction system is as follows:

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

Procedures to digest target vector:
(1) Add ingredients in 1.5 mL test tubes at a time according to the above table.

2. Mix and mark well.
3. 37℃ for 8-10 h.

**Part teo**
**PCR amplification of corresponding parts.**

PCR amplification for PST gene, FPS gene, and the HCF173 promoter. However, promoter
HCF173 first was not successfully amplified. After the second test, we successfully
amplified HCF173 promoter.

Procedure for PCR amplification:
(1) Mix up the recipe into 1.5 mL test tubes according to the following table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

### [Week 3 (7.4-7.10)](#id67)[¶](#week-3-7-4-7-10 "Permalink to this heading")

**Part one Construction of target vector.**

We construct the target vector by mixing pSP-GM2 vector with the genes we cloned. These
plasmids were later transformed into E.coli BL21.

**Part two Transformation of the plasmid to E.coli**

Procedure:
(1) Take 50 µL of E.coli cells and defrost E.coli with an ice bath for 25 min. Mixing
E.coli gently with 10 µL target recombinant plasmid.

2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for another
   3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix it upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of supernatant
   and resuspend the bacteria. The coating on the LB broth containing ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

### [Week 4 (7.11-7.17)](#id68)[¶](#week-4-7-11-7-17 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Colony PCR was used to select positive colonies, which was verified by sequencing and
then
in liquid LB.

General procedures:

1. Inoculate BL21 cells using a pipette tip, use 2 x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

### [Week 5 (7.18-7.24)](#id69)[¶](#week-5-7-18-7-24 "Permalink to this heading")

**Transformation of plasmid to Yeast**

**Procedure:**

1. select the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into a 1.5 mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into a 1.5 mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50 μL yeast cells, mix evenly, then add
   500 μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1 h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and coat
   them on a solid medium plate.

### [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#id70)[¶](#week-6-8-1-8-7-and-week-7-8-8-8-14 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Single yeast colonies were taken from stock plates and inoculated into 8 ml YPD. We carry
out PCR on these colonies and plan to test them again. Results showed that some plasmids
have been successfully transformed into Yeast.

### [Week 7 (8.15-8.27)](#id71)[¶](#week-7-8-15-8-27 "Permalink to this heading")

**Cultivation of recombinant yeasts in a Shaking Flask.**

The recombinant yeasts were precultured in 5 mL of YPD at 30℃, 220 rpm for 24 h.
Precultures were inoculated to 50 mL of YPGD or YPGL in 250 mL flasks at an initial
OD600 of 0.05 and were grown under the same condition. An overlay of 5 mL dodecane was
added to the flasks after 24 h and the light was opened.

**Analysis of Patchoulol**

Samples from the organic layer were centrifuged for 15 min at 6000 rpm to determine the
level of patchoulol during fermentation. The patchoulol was analyzed by gas
chromatography-mass spectrometry using the HP-5 column. The sample (1 μL) was injected
in splitless mode. The patchoulol standard was used to identify the substances.

## [Proof of concept](#id72)[¶](#proof-of-concept "Permalink to this heading")

### [Method development](#id73)[¶](#method-development "Permalink to this heading")

Two kinds of promoters were selected for gene expression, constitutive promoter PGK1 and
light-inducible promoter HCF173 (BBa\_K4270001). [1]

By fusing with FPS (farnesyl diphosphate synthase), PTS (patchoulol synthase), and
FPS-linker-PTS genes via PCR, they were linked into pSP-GM2 vector. After the
transformation of pSP-GM2 plasmids into E.coli, the DNA of each gene was amplified by
PCR and sequenced for verification. The recombinant vectors were then transformed into
yeast. Fermentation was then carried out and the target compounds were detected by
meteorological chromatography. A qualitative and quantitative analysis was carried out
for the production of patchoulol. The ones with the best performance were further
applied to real fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)

### [Plasmid construction](#id74)[¶](#id3 "Permalink to this heading")

The DNA sequence of PTS (encoding patchoulol synthase from P. cablin) was optimized
according to the preference of yeast for amino acid codons. Then FPS (BBa\_K517004), PTS
(BBa\_K4270004), and FPS-linker-PTS (BBa\_K4270010) were fused with promoter PGK1 or
light-inducible promoter HCF173 and constructed into pSP-GM2 plasmids separately, which
contains ampicillin resistance genes for colony selection.

The eight plasmids: pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS,
pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS,
pSP-GM2-HCF173-PTS-FPS were transformed into E.coli according to the protocol. The
colony PCR was conducted for verification of the correct plasmids (Figure 1 and Figure
2). Then the amplified DNAs were sequenced and compared with the designed DNA sequences
for further verification.

Then these vectors are all transformed into yeast. The colony PCR was conducted for
further verification of these recombinant vectors.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)

**Figure 1. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)

**Figure 2. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [Patchoulol detection](#id75)[¶](#patchoulol-detection "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 3). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)

**Figure 3 The detection of patchoulol in the supernatant. A-H. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS,
pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids,
respectively. I. The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id76)[¶](#id4 "Permalink to this heading")

[1] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Results](#id77)[¶](#id5 "Permalink to this heading")

**Overview**

Our results consist of our gene cloning, colony PCR results and Gas chromatography
results.

### [1. Codon Optimization](#id78)[¶](#codon-optimization "Permalink to this heading")

To improve the translation efficiency of PTS in yeast, we first optimized the Pogostemon
cablin PTS gene according to the codon preference of yeast. During optimization, the
amino acid sequence of the protein encoded by PTS remained unchanged (**Figure
1**) and the codon was adjusted.

[![https://static.igem.wiki/teams/4270/wiki/results1.png](https://static.igem.wiki/teams/4270/wiki/results1.png)](https://static.igem.wiki/teams/4270/wiki/results1.png)

**Figure 1. The original PTS protein sequence and optimized PTS protein
sequence.**

### [2. DNA Preparation](#id79)[¶](#dna-preparation "Permalink to this heading")

The FPS (ERG20) gene was PCR-amplified from the genome of S. cerevisiae 4741. The fusion
of FPS and PTS with a Gly-Ser-Gly (GSG) tag was constructed according to a previous
work,[1] which generated a fusion protein called PTS-linker-FPS. The FPS (ERG20) gene
was PCR-amplified from the genome of S. cerevisiae 4741. The light-inducible promoter
HCF173 (HIGH CHLOROPHYLL FLUORESCENCE173) was PCR-amplified from the genome of wild-type
Arabidopsis thaliana. [2] The results are shown in **Figure 2**.

[![https://static.igem.wiki/teams/4270/wiki/results2.png](https://static.igem.wiki/teams/4270/wiki/results2.png)](https://static.igem.wiki/teams/4270/wiki/results2.png)

**Figure 2A. The PCR results of FPS. B. The PCR results of PTS. C. The PCR results
of HCF173. D. The PCR results of PTS-linker-FPS.**

After HCF173 and FPS, PTS, PTS-linker-FPS were separately amplified, HCF173 and other
genes were overlapped and amplified using primers. The results are shown in **Figure
3**.

[![https://static.igem.wiki/teams/4270/wiki/results3.png](https://static.igem.wiki/teams/4270/wiki/results3.png)](https://static.igem.wiki/teams/4270/wiki/results3.png)

**Figure 3A. The PCR results of HCF173-FPS. B. The PCR results of HCF173-PTS. C.
The PCR results of HCF173- PTS-linker-FPS. D. The PCR results of PTS-FPS (namely
HCF173-FPS- terminator -HCF173- PTS- terminator)**

### [3. Construction of Plasmids](#id80)[¶](#construction-of-plasmids "Permalink to this heading")

We constructed 4 plasmids with the constitutive promoter (pGK1) separately connecting
FPS, PTS, FPS-linker-PTS. The plasmids containing promoter
pGK1-FPS-terminator-pGK1-PTS-terminator were also built. We also constructed 4 plasmids
with HCF173 as the promoter. The schematic diagram of these plasmids is shown in
**Figure 4**.

[![https://static.igem.wiki/teams/4270/wiki/results4.png](https://static.igem.wiki/teams/4270/wiki/results4.png)](https://static.igem.wiki/teams/4270/wiki/results4.png)

**Figure 4. The schematic diagram of plasmids. Pcp, constitutive promoter pGK1.
Plip, light-induced promoter HCF173. Tns, T ADH1 or T CYC1 terminator.**

The colony PCR was conducted for further verification of these recombinant vectors. The
results are shown in **Figure 5** and **Figure 6**.

[![https://static.igem.wiki/teams/4270/wiki/results5.png](https://static.igem.wiki/teams/4270/wiki/results5.png)](https://static.igem.wiki/teams/4270/wiki/results5.png)

**Figure 5A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/results6.png](https://static.igem.wiki/teams/4270/wiki/results6.png)](https://static.igem.wiki/teams/4270/wiki/results6.png)

**Figure 6A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [4. Patchoulol detection](#id81)[¶](#id6 "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 7). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/results7.png](https://static.igem.wiki/teams/4270/wiki/results7.png)](https://static.igem.wiki/teams/4270/wiki/results7.png)

**Figure 7. The detection of patchoulol in the supernatant. A-J. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2- FPS K197P, pSP -GM2- FPS K197H, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS,
pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids, respectively. I. The
detection of patchoulol in the standard substance containing patchoulol.**

### [Table of Contents](static/_static/index.html)

* [Team](team)
* Project
  + [Communication](#communication)
    - [Highschool
      Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
    - [Online
      Conferences](#online-conferences)
    - [Propaganda
      Speech](#propaganda-speech)
    - [Interview
      with Experts](#interview-with-experts)
    - [Interview
      with doctor](#interview-with-doctor)
  + [Contribution](#contribution)
    - [1.Characterization
      of previous iGEM parts](#characterization-of-previous-igem-parts)
    - [2.Add New
      Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
    - [3.Add New
      Parts](#add-new-parts)
    - [4.Improvement of
      Existing Parts](#improvement-of-existing-parts)
    - [Reference](#reference)
  + [Description](#description)
    - [Pogostemon cablin
      and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
    - [Patchoulol](#patchoulol)
    - [Plant
      extraction](#plant-extraction)
    - [Chemical
      synthesis](#chemical-synthesis)
    - [Microbial
      Fermentation](#microbial-fermentation)
    - [Patchoulol
      biosynthetic pathways](#patchoulol-biosynthetic-pathways)
    - [Vision](#vision)
    - [Reference](#id1)
  + [Engineering](#engineering)
    - [Abstract](#abstract)
    - [Experiment
      design](#experiment-design)
    - [Experiment structure
      and testing](#experiment-structure-and-testing)
  + [Experiments](#experiments)
    - [Protocol](#protocol)
  + [Implementation](#implementation)
    - [Background](#background)
    - [Target
      customers](#target-customers)
    - [Possible
      challenges](#possible-challenges)
    - [Reference](#id2)
  + [Notebook](#notebook)
    - [Week 1
      (6.20-6.26)](#week-1-6-20-6-26)
    - [Week 2
      (6.27-7.3)](#week-2-6-27-7-3)
    - [Week 3
      (7.4-7.10)](#week-3-7-4-7-10)
    - [Week 4
      (7.11-7.17)](#week-4-7-11-7-17)
    - [Week 5
      (7.18-7.24)](#week-5-7-18-7-24)
    - [Week 6 (8.1-8.7)
      and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
    - [Week 7
      (8.15-8.27)](#week-7-8-15-8-27)
  + [Proof of
    concept](#proof-of-concept)
    - [Method
      development](#method-development)
    - [Plasmid
      construction](#id3)
    - [Patchoulol
      detection](#patchoulol-detection)
    - [Reference](#id4)
  + [Results](#id5)
    - [1. Codon
      Optimization](#codon-optimization)
    - [2. DNA
      Preparation](#dna-preparation)
    - [3.
      Construction of Plasmids](#construction-of-plasmids)
    - [4. Patchoulol
      detection](#id6)
* [Parts](parts)
* [Safety](safety)
* [Human Practices](human-practices)
* [Model](model)
* [Education](education)

### Navigation

* [index](static/_static/genindex.html "General Index")
* [next](parts "Parts") |
* [previous](team "Team") |
* [GDSYZX v1.0 documentation](static/_static/index.html) »
* Project

© Copyright 2022, chenzhr23.
Created using [Sphinx](https://www.sphinx-doc.org/) 5.1.1.

### Navigation

* [index](static/_static/genindex.html "General Index")
* [next](parts "Parts") |
* [previous](team "Team") |
* [GDSYZX v1.0 documentation](static/_static/index.html) »
* Project

# Project[¶](#project "Permalink to this heading")

* [Communication](#communication)

  + [Highschool Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
  + [Online
    Conferences](#online-conferences)
  + [Propaganda
    Speech](#propaganda-speech)
  + [Interview
    with Experts](#interview-with-experts)
  + [Interview
    with doctor](#interview-with-doctor)
* [Contribution](#contribution)

  + [1.Characterization of previous iGEM parts](#characterization-of-previous-igem-parts)
  + [2.Add New
    Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
  + [3.Add New
    Parts](#add-new-parts)
  + [4.Improvement of Existing Parts](#improvement-of-existing-parts)

    - [4.1 Optimized of farnesyl diphosphate synthase (ERG20,
      FPS) (BBa\_K517004)](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004)
    - [4.2
      Plasmid Construction](#plasmid-construction)
    - [4.3
      Results](#results)
  + [Reference](#reference)
* [Description](#description)

  + [Pogostemon cablin and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
  + [Patchoulol](#patchoulol)
  + [Plant
    extraction](#plant-extraction)
  + [Chemical
    synthesis](#chemical-synthesis)
  + [Microbial
    Fermentation](#microbial-fermentation)
  + [Patchoulol biosynthetic pathways](#patchoulol-biosynthetic-pathways)
  + [Vision](#vision)
  + [Reference](#id1)
* [Engineering](#engineering)

  + [Abstract](#abstract)
  + [Experiment
    design](#experiment-design)

    - [8 type of expression vector](#type-of-expression-vector)
  + [Experiment structure and testing](#experiment-structure-and-testing)

    - [PCR amplification and estriction enzyme cutting
      expression vector pSP-GM2](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2)
    - [Likage of expression vector and chemical transformation
      of E.coli DH5α](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5)
    - [Screening of transformants](#screening-of-transformants)
    - [Plasmids
      extraction and extract sequencing](#plasmids-extraction-and-extract-sequencing)
    - [Transformation
      and screening of yeast](#transformation-and-screening-of-yeast)
    - [Transform
      procedure](#transform-procedure)
    - [Fermentation of
      modified strains](#fermentation-of-modified-strains)
    - [Extraction
      and determining of metabolite](#extraction-and-determining-of-metabolite)
    - [Learning and design again:](#learning-and-design-again)
* [Experiments](#experiments)

  + [Protocol](#protocol)

    - [1.
      The enzyme digestion reaction of pSP-GM2 vector](#the-enzyme-digestion-reaction-of-psp-gm2-vector)
    - [2. PCR and electropherosis](#pcr-and-electropherosis)
    - [3. DNA gel electrophoresis](#dna-gel-electrophoresis)
    - [4.
      Transformation of plasmid to E.coli](#transformation-of-plasmid-to-e-coli)
    - [5.
      Selecting colonies](#selecting-colonies)
    - [6. DNA
      sequencing](#dna-sequencing)
    - [7. Yeast transformation procedure](#yeast-transformation-procedure)
* [Implementation](#implementation)

  + [Background](#background)
  + [Target
    customers](#target-customers)
  + [Possible
    challenges](#possible-challenges)
  + [Reference](#id2)
* [Notebook](#notebook)

  + [Week 1
    (6.20-6.26)](#week-1-6-20-6-26)
  + [Week 2
    (6.27-7.3)](#week-2-6-27-7-3)
  + [Week 3
    (7.4-7.10)](#week-3-7-4-7-10)
  + [Week 4
    (7.11-7.17)](#week-4-7-11-7-17)
  + [Week 5
    (7.18-7.24)](#week-5-7-18-7-24)
  + [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
  + [Week 7
    (8.15-8.27)](#week-7-8-15-8-27)
* [Proof of concept](#proof-of-concept)

  + [Method
    development](#method-development)
  + [Plasmid construction](#id3)
  + [Patchoulol
    detection](#patchoulol-detection)
  + [Reference](#id4)
* [Results](#id5)

  + [1. Codon
    Optimization](#codon-optimization)
  + [2. DNA
    Preparation](#dna-preparation)
  + [3.
    Construction of Plasmids](#construction-of-plasmids)
  + [4. Patchoulol
    detection](#id6)

## [Communication](#id12)[¶](#communication "Permalink to this heading")

### [Highschool Teams HP Experience Exchange](#id13)[¶](#highschool-teams-hp-experience-exchange "Permalink to this heading")

We were invited by SYSU-CHINA to visit their laboratory and communicate about our
projects. They showed us their advanced and practical scientific research, and provided
us with experience and advice on the use of the equipment, which made great
contributions to the direction and operation of our team’s experiment. At the same time,
we learned each other’s speech skills, which provided us with valuable experience in
speaking and writing manuscripts.

**Lab Visit**[¶](#id7 "Permalink to this table")

|  |  |  |  |
| --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication1.png](https://static.igem.wiki/teams/4270/wiki/communication1.png) | ![https://static.igem.wiki/teams/4270/wiki/communication2.png](https://static.igem.wiki/teams/4270/wiki/communication2.png) | ![https://static.igem.wiki/teams/4270/wiki/communication3.png](https://static.igem.wiki/teams/4270/wiki/communication3.png) | ![https://static.igem.wiki/teams/4270/wiki/communication4.png](https://static.igem.wiki/teams/4270/wiki/communication4.png) |

### [Online Conferences](#id14)[¶](#online-conferences "Permalink to this heading")

Later, we also had a video conference with SYSU-CHINA team. We presented and shared our
story of patchoulol, questionary experiences, and failures to SYSU-CHINA team members.
They gave us some valuable suggestions. SYSU-CHINA team also presented their research
content about salidroside. The communication with SYSU-CHINA team gave us extraordinary
inspiration for our project.

[![https://static.igem.wiki/teams/4270/wiki/communication5.png](https://static.igem.wiki/teams/4270/wiki/communication5.png)](https://static.igem.wiki/teams/4270/wiki/communication5.png)

**Online Metting**[¶](#id8 "Permalink to this table")

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication6.png](https://static.igem.wiki/teams/4270/wiki/communication6.png) | ![https://static.igem.wiki/teams/4270/wiki/communication7.png](https://static.igem.wiki/teams/4270/wiki/communication7.png) | ![https://static.igem.wiki/teams/4270/wiki/communication8.png](https://static.igem.wiki/teams/4270/wiki/communication8.png) | ![https://static.igem.wiki/teams/4270/wiki/communication9.png](https://static.igem.wiki/teams/4270/wiki/communication9.png) | ![https://static.igem.wiki/teams/4270/wiki/communication10.png](https://static.igem.wiki/teams/4270/wiki/communication10.png) | ![https://static.igem.wiki/teams/4270/wiki/communication11.png](https://static.igem.wiki/teams/4270/wiki/communication11.png) |

### [Propaganda Speech](#id15)[¶](#propaganda-speech "Permalink to this heading")

In order to bring our project closer to the masses, our team held public talks to
introduce ourselves and our results. Adhering to the principle of making synthetic
biology enjoyable, we selected three speakers from our team to describe our team’s
struggles and research results along the way. After returning to campus in September, we
also held another club presentation to show new students our latest progress and basic
information. At the club festival of our school, we publicized our project in the
biology club, so that all the high school freshmen and part of the high school seniors
who participated in the activity knew about our project and activity, and learned
something from it. In all the lectures we held, we received a lot of compliments and
suggestions, so we would adjust our presentation each time and do it better next time to
give everyone a better understanding of the project.

**Public Speaking**[¶](#id9 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication12.png](https://static.igem.wiki/teams/4270/wiki/communication12.png) | ![https://static.igem.wiki/teams/4270/wiki/communication13.png](https://static.igem.wiki/teams/4270/wiki/communication13.png) | ![https://static.igem.wiki/teams/4270/wiki/communication14.png](https://static.igem.wiki/teams/4270/wiki/communication14.png) |

### [Interview with Experts](#id16)[¶](#interview-with-experts "Permalink to this heading")

Considering that the light-induced promoter is used in our project, and the professor’s
research field is about the regulation of photosynthesis, we asked the professor to give
us some suggestions for improving the knowledge of photobiology used in our project.

Initially, the professor says, you need to learn to screen when doing experiments.
Photobiology is a very cutting-edge branch of life science. There have been previous
achievements in this field. When we make use of it, we should screen the suitable
conditions for our experiments according to the actual situation. For example, the
light-induced promoter of HCF173 used in this experiment should be screened before use
to screen out the light-induced promoter with the best-carrying effect with our target
gene to ensure that its activity matches each other with the gene.

In addition, promoter optimization should be carried out in combination with the actual
situation. For example, the light we are using to activate now in our experiments, white
light, means that daily light can activate the promoter. However, in the actual
production, because white light is too easy to appear, it is impossible to achieve a
real light environment. To solve this problem, the professor suggested that the light we
used to induce could be selected from specific bands of light, such as those rare in
nature, so as to avoid the interference of light in nature and make the experiment more
accurate.

What is more, the promoter’s light requirements need to be feasible. If we have a light
promoter that has very high light intensity requirements, and you have to meet these
ultra-high requirements to start, then most of the time you can’t produce. And if very
strong light is required, it will greatly increase the cost of our production.

Considering the problems that arise when moving from the laboratory to mass production,
we also discussed the whole synthetic biology industry with the professor. The professor
pointed out that the laboratory environment is very different from the actual production
environment. For example, in this project, we have a light-induced promoter, and in the
lab, the volume that we synthesize is actually very small, maybe a thin layer in yeast.
However, in mass production, it is still very difficult to ensure that each yeast light
promoter can feel the light in the face of the large volume of the factory. For these
problems, the professor pointed out a clear way for us: we should first form a
theoretical basis in the laboratory, and when we find problems in large-scale
production, we should timely feedback, optimize and solve them in the laboratory, and
then put them into large-scale production for trial. And so on and so on until the
desired effect is achieved.

Finally, we asked professors how they could connect the projects they were working on to
society and make a real contribution. First of all, the professor affirmed our research
project on patchouli and patchoulol, which are very distinctive ingredients derived from
Lingnan’s traditional Chinese medicine. It is a contribution to society to bring
characteristic plants into play. As for the role of patchoulol, the professor pointed
out the following points: one is that patchoulol can be directly used as a component in
medicine, and the other is that patchoulol can be used as a chassis for other products,
providing a prerequisite for other drugs.

According to the suggestions made by the professor during the interview, we plan to make
the following improvements in the future: First, for the optical promoter itself, reduce
its requirements on light and increase the practicability of the product. Second, if in
the future will be put into large-scale production, the problems encountered in the
production should be timely improved in the laboratory.

### [Interview with doctor](#id17)[¶](#interview-with-doctor "Permalink to this heading")

Considering the particularity of the research objects of our project (patchouli and
patchouli alcohol), the team decided to interview some practitioners of traditional
Chinese medicine working on the frontline in the community clinic, and further discuss
the current situation of the use of patchouli and patchouli alcohol and the social value
of our project.

First, we asked doctors what kind of concoctions they typically prescribe to patients,
such as sliced or ground medicine. Doctors say there are only one or two medicines that
usually contain patchouli or patchoulol in hospitals, such as Huoxiang Zhengqi capsule
and Huoxiang Zhengqi liquid.

Then, we briefly introduced our project and discussed the impact of the increased
production of patchoulol on doctors’ consultations and even society as a whole. Doctors
say that if the production of patchoulol can be increased and more patchoulol becomes
more available, the price of patchoulol will fall accordingly. Among them, pharmacists
in the pharmacy told us that the price of Chinese patent medicine has been soaring. If
the price of patchoulol can be reduced, doctors will have fewer concerns when
prescribing medicine to patients, and it will also be able to ensure that some poor
people have access to medicine, which is of great significance to society.

**Interviewing Doctors**[¶](#id10 "Permalink to this table")

|  |  |  |
| --- | --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/communication15.png](https://static.igem.wiki/teams/4270/wiki/communication15.png) | ![https://static.igem.wiki/teams/4270/wiki/communication16.png](https://static.igem.wiki/teams/4270/wiki/communication16.png) | ![https://static.igem.wiki/teams/4270/wiki/communication17.png](https://static.igem.wiki/teams/4270/wiki/communication17.png) |

## [Contribution](#id18)[¶](#contribution "Permalink to this heading")

### [1.Characterization of previous iGEM parts](#id19)[¶](#characterization-of-previous-igem-parts "Permalink to this heading")

In this project, basic parts of previous projects are characterized, including pPGK1
promoter (BBa\_K122000), pTEF1 promoter (BBa\_K2765041), TADH1 terminator (BBa\_K122004),
and TCYC1 terminator (BBa\_K122003). Patchoulol synthase (PTS) gene and farnesyl
diphosphate synthase (ERG20, also named FPS) gene are expressed by this promoters and
terminators. The sequences were assembled into pSP-GM2 plasmids and constructed 4
recombinant vectors, namely pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS and
pSP-GM2-PTS-FPS (Figure 1-4). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 5**).

[![https://static.igem.wiki/teams/4270/wiki/contributions1.png](https://static.igem.wiki/teams/4270/wiki/contributions1.png)](https://static.igem.wiki/teams/4270/wiki/contributions1.png)

**Figure 1. The construction of pSP-GM2-FPS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions2.png](https://static.igem.wiki/teams/4270/wiki/contributions2.png)](https://static.igem.wiki/teams/4270/wiki/contributions2.png)

**Figure 2. The construction of pSP-GM2-PTS plasmid with pPGK1 as promoter and
TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions3.png](https://static.igem.wiki/teams/4270/wiki/contributions3.png)](https://static.igem.wiki/teams/4270/wiki/contributions3.png)

**Figure 3. The construction of pSP-GM2-PTS-linker-FPS plasmid with pPGK1 as
promoter and TADH1 as terminator.**

[![https://static.igem.wiki/teams/4270/wiki/contributions4.png](https://static.igem.wiki/teams/4270/wiki/contributions4.png)](https://static.igem.wiki/teams/4270/wiki/contributions4.png)

**Figure 4. The construction of pSP-GM2-PTS-FPS plasmid with pPGK1 and pTEF1 as
promoter, TADH1 and TCYC1 as terminators, respectively.**

[![https://static.igem.wiki/teams/4270/wiki/contributions5.png](https://static.igem.wiki/teams/4270/wiki/contributions5.png)](https://static.igem.wiki/teams/4270/wiki/contributions5.png)

**Figure 5 A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

### [2.Add New Documentations To An Existing Part](#id20)[¶](#add-new-documentations-to-an-existing-part "Permalink to this heading")

We will artificially cloned 2 new genes including HCF173 promoter and PTS. The patchoulol
synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces
more than 20 valuable sesquiterpenes including patchoulol by conversion of the natural
substrate FPP.[1-3] HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173),
which is required for Arabidopsis thaliana PSII biogenesis. HCF173 transcript levels and
expression were induced by light. HCF173 promoter contains ACE motif and G-box
element.[4] In our project, the CDS sequence of PTS was optimized according to yeast
codon preference. PCR was conducted first for cloning PTS, FPS (ERG20) and HCF173
(**Figure 6**). Later a gene fusion of PTS and FPS namely PTS-linker-FPS
are acquired by removing the stop codon of PTS and replacing it by a short peptide
(Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs.

[![https://static.igem.wiki/teams/4270/wiki/contributions6.png](https://static.igem.wiki/teams/4270/wiki/contributions6.png)](https://static.igem.wiki/teams/4270/wiki/contributions6.png)

**Figure 6. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

Later, we assembled HCF173 promoters, PTS gene and ERG20 (FPS) and constructed 4
recombinant vectors, namely pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS and pSP-GM2-HCF173-PTS-FPS (**Figure 7-10**).
The colony PCR were conducted for further verification of this recombinant vectors
(**Figure 11**).
.. image:: <https://static.igem.wiki/teams/4270/wiki/contributions7.png>

> width
> :   1000px
>
> align
> :   center

**Figure 7. The construction of pSP-GM2-HCF173-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions8.png](https://static.igem.wiki/teams/4270/wiki/contributions8.png)](https://static.igem.wiki/teams/4270/wiki/contributions8.png)

**Figure 8. The construction of pSP-GM2-HCF173-PTS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions9.png](https://static.igem.wiki/teams/4270/wiki/contributions9.png)](https://static.igem.wiki/teams/4270/wiki/contributions9.png)

**Figure 9. The construction of pSP-GM2-HCF173-PTS-linker-FPS plasmid with HCF173
as promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions10.png](https://static.igem.wiki/teams/4270/wiki/contributions10.png)](https://static.igem.wiki/teams/4270/wiki/contributions10.png)

**Figure 10. The construction of pSP-GM2-HCF173-PTS-FPS plasmid with HCF173 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions11.png](https://static.igem.wiki/teams/4270/wiki/contributions11.png)](https://static.igem.wiki/teams/4270/wiki/contributions11.png)

**Figure 11. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [3.Add New Parts](#id21)[¶](#add-new-parts "Permalink to this heading")

In our 2022 project, we provide 10 new parts:

> | part name | part number | website |
> | --- | --- | --- |
> | HCF173 promoter | BBa\_K4270001 | [http://parts.igem.org/Part:BBa\_K4270001](http://parts.igem.org/Part%3ABBa_K4270001) |
> | PTS (patchoulol synthase) | BBa\_K4270004 | [http://parts.igem.org/Part:BBa\_K4270004](http://parts.igem.org/Part%3ABBa_K4270004) |
> | erg20 K197P | BBa\_K4270007 | [http://parts.igem.org/Part:BBa\_K4270007](http://parts.igem.org/Part%3ABBa_K4270007) |
> | erg20 K197H | BBa\_K4270008 | [http://parts.igem.org/Part:BBa\_K4270008](http://parts.igem.org/Part%3ABBa_K4270008) |
> | FPS-linker-PTS | BBa\_K4270010 | [http://parts.igem.org/Part:BBa\_K4270010](http://parts.igem.org/Part%3ABBa_K4270010) |
> | pPGK1+PTS+TADH1 | BBa\_K4270011 | [http://parts.igem.org/Part:BBa\_K4270011](http://parts.igem.org/Part%3ABBa_K4270011) |
> | pPGK1+ERG20+TADH1 | BBa\_K4270012 | [http://parts.igem.org/Part:BBa\_K4270012](http://parts.igem.org/Part%3ABBa_K4270012) |
> | pTEF1+ERG20+TCYC1 | BBa\_K4270016 | [http://parts.igem.org/Part:BBa\_K4270016](http://parts.igem.org/Part%3ABBa_K4270016) |
> | HCF173+ ERG20+TADH1 | BBa\_K4270013 | [http://parts.igem.org/Part:BBa\_K4270013](http://parts.igem.org/Part%3ABBa_K4270013) |

### [4.Improvement of Existing Parts](#id22)[¶](#improvement-of-existing-parts "Permalink to this heading")

#### [4.1 Optimized of farnesyl diphosphate synthase (ERG20, FPS) (BBa\_K517004)](#id23)[¶](#optimized-of-farnesyl-diphosphate-synthase-erg20-fps-bba-k517004 "Permalink to this heading")

The ERG20 is a basic part comprised from a synthesized farnesyl diphosphate synthase
coding sequence. The basic part BBa\_K4270007 (erg20 K197P) and BBa\_K4270008 (erg20
K197H) are optimized versions of ERG20 (BBa\_K517004) that can be constructed into
pSP-GM2 plasmid. The resulted vectors are named pSP-GM2-FPS, pSP-GM2-FPS K197P and
pSP-GM2-FPS K197H respectively. Then this vectors were transformed into
Saccharomyces cerevisiae BY4741 to produce patchoulol.

#### [4.2 Plasmid Construction](#id24)[¶](#plasmid-construction "Permalink to this heading")

PCR was conducted for cloning FPS (ERG20), erg20 K197P and erg20 K197H. The purified
PCR products were assembled into pSP-GM2 plasmids and constructed 3 recombinant
vectors, namely pSP-GM2-FPS, pSP-GM2-FPS K197P and pSP-GM2-FPS K197H (**Figure
12-13**). Then these vectors are all transformed to Saccharomyces
cerevisiae BY4741. The colony PCR were conducted for further verification of this
recombinant vectors (**Figure 14**).

[![https://static.igem.wiki/teams/4270/wiki/contributions12.png](https://static.igem.wiki/teams/4270/wiki/contributions12.png)](https://static.igem.wiki/teams/4270/wiki/contributions12.png)

**Figure 12. The construction of pSP-GM2-FPS K197P plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions13.png](https://static.igem.wiki/teams/4270/wiki/contributions13.png)](https://static.igem.wiki/teams/4270/wiki/contributions13.png)

**Figure 13. The construction of pSP-GM2-FPS K197H plasmid with pPGK1 as
promoter, TADH1 as terminators.**

[![https://static.igem.wiki/teams/4270/wiki/contributions14.png](https://static.igem.wiki/teams/4270/wiki/contributions14.png)](https://static.igem.wiki/teams/4270/wiki/contributions14.png)

**Figure 14. The colony PCR results of pSP-GM2-FPS K197P (left side) and
pSP-GM2-FPS K197H (right side).**

#### [4.3 Results](#id25)[¶](#results "Permalink to this heading")

Gas chromatography (GC) method to observe the patchoulol in Yeast fermentation broth.
Unfortunately, we did not detect patchoulol in the supernatant but detected
patchoulol in the standard substance containing patchoulol (Figure 15). The reason
we did not detect patchoulol in the supernatant may due to the low yield of
patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/contributions15.png](https://static.igem.wiki/teams/4270/wiki/contributions15.png)](https://static.igem.wiki/teams/4270/wiki/contributions15.png)

**Figure 15. The detection of patchoulol in the supernatant. A-C. The
detection of patchoulol in Yeast fermentation broth from Yeast transformed with
pSP-GM2-FPS, pSP-GM2- FPS K197P, pSP-GM2- FPS K197H plasmids, respectively. D.
The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id26)[¶](#reference "Permalink to this heading")

[1] Albertsen L, Chen Y,Bach L. S, Rattleff S, et al. Diversion of flux toward
sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous
enzymes. Appl. Environ. Microbiol. 2011, 77, 1033−1040.

[2] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid precursors elevates terpene production in plants. Nature Biotechnology, 2006,
24: 1441-47.

[3] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[4] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Description](#id27)[¶](#description "Permalink to this heading")

### [Pogostemon cablin and patchouli oil](#id28)[¶](#pogostemon-cablin-and-patchouli-oil "Permalink to this heading")

Pogostemon cablin is a kind of medicinal aromatic herb and one of the Top 10 drugs in
Guangdong province. P. cablin is mainly grown in Indonesia, Singapore, Vietnam,
Malaysia, China, and other areas.[1] Patchouli oil is made from the dried ground part of
P. cablin by distillation and is mainly used in two aspects.[2] First, it is used as a
drug because of its anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other
pharmacological effects.[3-5] On the other hand, its fragrance is unique and used as a
spices and food additive.[6] Benefiting from its good efficacy, patchouli oil is widely
used in the daily chemical, pharmaceutical, and food industries and has a broad market
prospect. The price of high-quality patchouli oil can be as high as ¥1300 per kilogram.
In terms of the retail price, it can rank 10th among the important essential oils in the
world and has obvious economic benefits. [7, 8]

**Pogostemon cablin**[¶](#id11 "Permalink to this table")

|  |  |
| --- | --- |
| ![https://static.igem.wiki/teams/4270/wiki/description1.png](https://static.igem.wiki/teams/4270/wiki/description1.png) | ![https://static.igem.wiki/teams/4270/wiki/description2.png](https://static.igem.wiki/teams/4270/wiki/description2.png) |

### [Patchoulol](#id29)[¶](#patchoulol "Permalink to this heading")

Patchoulol, a sesquiterpene, is one of the most abundant components in patchouli oil. [6,
9] As an important index for evaluating the quality of P. cablin and patchouli oil in
Chinese Pharmacopoeia, patchoulol is also considered the main component contributing to
the pharmacological effects and aroma of patchouli oil. [10] Nowadays, there is a
serious imbalance between the supply and demand of patchoulol and patchouli oil in the
world. The global annual demand for patchoulol and patchouli oil can reach over 2 000 t,
but the annual output is about 1300 t. Indonesia supplies 90 percent of all annual
output, while China produces less than 10 percent of that, which has plenty of room for
growth. [11, 12] Increasing the yield of patchoulol has become a key point in the study
of p. cablin.

At present, patchoulol is obtained in three main ways, namely, plant extraction, chemical
synthesis, and microbial fermentation.

[![https://static.igem.wiki/teams/4270/wiki/description3.png](https://static.igem.wiki/teams/4270/wiki/description3.png)](https://static.igem.wiki/teams/4270/wiki/description3.png)

### [Plant extraction](#id30)[¶](#plant-extraction "Permalink to this heading")

Typically, patchoulol is extracted from the leaves of P.cablin. However, the low content
of patchoulol in P. cablin is the bottleneck of its large-scale application. [2]

### [Chemical synthesis](#id31)[¶](#chemical-synthesis "Permalink to this heading")

Although the chemical synthesis method can support patchoulol with high purity, it needs
many steps, complex operation, high synthesis cost, and the yield are low, so it cannot
be applied to large-scale industrial production. [13]

### [Microbial Fermentation](#id32)[¶](#microbial-fermentation "Permalink to this heading")

Compared with plant extraction and chemical synthesis, microbial fermentation synthesis
exhibits several advantages in producing patchoulol, such as fast-growing, land-saving,
and controllable culture conditions. With the development of metabolic engineering and
synthetic biology, many valuable plant-derived terpenoids also have been produced in
microbial cell factories, such as artemisinic acid [14], ginsenosides [15], and
tanshinones [16]. Although naturally produced patchoulol can be obtained from plants,
patchoulol production using biobased microbial platforms is an economical and
sustainable alternative.

### [Patchoulol biosynthetic pathways](#id33)[¶](#patchoulol-biosynthetic-pathways "Permalink to this heading")

There are two biosynthetic pathways for patchoulol formation, namely the mevalonic acid
(MVA) pathway located in the cytoplasm and the methylerythritol 4-phosphate
(MEP) pathway located in plastids. [17, 18] In general, patchoulol is mainly synthesized
through the MVA pathway. There are 3 key enzymes in the synthesis of patchoulol, namely
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FPS),
and patchoulol synthase (PTS). HMGR catalyzes the irreversible formation of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into MVA. Because it can affect the
yield of IPP (isopentenyl diphosphate), the precursors of patchoulol, HMGR is the key to
the metabolism of patchoulol. FPS catalyzes IPP to FPP (farnesyl diphosphate). PTS is a
key rate-limiting enzyme in the formation of the patchoulol, which catalyzes the
formation of patchoulol skeleton from the FPP precursor, and the skeleton is modified by
secondary enzymes to form patchoulol.

[![https://static.igem.wiki/teams/4270/wiki/description4.png](https://static.igem.wiki/teams/4270/wiki/description4.png)](https://static.igem.wiki/teams/4270/wiki/description4.png)

### [Vision](#id34)[¶](#vision "Permalink to this heading")

Patchoulol production using biobased microbial platforms is an economical and sustainable
alternative. Recently, based on the endogenous mevalonate (MVA) pathway or
2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, heterologous production of patchoulol
has been accomplished by introducing the patchoulol synthase gene in Saccharomyces
cerevisiae [8] and Corynebacterium glutamicum [19], as well as in the moss
Physcomitrella patens[20] and eukaryotic microalga Chlamydomonas reinhardtii [21].
Nevertheless, the yield is still too low for industrial production. Therefore,
significant engineering is required to achieve sufficient yields for industrial
applications. Scientists see the use of environmental signals (light, temperature) and
endogenous signals (quorum sensing, pressure) to establish tightly controlled patchoulol
biological production systems as the future trend.

Promoters are crucial elements in controlling the expression of target genes, and the
emergence of inducible promoters provides the possibility to accurately and flexibly
regulate the expression of foreign genes, which has become one of the hot spots in the
research and development of genetic engineering. [22] Therefore, in the present study,
two metabolic engineering strategies were conducted to enhance the production of
patchoulol in yeast. First, ERG20 (encoding FPP synthase from yeast) and PTS (encoding
patchoulol synthase from P. cablin) were used to increase the utilization of the FPP
precursor. Second, the constituted promoter and light-inducible promoter are
respectively used to drive the overexpression of the critical gene FPS and PTS. In
addition, studies have reported that high-level patchoulol production in tobacco plants
was achieved by fusing FPS and PTS genes. [17] Hence, vectors consisting of a gene
fusion of PTS and FPS (PTS-FPS) inserted downstream of the constitutive and
light-inducible promoter were also constructed. Finally, we carried out fermentation in
yeast and envisioned our final product by meteorological chromatography.

[![https://static.igem.wiki/teams/4270/wiki/description5.png](https://static.igem.wiki/teams/4270/wiki/description5.png)](https://static.igem.wiki/teams/4270/wiki/description5.png)

### [Reference](#id35)[¶](#id1 "Permalink to this heading")

[1] Yao G, Drew B T, Yi T S, et al. Phylogenetic relationships, character evolution and
biogeographic diversification of Pogostemon s.l. (Lamiaceae) [J]. Mol Phylogenet Evol,
2016, 98(184-200.

[2] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[3] Zhang R, Yan P, Li Y, et al. A pharmacokinetic study of patchouli alcohol after a
single oral administration of patchouli alcohol or patchouli oil in rats [J]. European
Journal of Drug Metabolism and Pharmacokinetics, 2016, 41(4): 441-8.

[4] Wu Z, Zeng H, Zhang L, et al. Patchouli alcohol: a natural sesquiterpene against both
inflammation and intestinal barrier damage of ulcerative colitis [J]. Inflammation,
2020, 43(4): 1423-35.

[5] 闵琼, 曾海荣, 陆翠燕, et al. 广藿香醇通过调控上皮间质转化抑制人胃癌细胞HGC-27的侵袭和转移 [J]. 药学服务与研究, 2020, 20(01):
6-11.

[6] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[7] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Ind Crop Prod, 2016, 87(161-76.

[8] Ma B, Liu M, Li Z H, et al. Significantly enhanced production of patchoulol in
metabolically engineered Saccharomyces cerevisiae [J]. J Agric Food Chem, 2019, 67(31):
8590-8.

[9] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[10] 徐雯, 吴艳清, 丁浩然, et al. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[11] Swamy M K, Mohanty S K, Sinniah U R, et al. Evaluation of patchouli (Pogostemon
cablin Benth.) cultivars for growth, yield and quality parameters [J]. Journal of
Essential Oil-Bearing Plants, 2015, 18(4): 826-32.

[12] Swamy M K, Sinniah U R. Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology
and biotechnological aspects [J]. Industrial Crops & Products, 2016, 87(161-76.

[13] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of ()-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[14] Westfall P J, Pitera D J, Lenihan J R, et al. Production of amorphadiene in yeast,
and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent
artemisinin [J]. P Natl Acad Sci USA, 2012, 109(3): 655-6.

[15] Wei W, Wang P, Wei Y, et al. Characterization of Panax ginseng
UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive
ginsenosides F1 and Rh1 in metabolically engineered yeasts [J]. Molecular Plant,
2015,

[16] Guo J, Zhou Y J, Hillwig M L, et al. CYP76AH1 catalyzes turnover of miltiradiene in
tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts
[J]. Pnas, 2013, 110(29): 12108-13.

[17] Shuiqin W, Michel S, Anthony C, et al. Redirection of cytosolic or plastidic
isoprenoid
precursors elevates terpene production in plants. Nature Biotechnology, 2006, 24:
1441-47.

[18] Tang Y, Zhong L, Wang X, et al. Molecular identification and expression of
sesquiterpene pathway genes responsible for patchoulol biosynthesis and regulation in
Pogostemon cablin［J］.
Bot Stud, 2019, 60：11.

[19] Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered Corynebacterium glutamicum［J］. Genes (Basel), 2018, 9(4): E219.

[20] Zhan X, Zhang YH, Chen DF, et al. Metabolic engineering of the moss Physcomitrella
patens to produce the sesquiterpenoids patchoulol and α/β-santalene［J］. Front Plant Sci,
2014, 5 ：636.

[21] Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a
high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii［J］.
Metab Eng, 2016,
38: 331-343.

[22] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Engineering](#id36)[¶](#engineering "Permalink to this heading")

### [Abstract](#id37)[¶](#abstract "Permalink to this heading")

Patchoulol is one of the main active components of Pogostemon cablin, and it is also an
important indicator for evaluating the medicinal materials of P. cablin in the Chinese
Pharmacopoeia. Patchoulol can be obtained by natural extraction, chemical synthesis, and
synthetic biology engineering. Due to the low content of patchoulol in P. cablin and the
low yield of chemical synthesis, the study of combinatorial biosynthesis and molecular
regulation can provide the basis for obtaining high-content patchoulol. In this study,
on the basis of the biosynthetic pathway, molecular regulation mechanism, and synthetic
biology engineering of patchoulol, the key genes for its biosynthesis were combined and
expressed in heterologous host yeast to produce patchoulol, which provides the technical
foundations of the development and application of patchoulol.

### [Experiment design](#id38)[¶](#experiment-design "Permalink to this heading")

#### [8 type of expression vector](#id39)[¶](#type-of-expression-vector "Permalink to this heading")

We designed eight plasmids: The strong constitutive promoter (pGK1) was separately
used to connect FPS (BBa\_K517004), PTS (BBa\_K4270004), and FPS-linker-PTS
(BBa\_K4270010), and plasmids containing pGK1, FPS, terminator, pGK1, PTS terminator
were constructed .In the following four designs, all the above strong promoters were
replaced with light-induced promoters HCF173, and then the test was carried out to
verify (Figure 1).

[![https://static.igem.wiki/teams/4270/wiki/engineering1.png](https://static.igem.wiki/teams/4270/wiki/engineering1.png)](https://static.igem.wiki/teams/4270/wiki/engineering1.png)

### [Experiment structure and testing](#id40)[¶](#experiment-structure-and-testing "Permalink to this heading")

#### [PCR amplification and estriction enzyme cutting expression vector pSP-GM2](#id41)[¶](#pcr-amplification-and-estriction-enzyme-cutting-expression-vector-psp-gm2 "Permalink to this heading")

Amplified by PCR, the plasmids were double digestion by the restriction enzymes NotI
and SacI. Plenty of strands of DNA were obtained. Plasmid DNA, restriction enzyme,
reaction buffer, and ddH2O were added successively in a 1.5 mL sterilized centrifuge
tube according to the table below. Mix and mark well. The mixture reacted at 37℃ for
eight to ten hours. Agarose gel electrophoresis was used to detect the product size
and determine whether the enzyme was successfully cut.

**Table 1 Enzyme digestion reaction system**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | Not I | 0.5 |
> | Sac I | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

#### [Likage of expression vector and chemical transformation of E.coli DH5α](#id42)[¶](#likage-of-expression-vector-and-chemical-transformation-of-e-coli-dh5 "Permalink to this heading")

In vitro assembly gene, including promoter, control of the corresponding enzyme
synthesis gene, sequence of the terminator, DNA ligase access strip DNA, forming a
circular recombinant vector. Add 10 ng vector recombinant DNA solution to 50 μL
competent E.coli, mix well, and let stand in an ice bath for 30 min. By heat pulse
method, the mixture was shocked in a water bath at 42℃ for 30 s, then placed on ice
for 3-5 minutes. 1 mL fresh medium was added and cultured at 37℃ for 1 h for
amplification. Add 500 μL to sterilized LB medium into a centrifuge tube, mix
evenly, and culture for 1 h at 37℃ and 200 rpm. Adjust the speed to 5000rpm,
centrifuge at room temperature for 1min, take out the supernatant and pour it out
until there was 100 μL of thallus remaining.

#### [Screening of transformants](#id43)[¶](#screening-of-transformants "Permalink to this heading")

Pick a single bacteria placement from the conversion plate and place it on the new LB
solid plate containing antibiotics, culture it at 37℃ for 2-4 h and then pick up the
colonies. Put them into the centrifuge tube of the corresponding PCR reaction
solution. Take the colony as the template of PCR reaction and amplify the colony by
T5 Super PCR Mix.

**Table 2 Colony PCR reaction system**

> | Reagent | Dosage |
> | --- | --- |
> | 2×T5 Super PCR Mix | 5 μL |
> | M13F（10 μM） | 0.4 μL |
> | M13R（10 μM） | 0.4 μL |
> | ddH2O | 4.2 μL |

**Table 3 Colony PCR reaction procedure**

> | Temperature | Time | Cycle |
> | --- | --- | --- |
> | 98℃ | 10 min | 1 |
> | 98℃ | 10 s | 35 |
> | 50℃ | 10 s |  |
> | 72℃ | 30 s |  |
> | 72℃ | 2 min | 1 |

#### [Plasmids extraction and extract sequencing](#id44)[¶](#plasmids-extraction-and-extract-sequencing "Permalink to this heading")

The transformed E.coli was inoculated with 0.5% glycerin and ampicillin in sterilized
TB culture medium. Culture at 220 rpm for 12 h at 37℃ until saturated. Adjust the
speed to 10000 rpm, centrifuge at room temperature for 6 min, remove and pour off
the supernatant. The centrifugation process was repeated until the plasmid was
successfully extracted. After gene sequencing, verify whether the vector is
successfully transformed and consistent with the design sequence.

#### [Transformation and screening of yeast](#id45)[¶](#transformation-and-screening-of-yeast "Permalink to this heading")

About 16 colonies growing in LB solid medium containing ampicillin were selected and
verified by 2x Easy Taq enzyme. The PCR system is shown below.

**Table 4 PCR reaction (20 μL)**

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | buffer | 8 |

The reaction time and temperature were set according to the table below, and
electrophoresis was performed after amplification.

**Table 5 experimental set**

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

The monoclones were selected into 5 mL of the corresponding defective medium (BY4741
into YPD containing glucose). The culture was incubated overnight at 28℃ at 200-220
rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial solution
into 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out the
supernatant. The monoclones were selected into 5mL of corresponding defective medium
(BY4741 into YPD containing glucose). The culture was incubated overnight at 28℃ at
200-220 rpm until the OD600 value was 0.5-0.6. Take 500 μL of cultured bacterial
solution into a 1.5 mL EP tube, centrifuge at 300-500 rpm for 3-5 min, and pour out
the supernatant. Add 500 μL of EZ1 and resuspend thallus. Centrifuge 300-500 rpm for
3-5 min to remove the supernatant. Add 50μL of EZ2, flick, and resuspended the
thallus to obtain a competent state.

#### [Transform procedure](#id46)[¶](#transform-procedure "Permalink to this heading")

3-5 μL plasmid was added to 50 μL competent state, mixed evenly, then 500 μL EZ3 was
added, then evenly and lightly beaten with a blue gun since EZ3 is sticky. Incubated
in a water bath at 30℃ for 45 min-1 h. After centrifugation at 500 rpm for 5 min,
flocculent accumulation of thalli could be seen near the bottom of the tube. After
centrifugation, 400 μL centrifuged EZ3 was sucked off, leaving 100 μL resuspended
thallus and applied directly to the plate.

The vector was transformed into yeast and screened for nutritional deficiency.
Successfully transformed recipient cells can be grown on nutritionally deficient
media. The successfully transformed yeast cells were taken and examined again.

#### [Fermentation of modified strains](#id47)[¶](#fermentation-of-modified-strains "Permalink to this heading")

Provide suitable nutrients and environment for fermentation, so that the successful
transformation of yeast fermentation at room temperature.

#### [Extraction and determining of metabolite](#id48)[¶](#extraction-and-determining-of-metabolite "Permalink to this heading")

The content of patchoulol, a volatile sesquiterpene, was analyzed by gas
chromatography.

#### [Learning and design again:](#id49)[¶](#learning-and-design-again "Permalink to this heading")

We further optimized the original improved carrier. On this basis, the experimental
scheme was further optimized.

## [Experiments](#id50)[¶](#experiments "Permalink to this heading")

### [Protocol](#id51)[¶](#protocol "Permalink to this heading")

#### [1. The enzyme digestion reaction of pSP-GM2 vector](#id52)[¶](#the-enzyme-digestion-reaction-of-psp-gm2-vector "Permalink to this heading")

Material: pSP-GM2 vector, restriction enzyme, 10 x Cutsmart buffer, ddH2O.

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

**Procedure：**

1. Add ingredients in 1.5 mL test tubes at a time according to the above
   table.
2. Mix and mark well.
3. 37℃ for 8-10 h.

#### [2. PCR and electropherosis](#id53)[¶](#pcr-and-electropherosis "Permalink to this heading")

Material: DNA template、primer、dNTPs、5 × Q5 reaction buffer、Q5 DNA polymerase.

Procedure: (1) Mix up the recipe into 1.5 mL test tubes according to the following
table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

#### [3. DNA gel electrophoresis](#id54)[¶](#dna-gel-electrophoresis "Permalink to this heading")

(1) Preparation of 1% DNA gel
Weigh 1 g of agarose into a cone-shaped flask, and then add 100 mL of 10xTAE buffer
into this cone-shaped flask. Transfer the cone to the microwave and heat until
boiling (2-3 times). Cooling the cone-shaped flask to about 50℃, and then add the
nucleic acid dye into it. Mixing agarose and nucleic acid well and pour them into
the rubber rack. After 1 hour later, 1% DNA gel is ready.

(2) point sample
Mixing the DNA sample and the adding buffer (4:1). Then adding the mixture to the
sample tank with a micropipette gun. Each tank addes 20 μL mixture. Recording the
order of spot sampling and the amount of adding sample.

(3) Gel electrophoresis
After installing the electrode wire, connect one end of the sample hole to the
negative electrode and the other end to the positive electrode. Then switching on
the power supply and adjust the voltage to 120 V. After electrophoresis for 20-30
min, stop electrophoresis.

#### [4. Transformation of plasmid to E.coli](#id55)[¶](#transformation-of-plasmid-to-e-coli "Permalink to this heading")

1. Take 50 µL of E.coli cells and defrost E.coli with ice bath for 25 min.
   Mixing E.coli gently with 10 µL target recombinant plasmid.
2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for
   another 3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of
   supernatant and resuspend the bacteria. Coating on the LB broth containing
   ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

#### [5. Selecting colonies](#id56)[¶](#selecting-colonies "Permalink to this heading")

**Colony PCR**

1. Inoculate BL21 cells using a pipette tip，use 2x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run
   the PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

#### [6. DNA sequencing](#id57)[¶](#dna-sequencing "Permalink to this heading")

Expand and culture the positive colonies in LB liquid medium containing ampicillin at
37℃ for 10-12 h. Extract the plasmid by using the plasmid extraction kit. Sent 10 μL
plasmid to sequencing company for sequencing. The correctly sequenced plasmid was
stored at -20°C and the corresponding bacterial solution was stored at -80°C.

#### [7. Yeast transformation procedure](#id58)[¶](#yeast-transformation-procedure "Permalink to this heading")

1. selecte the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into 1.5mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into 1.5mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50μL yeast cells, mix evenly, then
   add 500μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1
   h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and
   coate them on a solid medium plate.

## [Implementation](#id59)[¶](#implementation "Permalink to this heading")

### [Background](#id60)[¶](#background "Permalink to this heading")

Patchoulol is one of the most abundant components in patchouli oil[1, 2] and is also
considered the main component contributing to the pharmacological effects and aroma of
patchouli oil[3], which is widely used in medicine, daily chemicals, food, and other
industries, with broad market prospects. However, there is a serious imbalance between
the supply and demand of patchoulol and patchouli oil in the world. There are three ways
to prepare patchouli so far. Typically, patchoulol is extracted from the leaves of
Pogostemon cablin. However, the low content of patchoulol in P. cablin is the bottleneck
of its large-scale application [4]. Although the chemical synthesis method can support
patchoulol with high purity, it has many drawbacks, such as too many steps, complex
operation, high synthesis cost, and low yield, so it is may not suitable for large-scale
industrial production [5]. Compared with plant extraction and chemical synthesis,
microbial fermentation synthesis exhibits several advantages in producing patchoulol,
such as fast-growing, land-saving, and controllable culture conditions. With the
development of synthetic biology, patchoulol has been produced in yeast under the
control of a constitutive promoter. [6] However, the production of patchoulol is
uncontrollable and yeast cells may produce too much patchoulol at a time, which is toxic
to yeast cells. Herein, our team has innovatively applied the light-induced promoter in
the biosynthesis of patchoulol in yeast, which makes the production of patchoulol
controllable. When the light is on, yeast cells produce patchoulol. Otherwise, it is not
produced patchoulol. We believe the increasing production of patchoulol will lower the
price of patchoulol. Furthermore, the lower price of patchoulol can effectively help
more people particularly those in outlying poverty-stricken areas.

### [Target customers](#id61)[¶](#target-customers "Permalink to this heading")

**1. Patchouli essential oil factory**

Patchouli essential oil is in great demand in the market because of its long history of
use, wide range of uses, and good fragrance fixing effect. Patchouli essential oil is
beneficial to the body and mind. For example, it can be mentally refreshing, relieve
anxiety, and make people feel excited. Physiologically, it has a good bactericidal
effect, promotes skin regeneration and reduces inflammation, and so on. These properties
have made patchouli popular. As we all know, patchoulol is one of the main ingredients
in patchouli oil. If our project can produce patchoulol on a large-scale industrial
production, it will generate a lot of business value.

**2. Pharmaceutical factory**

Patchoulol is also considered the main component contributing to the pharmacological
effects of patchouli oil. It is often used as raw materials for drugs because of its
anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other pharmacological
effects[7]. Nowadays, The market demand for patchoulol is increasing. For example,
Huoxiang Zhengqi Shui (KangShengTang), a kind of medicine that is always available in
every household in China, contains a huge amount of patchoulol. Therefore, when our
project achieves the efficient production of patchoulol, we can sell patchoulol to
pharmaceutical factories and profit from it.

**3. Customers who need environment-friendly pesticides**

Because of its insecticidal biological activity, patchoulol can be made into insecticides
to control some specific species of pests. And because it has the characteristics of low
toxicity, easy degradation, safety, and environmental friendliness, it is a good choice
to use as an insecticide. Therefore, we can also consider producing a large amount of
patchoulol by ourselves, preparing it into an environment-friendly insecticide, and
selling it to customers who need it in this regard.[8]

### [Possible challenges](#id62)[¶](#possible-challenges "Permalink to this heading")

Although our project has achieved initial results in the laboratory environment,
considering the difference between the laboratory environment and the actual large-scale
production environment, we are worried that there will be some problems during the
large-scale production. For example, the accumulation of too much yeast may lead to an
untimely and/or insensitive response of the light-induced prompter in yeast to light
conditions, and even cause the light-induced promoter to lose its original function,
making the whole biosynthesis process uncontrollable. These accidents that may occur in
the mass production process will greatly affect the mass production of patchoulol, which
may limit the availability of our product to the market. So for us, problems that arise
during mass production can be a challenge.

### [Reference](#id63)[¶](#id2 "Permalink to this heading")

[1] Van Beek T A, Joulain D. The essential oil of patchouli, Pogostemon cablin: A review
[J]. Flavour and Fragrance Journal, 2018, 33(1): 6-51.

[2] Hybertson B M. Solubility of the sesquiterpene alcohol patchoulol in supercritical
carbon dioxide [J]. J Chem Eng Data, 2007, 52(1): 235-8.

[3] 徐雯, 吴艳清, 丁浩然, 等. 广藿香的药理作用及机制研究进展 [J]. 上海中医药杂志, 2017, 51(10): 4.

[4] Henke N A, Wichmann J, Baier T, et al. Patchoulol production with metabolically
engineered corynebacterium glutamicum [J]. Genes (Basel), 2018, 9(4): 219.

[5] Srikrishna A, Satyanarayana G. An enantiospecific total synthesis of (-)-patchouli
alcohol [J]. Tetrahedron Asymmetry, 2005, 16(24): 3992-7.

[6] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene
production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J].
Appl Environ Microbiol, 2011, 77(3): 1033-40.

[7] 魏晓露, 彭成, 万峰. 广藿香醇体外抗呼吸道病毒作用研究 [J]. 中药药理与临床, 2013, 29(1): 4.

[8] 陈义娟, 范能能, 蒋杰贤, et al. 百秋李醇的用途及杀虫剂 [M]. 2020.

## [Notebook](#id64)[¶](#notebook "Permalink to this heading")

### [Week 1 (6.20-6.26)](#id65)[¶](#week-1-6-20-6-26 "Permalink to this heading")

We inoculated and cultured Escherichia coli containing the target vector in a liquid LB
growth medium. Then we waited for the cultures to grow and develop. We extracted the
target vector and designed primers needed for the construction of our vectors and sent
them to the company for synthesis.

We purchased the reagents needed for our experiments.

### [Week 2 (6.27-7.3)](#id66)[¶](#week-2-6-27-7-3 "Permalink to this heading")

**Part one**
**First attempt to digest target vector.**

We used the target pSP-GM2 vector, restriction enzyme, reaction buffer, and ddH2O to
digest
the target vector. The reaction system is as follows:

> | Reagent | Volume (μL) |
> | --- | --- |
> | 10 x Cutsmart buffer | 5 |
> | restriction enzyme 1 | 0.5 |
> | restriction enzyme 2 | 0.5 |
> | pSP-GM2 vector | 1 |
> | ddH2O | 43 |

Procedures to digest target vector:
(1) Add ingredients in 1.5 mL test tubes at a time according to the above table.

2. Mix and mark well.
3. 37℃ for 8-10 h.

**Part teo**
**PCR amplification of corresponding parts.**

PCR amplification for PST gene, FPS gene, and the HCF173 promoter. However, promoter
HCF173 first was not successfully amplified. After the second test, we successfully
amplified HCF173 promoter.

Procedure for PCR amplification:
(1) Mix up the recipe into 1.5 mL test tubes according to the following table.

> | Reagent | Volume (μL) |
> | --- | --- |
> | Q5 DNA polymerase | 0.5 |
> | 5 × Q5 reaction buffer | 10 |
> | 10 mM dNTPs | 1 |
> | forward primer | 2.5 |
> | downstream primer | 2.5 |
> | DNA template | 1 |
> | ddH2O | 32.5 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | procedure | temperature/℃ | time/s | repeat |
> | --- | --- | --- | --- |
> | preprocessing | 98 | 30 | 1 cycle |
> | denaturation | 98 | 30 | 35 cycle |
> | annealing | 55 | 30 |  |
> | extension | 72 | 60 |  |
> | Final extension | 72 | 300 | 1 cycle |
> | Hold | 4 | ∞ | 1 cycle |

### [Week 3 (7.4-7.10)](#id67)[¶](#week-3-7-4-7-10 "Permalink to this heading")

**Part one Construction of target vector.**

We construct the target vector by mixing pSP-GM2 vector with the genes we cloned. These
plasmids were later transformed into E.coli BL21.

**Part two Transformation of the plasmid to E.coli**

Procedure:
(1) Take 50 µL of E.coli cells and defrost E.coli with an ice bath for 25 min. Mixing
E.coli gently with 10 µL target recombinant plasmid.

2. Heat shock at 42℃ for 90 s, then place it immediately in the ice bath for another
   3-5 min.
3. Add 500 µL LB broth (not containing antibiotics), mix it upside down, and put it
   in the shaker to recover at 200 rpm, 37℃ for 60 min.
4. Centrifuge at 5,000 rpm, room temperature, for 1 min. Keep 100 µL of supernatant
   and resuspend the bacteria. The coating on the LB broth containing ampicillin.
5. Place upside down to incubate at 37℃ for 10-12 h in the incubator.

### [Week 4 (7.11-7.17)](#id68)[¶](#week-4-7-11-7-17 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Colony PCR was used to select positive colonies, which was verified by sequencing and
then
in liquid LB.

General procedures:

1. Inoculate BL21 cells using a pipette tip, use 2 x Easy Taq enzyme for colonies
   selecting：

> | Reagent | Volume (μL) |
> | --- | --- |
> | 2x Easy Taq Mix | 10 |
> | colony | 0 |
> | forward primer | 1 |
> | Downstream primer | 1 |
> | ddH2O | 8 |

2. The reaction time and temperature were set according to the table below. Run the
   PCR program.

> | Procedure | Temperature/℃ | Time/S | Repeat |
> | --- | --- | --- | --- |
> | preprocessing | 94 | 30 | 1 cycle |
> | denaturation | 94 | 10 | 35 cycles |
> | annealing | 55 | 30 |  |
> | extension | 72 | 30 |  |
> | final extension | 72 | 60 | 1 cycle |
> | hold | 4 | ∞ | 1 cycle |

### [Week 5 (7.18-7.24)](#id69)[¶](#week-5-7-18-7-24 "Permalink to this heading")

**Transformation of plasmid to Yeast**

**Procedure:**

1. select the monoclones into 5mL corresponding defective medium (containing
   glucose)).
2. Incubate at 28℃ overnight at 200-220 rpm until OD600 value is 0.5-0.6.
3. Take 500 μL cultured yeast solution into a 1.5 mL EP tube, and centrifuge at
   300-500 rpm for 3-5 min. Remove the supernatant.
4. Add 500 microliters of EZ1 into a 1.5 mL EP tube and resuspend yeast cells.
5. Centrifuge at 300-500 rpm for 3-5 min and remove the supernatant.
6. Add 50 μL EZ2 into 1.5mL EP tube, flick, and resuspend yeast cells.
7. Transformation: add 3-5 μL plasmid into 50 μL yeast cells, mix evenly, then add
   500 μL EZ3 into it. Incubate yeast cells in a water bath at 30℃ for 45 min-1 h.
8. Centrifugate at 500 rpm for 5 min.
9. After centrifugation, remove 400μL supernate. Resuspend the yeast cells and coat
   them on a solid medium plate.

### [Week 6 (8.1-8.7) and Week 7 (8.8-8.14)](#id70)[¶](#week-6-8-1-8-7-and-week-7-8-8-8-14 "Permalink to this heading")

**Selecting colonies to inoculate and culture**

Single yeast colonies were taken from stock plates and inoculated into 8 ml YPD. We carry
out PCR on these colonies and plan to test them again. Results showed that some plasmids
have been successfully transformed into Yeast.

### [Week 7 (8.15-8.27)](#id71)[¶](#week-7-8-15-8-27 "Permalink to this heading")

**Cultivation of recombinant yeasts in a Shaking Flask.**

The recombinant yeasts were precultured in 5 mL of YPD at 30℃, 220 rpm for 24 h.
Precultures were inoculated to 50 mL of YPGD or YPGL in 250 mL flasks at an initial
OD600 of 0.05 and were grown under the same condition. An overlay of 5 mL dodecane was
added to the flasks after 24 h and the light was opened.

**Analysis of Patchoulol**

Samples from the organic layer were centrifuged for 15 min at 6000 rpm to determine the
level of patchoulol during fermentation. The patchoulol was analyzed by gas
chromatography-mass spectrometry using the HP-5 column. The sample (1 μL) was injected
in splitless mode. The patchoulol standard was used to identify the substances.

## [Proof of concept](#id72)[¶](#proof-of-concept "Permalink to this heading")

### [Method development](#id73)[¶](#method-development "Permalink to this heading")

Two kinds of promoters were selected for gene expression, constitutive promoter PGK1 and
light-inducible promoter HCF173 (BBa\_K4270001). [1]

By fusing with FPS (farnesyl diphosphate synthase), PTS (patchoulol synthase), and
FPS-linker-PTS genes via PCR, they were linked into pSP-GM2 vector. After the
transformation of pSP-GM2 plasmids into E.coli, the DNA of each gene was amplified by
PCR and sequenced for verification. The recombinant vectors were then transformed into
yeast. Fermentation was then carried out and the target compounds were detected by
meteorological chromatography. A qualitative and quantitative analysis was carried out
for the production of patchoulol. The ones with the best performance were further
applied to real fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept1.png)

### [Plasmid construction](#id74)[¶](#id3 "Permalink to this heading")

The DNA sequence of PTS (encoding patchoulol synthase from P. cablin) was optimized
according to the preference of yeast for amino acid codons. Then FPS (BBa\_K517004), PTS
(BBa\_K4270004), and FPS-linker-PTS (BBa\_K4270010) were fused with promoter PGK1 or
light-inducible promoter HCF173 and constructed into pSP-GM2 plasmids separately, which
contains ampicillin resistance genes for colony selection.

The eight plasmids: pSP-GM2-FPS, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS,
pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS,
pSP-GM2-HCF173-PTS-FPS were transformed into E.coli according to the protocol. The
colony PCR was conducted for verification of the correct plasmids (Figure 1 and Figure
2). Then the amplified DNAs were sequenced and compared with the designed DNA sequences
for further verification.

Then these vectors are all transformed into yeast. The colony PCR was conducted for
further verification of these recombinant vectors.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept2.png)

**Figure 1. A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept3.png)

**Figure 2. A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [Patchoulol detection](#id75)[¶](#patchoulol-detection "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 3). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)](https://static.igem.wiki/teams/4270/wiki/proofofconcept4.png)

**Figure 3 The detection of patchoulol in the supernatant. A-H. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS, pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS,
pSP-GM2-HCF173-PTS, pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids,
respectively. I. The detection of patchoulol in the standard substance containing
patchoulol.**

### [Reference](#id76)[¶](#id4 "Permalink to this heading")

[1] Xue L, Hong-Bin W, and Hong-Lei J. Light signaling-dependent regulation of PSII
biogenesis and functional maintenance. Plant Physiology, 2020, 183, 1855-1868.

## [Results](#id77)[¶](#id5 "Permalink to this heading")

**Overview**

Our results consist of our gene cloning, colony PCR results and Gas chromatography
results.

### [1. Codon Optimization](#id78)[¶](#codon-optimization "Permalink to this heading")

To improve the translation efficiency of PTS in yeast, we first optimized the Pogostemon
cablin PTS gene according to the codon preference of yeast. During optimization, the
amino acid sequence of the protein encoded by PTS remained unchanged (**Figure
1**) and the codon was adjusted.

[![https://static.igem.wiki/teams/4270/wiki/results1.png](https://static.igem.wiki/teams/4270/wiki/results1.png)](https://static.igem.wiki/teams/4270/wiki/results1.png)

**Figure 1. The original PTS protein sequence and optimized PTS protein
sequence.**

### [2. DNA Preparation](#id79)[¶](#dna-preparation "Permalink to this heading")

The FPS (ERG20) gene was PCR-amplified from the genome of S. cerevisiae 4741. The fusion
of FPS and PTS with a Gly-Ser-Gly (GSG) tag was constructed according to a previous
work,[1] which generated a fusion protein called PTS-linker-FPS. The FPS (ERG20) gene
was PCR-amplified from the genome of S. cerevisiae 4741. The light-inducible promoter
HCF173 (HIGH CHLOROPHYLL FLUORESCENCE173) was PCR-amplified from the genome of wild-type
Arabidopsis thaliana. [2] The results are shown in **Figure 2**.

[![https://static.igem.wiki/teams/4270/wiki/results2.png](https://static.igem.wiki/teams/4270/wiki/results2.png)](https://static.igem.wiki/teams/4270/wiki/results2.png)

**Figure 2A. The PCR results of FPS. B. The PCR results of PTS. C. The PCR results
of HCF173. D. The PCR results of PTS-linker-FPS.**

After HCF173 and FPS, PTS, PTS-linker-FPS were separately amplified, HCF173 and other
genes were overlapped and amplified using primers. The results are shown in **Figure
3**.

[![https://static.igem.wiki/teams/4270/wiki/results3.png](https://static.igem.wiki/teams/4270/wiki/results3.png)](https://static.igem.wiki/teams/4270/wiki/results3.png)

**Figure 3A. The PCR results of HCF173-FPS. B. The PCR results of HCF173-PTS. C.
The PCR results of HCF173- PTS-linker-FPS. D. The PCR results of PTS-FPS (namely
HCF173-FPS- terminator -HCF173- PTS- terminator)**

### [3. Construction of Plasmids](#id80)[¶](#construction-of-plasmids "Permalink to this heading")

We constructed 4 plasmids with the constitutive promoter (pGK1) separately connecting
FPS, PTS, FPS-linker-PTS. The plasmids containing promoter
pGK1-FPS-terminator-pGK1-PTS-terminator were also built. We also constructed 4 plasmids
with HCF173 as the promoter. The schematic diagram of these plasmids is shown in
**Figure 4**.

[![https://static.igem.wiki/teams/4270/wiki/results4.png](https://static.igem.wiki/teams/4270/wiki/results4.png)](https://static.igem.wiki/teams/4270/wiki/results4.png)

**Figure 4. The schematic diagram of plasmids. Pcp, constitutive promoter pGK1.
Plip, light-induced promoter HCF173. Tns, T ADH1 or T CYC1 terminator.**

The colony PCR was conducted for further verification of these recombinant vectors. The
results are shown in **Figure 5** and **Figure 6**.

[![https://static.igem.wiki/teams/4270/wiki/results5.png](https://static.igem.wiki/teams/4270/wiki/results5.png)](https://static.igem.wiki/teams/4270/wiki/results5.png)

**Figure 5A. The colony PCR results of pSP-GM2-FPS. B. The colony PCR results of
pSP-GM2-PTS. C. The colony PCR results of pSP-GM2-PTS-linker-FPS. D. The colony PCR
results of pSP-GM2-PTS-FPS.**

[![https://static.igem.wiki/teams/4270/wiki/results6.png](https://static.igem.wiki/teams/4270/wiki/results6.png)](https://static.igem.wiki/teams/4270/wiki/results6.png)

**Figure 6A. The colony PCR results of pSP-GM2-HCF173-FPS. B. The colony PCR
results of pSP-GM2-HCF173-PTS. C. The colony PCR results of
pSP-GM2-HCF173-PTS-linker-FPS. D. The colony PCR results of
pSP-GM2-HCF173-PTS-FPS.**

### [4. Patchoulol detection](#id81)[¶](#id6 "Permalink to this heading")

After breeding and fermentation, Yeast fermentation broth was used for patchoulol
detection via the Gas chromatography (GC) method. Unfortunately, we did not detect
patchoulol in the supernatant but detected patchoulol in the standard substance
containing patchoulol (Figure 7). The reason we did not detect patchoulol in the
supernatant may due to the low yield of patchoulol and not enough fermentation.

[![https://static.igem.wiki/teams/4270/wiki/results7.png](https://static.igem.wiki/teams/4270/wiki/results7.png)](https://static.igem.wiki/teams/4270/wiki/results7.png)

**Figure 7. The detection of patchoulol in the supernatant. A-J. The detection of
patchoulol in Yeast fermentation broth from Yeast transformed with pSP-GM2-FPS,
pSP-GM2- FPS K197P, pSP -GM2- FPS K197H, pSP-GM2-PTS, pSP-GM2-PTS-linker-FPS,
pSP-GM2-PTS-FPS, pSP-GM2-HCF173-FPS, pSP-GM2-HCF173-PTS,
pSP-GM2-HCF173-PTS-linker-FPS, pSP-GM2-HCF173-PTS-FPS plasmids, respectively. I. The
detection of patchoulol in the standard substance containing patchoulol.**

### [Table of Contents](static/_static/index.html)

* [Team](team)
* Project
  + [Communication](#communication)
    - [Highschool
      Teams HP Experience Exchange](#highschool-teams-hp-experience-exchange)
    - [Online
      Conferences](#online-conferences)
    - [Propaganda
      Speech](#propaganda-speech)
    - [Interview
      with Experts](#interview-with-experts)
    - [Interview
      with doctor](#interview-with-doctor)
  + [Contribution](#contribution)
    - [1.Characterization
      of previous iGEM parts](#characterization-of-previous-igem-parts)
    - [2.Add New
      Documentations To An Existing Part](#add-new-documentations-to-an-existing-part)
    - [3.Add New
      Parts](#add-new-parts)
    - [4.Improvement of
      Existing Parts](#improvement-of-existing-parts)
    - [Reference](#reference)
  + [Description](#description)
    - [Pogostemon cablin
      and patchouli oil](#pogostemon-cablin-and-patchouli-oil)
    - [Patchoulol](#patchoulol)
    - [Plant
      extraction](#plant-extraction)
    - [Chemical
      synthesis](#chemical-synthesis)
    - [Microbial
      Fermentation](#microbial-fermentation)
    - [Patchoulol
      biosynthetic pathways](#patchoulol-biosynthetic-pathways)
    - [Vision](#vision)
    - [Reference](#id1)
  + [Engineering](#engineering)
    - [Abstract](#abstract)
    - [Experiment
      design](#experiment-design)
    - [Experiment structure
      and testing](#experiment-structure-and-testing)
  + [Experiments](#experiments)
    - [Protocol](#protocol)
  + [Implementation](#implementation)
    - [Background](#background)
    - [Target
      customers](#target-customers)
    - [Possible
      challenges](#possible-challenges)
    - [Reference](#id2)
  + [Notebook](#notebook)
    - [Week 1
      (6.20-6.26)](#week-1-6-20-6-26)
    - [Week 2
      (6.27-7.3)](#week-2-6-27-7-3)
    - [Week 3
      (7.4-7.10)](#week-3-7-4-7-10)
    - [Week 4
      (7.11-7.17)](#week-4-7-11-7-17)
    - [Week 5
      (7.18-7.24)](#week-5-7-18-7-24)
    - [Week 6 (8.1-8.7)
      and Week 7 (8.8-8.14)](#week-6-8-1-8-7-and-week-7-8-8-8-14)
    - [Week 7
      (8.15-8.27)](#week-7-8-15-8-27)
  + [Proof of
    concept](#proof-of-concept)
    - [Method
      development](#method-development)
    - [Plasmid
      construction](#id3)
    - [Patchoulol
      detection](#patchoulol-detection)
    - [Reference](#id4)
  + [Results](#id5)
    - [1. Codon
      Optimization](#codon-optimization)
    - [2. DNA
      Preparation](#dna-preparation)
    - [3.
      Construction of Plasmids](#construction-of-plasmids)
    - [4. Patchoulol
      detection](#id6)
* [Parts](parts)
* [Safety](safety)
* [Human Practices](human-practices)
* [Model](model)
* [Education](education)

### Navigation

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* [previous](team "Team") |
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