iGEM 2022 — Team scut-china

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Notebook

October

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November

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December

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January

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March

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April

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May

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June

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July

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August

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September

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October

### Team:

-Our team was officially formed, initially consisting of wet-lab group, dry-lab group and HP group.

-All members began to learn the excellent projects of the previous iGEM teams.

### Brainstorm:

-Our team started the first round of brainstorming, in which the themes include the production of natural
sugar substitutes, the biodegradation of metformin in the environment, high-precision neural/vascular
binding, and the biosynthesis of atropine.

### Brainstorm:

-We determined the research direction of our project, that is, the synthesis and application of
terpenoids, and began to investigate and select terpenoids for microbial synthesis, including geraniol,
limonene, **patchoulol**, medroxone, squalene, astaxanthin, **lycopene**, etc.

-At the end of November (11.29), we basically determined the framework of the project, that is, to
biosynthesize lycopene and patchoulol using *Pichia pastoris* as the chassis, and apply them to
silk as terpenoid dyes.

### Team:

-The team regularly held brainstorming sharing every week. The theme included the characteristics of silk
and its industrial development status, and the research progress of lycopene and patchoulol
biosynthesis.

### Wet-lab:

-All the iGEMers from our team learned laboratory fundamental qualities and received experimental skills
training in molecular biology and microbiology.

-We carried out the lycopene dyeing experiment on silk, observed the dyeing effect and improved the
dyeing conditions.

### HP:

-We started to operate a Wechat official account dedicated to SUCT-China 2022.

### Team:

-We recruited students from South China University of Technology with design, modeling or web building
skills, and the new teammates became one of the members of the art group or web/modeling group.

### Education:

-From 1.15 to 1.16, we held winter vacation biotechnology skill training for the School of Bioscience and
Engineering of SCUT.

-We released a series of popular science push on WeChat official account, with the theme of wet
experimental teaching of molecular biology and popular science of synthetic biology.

### Wet-lab:

-We conducted a preliminary experiment on *PTS* mutation

-[BBa\_K4263007](http://parts.igem.org/Part%3ABBa_K4263007) was amplified from
the genome of *Pichia pastoris* GS115.

-[BBa\_K4263008](http://parts.igem.org/Part%3ABBa_K4263008) was amplified from
the plasmid available in our laboratory.

### Team:

-The members of the art team completed the design of team elements, such as team logo, team uniform and
mascot of FUNDYES.

### Education:

-A questionnaire on the social acceptance of FUNCDYES was released.

-We started planning the "Run Plasmid" orienteering activity.

### Education:

-On 3.13, we carried out the synthetic biology popularization activity, "Running Plasmid".

### Wet-lab:

-On 3.17, [BBa\_K4263002](http://parts.igem.org/Part%3ABBa_K4263002) was
amplified from the genome of *P.pastoris* GS115.

-On 3.20, successful construction of plasmid pPICZA-P*PET9*-*EGFP*-His.

-On 3.25, [BBa\_K4263000](http://parts.igem.org/Part%3ABBa_K4263000), [BBa\_K4263003](http://parts.igem.org/Part%3ABBa_K4263003), [BBa\_K4263004](http://parts.igem.org/Part%3ABBa_K4263004) and [BBa\_K4263006](http://parts.igem.org/Part%3ABBa_K4263006) were extracted from
the genome of *P.pastoris* GS115 and amplified.

### Team:

-After communicating with Prof Jianwen Ye on project design, the team decided not to limit to single
static regulation, but to start the design of dynamic regulation.

### Wet-lab:

-On 4.2, successful construction of plasmid pPICZA-P*0547*-*EGFP*-His,
pPICZA-P*DAS2*-*EGFP*-His, pPICZA-P*PGK1*-*EGFP*-His
and pPICZA-P*GAP*-*EGFP*-His.

-On 4.8, we linearized the above plasmids and electrotransform them into *P.pastoris* GS115
strain.

-On 4.22, [BBa\_K4263001](http://parts.igem.org/Part%3ABBa_K4263001) and [BBa\_K4263005](http://parts.igem.org/Part%3ABBa_K4263005) were amplified from
the genome of *P.pastoris* GS115.

-From 4.25 to 4.30, shaker fermentation for 120 hours, and use the microplate reader to characterize the
fluorescence of the promoter.

### Brainstorm:

-Based on the photosensitive protein EL222, we hope to introduce the optogenetic system to improve our
regulation means, that is, dynamic regulation.

### Team:

-Our team was formally accepted by iGEM

### Wet-lab:

-The first round of fermentation of engineering yeast containing patchoulol and lycopene synthesis
pathway was carried out, and two terpenoids were analyzed by GC and HPLC.

### Education:

-We finished the design of “Puzzle games!” with "Mr. yeast's adventure" as the background, and used vivid
language to tell the public the rules of the game.You can play our game in our Wiki.

### Team:

-We participated in this year's Mentorship Program on May 1st.

-We completed the design of the gene circuit of the optogenetic system in *P.pastoris* strain.

### HP:

-On 6.3, our team attended the 6th Southern China Regional meeting. During the meeting, we
established contact with SUSTech\_Shenzhen.

-Exchange meeting with SUSTech\_Shenzhen on 6.3.

### Team:

-On 6.24, we submitted preliminary project safety form.

### Wet-lab:

-We carried out the manganese ion concentration exploration experiment of *PTS* mutation.

-On 7.10, we submitted the sequence of [BBa\_K4263009](http://parts.igem.org/Part%3ABBa_K4263009) and [BBa\_K4263010](http://parts.igem.org/Part%3ABBa_K4263010) to Tsingke Biotechnology Co., Ltd. for DNA synthesis.

-On 7.19, successful construction of plasmid pPICZA-P*ACC1*-*EGFP*-His and
pPICZA-P*ERG9*-*EGFP*-His, and we linearized the above plasmids and
electrotransform them into *P.pastoris* GS115 strain.

-On 7.21, successful construction of plasmid pPICZA-P*ACC1*-*EGFP*-His,
pPICZA-P*ERG9*-*EGFP*-His, pPICZA-P*GAP*-*EGFP*-His,
pPICZA-P*DAS2*-*EGFP*-His, pPICZA-P*PGK1*-*EGFP*-His,
pPICZA-P*0547*-*EGFP*-His, pPICZA-P*PET9*-*EGFP*-His
and pPICZA-P*AOX1*-*EGFP*-His.

-From 7.24 to 7.28, we carried out the second round of fermentation of engineering yeast with
*EGFP* as the reporter gene.

### HP:

-Exchange meeting with GYHS team from Guangya Middle School on 7.15.

-Interview for SCUT-China 2023 on 7.20.

-Exchange meeting with NPU-China on 7.21.

### Wet-lab:

-From 8.04 to 8.08, we carried out the third round of fermentation of engineering yeast with
*EGFP* as the reporter gene, and obtained the data of promoter strength by analyzing the relative
fluorescence unit.

-From 8.10 to 8.14, the second round of fermentation of engineering yeast 2OZPP containing patchoulol and
lycopene synthesis pathway was carried out, and two terpenoids were analyzed by GC and HPLC. We obtained
the production and titer of patchoulol and lycopene of engineering yeast corresponding to different
promoters.

-Color complementary analysis of engineering yeast on 8.15.

### HP:

-On 8.19, our team attended the Conference of China iGEMer Community.

-Exchange meeting with BiocreAtech (Shenzhen) on 8.23.

-Exchange meeting with USTC-China on 8.9.

### Education:

-On 8.30, we went to Kangyuan Therapy Station with LZU-China for volunteer activities to help the
disabled.

### Wet-lab:

-On 9.9, we successfully constructed
pPICZA-(C120)5-P*minCYC*-*PTS*-P*GAP*-TF-His.

-Replaced the upstream promoter of *PTS* mutation, and determined the appropriate manganese ion
concentration. Carry out color complementary analysis and preliminarily screen *PTS* mutants.

-From 9.14 to 9.15, the last round of fermentation of engineering yeast 2OZPP containing optogenetic
system was carried out, and two terpenoids were analyzed by GC and HPLC. In this round of fermentation,
we obtained data to verify the blue light induced activation.

### Education:

-Science Popularization Activity of Synthetic Biology in Guangzhou Guangya Middle School on 9.16.

### HP:

-Meeting with NFLS\_Nanjing & Nanjing-China on 9.30.

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Engineering

#### Ⅰ Overview

#### Ⅱ Promoter screening

#### Ⅲ PTS mutation

#### Ⅳ Dynamic regulation

#### Ⅴ Dowmstream-dyeing

## Overview

Synthetic biology is often described as the design and construction of new biological elements, devices, and
systems, as well as the redesign of existing natural biological systems for application purposes (Nature, 2021).
As in any other engineering field, synthetic biologists follow iterations of the DBTL cycle (Design, Build,
Test, Learn) to successfully develop innovative biological systems (Fig.1).

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/overview-fig1.png)

##### Fig.1 Iterative design-build-test-learn (DBTL) cycle in Synthetic Biology (Waldby et al. 2018).

Design: Using acquired knowledge either through experimentation or literature, a rational plan
is devised for implementing our system.

Build: High efficiency cloning methods are utilized to forge genetic constructs and develop
strains to test our systems.

Test: Systems are characterized and quantified with the scientific method.

Learn: Conclusions are drawn on our design and used to inform our next iteration of the cycle.

After an early brainstorming session to identify the problem of "silk spoilage", we wanted to find a bacterial
inhibitor that could be applied to silk as a special material. Then we found that many terpenoids were good
choices. In the process of researching various terpenoids, we found not only a class of terpenoids with
refreshing, antiseptic and antibacterial properties, such as patchoulol and nerolidol, but also many colorful,
antioxidant terpenoids, such as lycopene and astaxanthin.

Because terpenoids are complex compounds, fungi possess more complex organelles for protein expression that
promise terpene synthesis. The yeast *picha pastoris* has become an important industrial microorganism
due to its rapid growth, high density fermentation, simple genetic manipulation, and ability to perform
post-translational modifications. The sequencing of the whole genome of *P.pastoris* has led to its
application not only as an expression system for exogenous proteins, but also in synthetic biology and metabolic
engineering.

We further understood the biosynthetic pathways of both, and selected *P.pastoris* as an engineered
strain based on the current research status.

Based on the laboratory conditions and resources, we then chose patchoulol and lycopene, which can be used as
bacterial inhibitor and colorant, respectively, in silk staining. Producing them in the same strain reduces the
energy consumption and time of fermentation, so we wanted to use a strain with lycopene production pathway
already constructed in the lab and introduce the patchoulol synthase gene to achieve co-expression of terpenes.
Patchoulol and lycopene share the same precursor FPP(farnesyl pyrophosphate), so, presume FPP stay nearly
constant during the same fermentation environment, an increase in the yield of one will result in a
corresponding decrease in the yield of the other, further regulating the ratio of the two terpene products by
regulating the synthesis of patchoulol as a pathway.

At the same time, in order to achieve a high yield of both products simultaneously, we performed a screening
for the endogenous promoter of *P.pastoris*; meanwhile, in order to reduce the yield of by-products
produced during fermentation by a key enzyme in patchoulol production (patchoulol synthase), we performed a
random mutation to achieve an increase in the catalytic activity of the enzyme and its specificity to the
substrate. These method above are only static control system. In order to realize dynamic control of both, we
introduced a blue light-induced dynamic regulation system to achieve a change in the metabolic flow through a
blue light switch to maintain maximum growth of the organism while regulating the product yield.

In our downstream application, we explored dyeing conditions and create some beautiful handmakes.

In summary, the introduction of the 4 systems is the result of several engineering design communities. Below
are some of the engineering design circles in each system.

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/overview-fig2.png)

## Cycle Ⅰ-1: the basic strength of promoters

### Design: promoter selection

There are four commonly used strain types of *Pichia pastoris* host bacteria (Fig.1), namely X33, GS115,
KM71H, and SMD116. Among them, X33 is a wild-type strain, while GS115 and KM71H, both of which have HIS4
nutrient-deficient markers. Strain GS115 has the AOX1 gene and belongs to Mut+, referring to the normal type of
methanol utilization; KM71 H strain with AOX1 locus inserted by ARG4 gene belongs to Muts, referring to slow
methanol utilization type, and both strains are suitable for general yeast transformation methods. SMD116 (his4
pep4) belongs to the protease deletion type strain, which is able to express some uneven distribution of
expression products due to protein degradation. However, the slow growth of SMD116 (his4 pep4) strain resulted
in low yield of expressed exogenous protein(Tbl.1). Based on this, we selected strain GS115 as the screening
vector for the endogenous promoter.

|  |  |  |
| --- | --- | --- |
| Strain | Genotype | Phenotype(Pichia only) |
| X33 | wild type | Mut+ |
| GS115 | his4 | His-,Mut+ |
| KM71H | arg4 aox1::ARG4 | Muts,Arg+ |
| SMD116 | his4 pep4 | Mut+ |

##### Tbl.1 Four strains commonly used by Pichia pastoris host bacteria

Through research, we found six constructive promoters and two inducible promoters that are endogenous of
*P.pastoris*. The constructive promoters contain: P*ERG9*, P*GAP*,
P*PGK1*, P*PET9*, P*0547* and P*ACC1*.
The inducible promoters contain P*AOX1* and P*DAS2*.

### Build: construction of vectors

To construct the vectors, the P*ERG9*, P*GAP*, P*PGK1*,
P*PET9*, P*0547*, P*ACC1*, P*AOX1* and
P*DAS2* genes were amplified from genomic DNA of *P.pastoris*. What's next, we use
enhanced green fluorescent protein (EGFP) as report element to characterization the strength of promoters.

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/1-1.png)

##### Fig.1 Construction of pPICZA-promoters-EGFP-HIS in GS115. Line 1 are inducible promoters. Line 2 are constructive promoters.

### Test: the fluorescence intensity

The constructed plasmid with promoters and EGFP was subsequently inserted into the yeast genome to build the
report system. After incubated at 30°C for 120h, we used microplate reader to test the fluorescence intensity
(Fig.2).

![](https://static.igem.wiki/teams/4263/wiki/images/contribution-images/fluorescence.jpg)

##### Fig.2 Fluorescence intensity of promoters

### Learn:

After quantifying fluorescence using the microplate reader and measuring od at 600nm, we preliminarily ranked
the promoter strength according to the fluorescence per OD (Fig.3). The order of the promoters from strong to
weak is: P*DAS2*, P*AOX1*, P*0547*,
P*GAP*, P*PET9*, P*ACC1*, P*PGK1*,
P*ERG9*. The above results also paved the way for us to replace EGFP gene in
*P.pastoris* genome with PTS gene in the next cycle.

## Cycle Ⅰ-2: productivity of promoters in terpenoids production

### Design:

Thanks to Xinying Zhang, Ph.D., We got the strain which can already produce lycopene, to be our chassis, and we
named it 2OZPP. What's more, 2OZPP here means two copies of gene *OcrtE* + *OcrtB* +
*OcrtI* were inserted into the yeast geonomy(Fig.3).

![](https://static.igem.wiki/teams/4263/wiki/images/description-images/process.jpg)

##### Fig.3 Metabolic pathway of FUNCDYES.

### Build:

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/1-4.png)

##### Fig.4 Construction of PpICZA-promoters-EGFP-HIS in 2OZPP.

Based on the strain, we inserted patchoulol synthase (PTS) into the geonomy. Thus, we got eight different
stains with eight promoters at the same time(Fig.4).

### Test:

After cultured in a shaking incubator under the same conditions for 120 h, we tested the titters of two
terpenoids. The quantification of lycopene was performed on a Waters 2695 series HPLC system (Waters
Technologies, Milford, MA, USA). The quantification of patchoulol was performed on a GC system (Agilent 7820 a,
USA) equipped with HP-5 column (30 m×0.25 mm, 0.25μM U.M film thickness) and a flame ionization detector (FID).

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/lightbar.jpg)

##### Fig.5 The production of two terpenoids

### Learn:

We learn from the test above that we constructed the PTS expression pathway successfully. Furthermore, we would
try more ways such as PTS mutation to amplify the static control system. And these results would help us to
build a rapid selection system of PTS mutations.

## CycleⅡ-1: construction of PTS mutation system

### Design-1:

In vitro selection coupled with directed evolution represents a powerful method for generating nucleic acids
and proteins with desired functional properties. Creating high-quality libraries of random sequences is an
important step in this process as it allows variants of individual molecules to be generated from a
single-parent sequence. These libraries are then screened for individual molecules with interesting, and
sometimes very rare, phenotypes. Here, we describe a general method to introduce random nucleotide mutations
into a parent sequence that takes advantage of the error-prone polymerase chain reaction (epPCR). Our goal is to
realize 2-3bp mutations per 1000bp. What's more, through our research, we found that PTS has poor specificity
and produces many by-products during the culture procedure. Thus, we intended to use this method to selected
positive mutants which can produce high titer of patchoulol.

### Build-1:

Thanks to our promoter screening, we surprisingly found that there are two promoters that obey a so-called
proportional relationship (Fig.1). Once the yeast produces more patchoulol, the production of lycopene reduces.
These two promoters are P*0547* and P*PGK1*.

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/2-1.png)

##### Fig.1 The relationship between patchoulol and lycopene.

We chose manganese ions as mutagen for patchouli alcohol synthase gene to produce random mutations through
epPCR. In this cycle, we selected 0.4μL, 0.8μL, 1μL and 2μL of 2mM MnSO4 for the first try (Fig.2). The total
system for PCR is 50µL.

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/2-2.png)

##### Fig.2 Construction of PTS mutation vector.

### Test-1:

*Escherichia coli* TOP10F' (Invitrogen, Carlsbad, CA, USA) was used as the host for DNA manipulations
and the cells were cultivated in at 37°C. Next, we extracted the DNA from the cell and check the length of
fragments through agarose gel electrophoresis.

### Learn-1:

It's so sad that we failed to sequencing. We speculated that the concentration of MnSO4 is low than
the threshold value, so in the next cycle, we would turn up the dosage of MnSO4.

### Design-2 & Learn-2:

In this cycle, we selected 1μL, 2μL, 3μL and 4μL of 8mM MnSO4 for the mutation system. At the same time, we use
higher concentration for the third try. They are 5μL, 6μL, 7μL and 8μL of 2mM MnSO4.

We failed again. This led us to next cycle of the system by doing loads of research to confirm the mutagen and
the concentration.

### Design-3 & Learn-3:

We use 2μL and 6μL of 8mM MnCl2 instead of 8mM MnSO4 for this cycle.

To our surprise, we successfully finished the agarose gel electrophoresis and DNA sequencing. However, there
are much more mutations than we expected and the template which add 6uL MnCl2 even fail to combine
the primers. So, in the next cycle, we will reduce the usage of mutagen.

### Design-4 & Learn-4:

We use 0.5μL 0.75μL and 1.25μL of 8mM MnCl2 for this cycle.

Ultimately, we found that the 0.75uL can realize our goal. Followed by, we would use this concentration to
construct our mutation library (Fig.3).

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/2-3.png)

##### Fig.3 Result of the mutation in cycle 4. The total system for PCR is 50µL.

Prospect: fixed point mutations

We used modeling method to find the active pocket of PTS(Fig.4). And next step we planned to use fixed point
mutations to realize the mutation of these sites. But due to the time limitation, we haven't finished this part
of experiment.

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/2-4.png)

##### Fig.4 Active pocket of PTS (the yellow part of the picture).

## Cycle Ⅱ-2: construction of a fast-screening method

### Design:

Based on the relationship between lycopene and patchoulol production in *P.pastoris* we have already
mentioned, we developed a visual method to screen for efficient mutations in *PTS*.

### Build:

Firstly, We performed epPCR on *PTS* at the identified appropriate Mn2+ concentration and
obtained mutated *PTS* libraries. We have obtained a lycopene producing strain of *P.pastoris*
which names 2OZPP from our advisor Xinying Zhang. Then We completed the construction of mutant *PTS*
yeast library by transferring the mutant *PTS* into 2OZPP by homologous recombination.

### Test:

*P.pastoris* strain 2OZPP and strains with unmutated *PTS* constructed in the promoter screening
assay were used as controls, and yeast with mutated *PTS* were inoculated on solid plates and given 1%
methanol daily. After 96h of incubation, we observed colonies of different colors (Fig.5).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/pts-fig2-min.jpg)

##### Fig.5 First round of PTS visualization screening.

### Learn:

A clear color change of the colonies can be found in the results, and such a screening method is effective.
However, we observed that colonies growing on the edges of the plates were generally lighter in color, and we
believe that this situation is due to the edge effect caused by nutrient limitation at the edges of the plates,
which can mislead the judgment of the *PTS* mutation effect. Therefore, we optimized it in the next round
of DBTL.

## Cycle Ⅱ-3: edge effect's solution

### Design:

In order to reduce the misleading effect of edges on the experimental results, we chose a smaller plate in this
round of experiments and tried to make the experimental group positioned in the center of the plate.

### Build & Test:

We selected strains that showed lighter colors in the previous round of experiments, re-inoculated them on the
plates, and re-arranged the positions of the colonies to obtain better experimental results (Fig.5).

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/2-7.jpg)

##### Fig.6 Second round of PTS visualization screening.

### Learn:

We did find some strains that were lighter in color than other transformants, which gave us great confidence.
Although we have not quantitatively validated the lighter colored colonies due to time constraints, and we did
not find enough positive mutant strains due to limitations in the number of mutant libraries, we are confident
that this method must be feasible.

## Cycle Ⅲ-1: constructing dynamic regulation with light-control system

The idea of constructing a light-control module in our host is derived from our previous wet lab results. As we
discovered that by applying mere static control our function of interest cannot be reach, we turn to seek a
dynamic control strategy to meet our need of changing the proportion of the two terpenoids at our will.

### Design:

Inspired from certain parts in the database and a recent literature which demonstrated the application of a
blue light-induced system in *P.pastoris*, we establish blue light control system consist of a
constitutive or inducible promoter and its downstream gene that encoded light-activated transcriptional factor
protein EL222, a promoter including a (C120)5 region where EL222 binds to, our target gene and so on.
EL222 is expressed as monomer, and turn to dimer as the blue light (465 nm) was on. Then, activated EL222 binds
to the (C120)5 region and initiate the expression of our target gene, *PTS*. (Fig.1)

![](https://static.igem.wiki/teams/4263/wiki/images/engineering-images/3-1.png)

##### Fig.1 Design of the optogenetic system

### Build:

We synthesize the parts, [BBa\_K4263009](http://parts.igem.org/Part%3ABBa_K4263009) and
[BBa\_K4263010](http://parts.igem.org/Part%3ABBa_K4263010), and construct them on the
same vector pPIcza. Beside, Sequence optimization of our dynamically regulated light-control part [BBa\_K4263009](http://parts.igem.org/Part%3ABBa_K4263009) was done to allow it to better
play a regulatory role in *P.pastoris*.(reference to the existed part [BBa\_K4263021](http://parts.igem.org/Part%3ABBa_K4263021))

Four groups of our host, three parallel tests each, are set with difference in the promoter which control the
expression of EL222 in 250ml shake flasks. All of the flasks are cultured for 48h, and their OD were checked
every 24h, then methanol was added.

We also build up a LED device to spray blue light. LEDs (5W, 465 nm) were hung above the flasks to create a
blue light environment. The incubator was sealed with black cloth to block the blue light, preventing it from
doing harm to the people passing by and reducing the influence from the outside. (Fig.2)

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dynamic-1-1-min.png)![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dynamic-1-2-min.png)

##### Fig.2 The incubator with blue light device

### Test:

The product from each parallel is examined by GC and HPLC. (Fig.3)

|  |  |  |  |
| --- | --- | --- | --- |
| Names | Dark | Blue light | Strain |
| Control-1 | 0 | 48h | 2OZPP |
| Control-2 | 0 | 48h | 2OZPP+PTS |
| Longer blue light duration | 0 | 48h | 2OZPP+PGAP-TF-C(120)5-PTS |
| Shorter blue light duration | 16h | 32h | 2OZPP+PGAP-TF-C(120)5-PTS |

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dynamic-2-2-min.jpg)![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/lightbar.jpg)

##### Fig.3 Titer and production of lycopene and patchoulol of each parallel

### Learn:

From the results, we are aware that lycopene and patchoulol are produced when blue light is on, which means our
blue light-induced system is able to work effectively. Besides, the expression level is related to the dose of
the blue light. The longer exposure to blue light, the higher titer of patchoulol.

## Cycle Ⅲ-2: modifying dynamic regulation with light-control system

### Design:

Our next question is, naturally, whether the system efficient or not. How can we evaluate the performance and
what can we do to improve it? Thus, we use *EGFP* as the target gene to figure out certain parameters of
the system. However, limited time of wet lab disable us from continuing on our design, and we are not ready to
present our progress here. The characterization of the parts may be done in the future.

After our design, we got the great cell factory that produces FUNCDYES! In the next DBTL cycles, we carried out
experiments on dyeing.

In the industrial production scenario, we hope to use volatile solvent as the extractant to extract patchoulol
during fermentation and then concentrate it for dyeing. However, in the laboratory, we used n-dodecane as the
extraction agent in the fermentation process, which is not easy to concentrate. Therefore, in the subsequent
dyeing experiments, we used store-bought patchoulol for dyeing experiment.

At the end of fermentation, the fermentation broth was collected and centrifuged to obtain lycopene containing
cells, which were then broken to obtain lycopene dissolved in the extractant - ethyl acetate. The dye containing
lycopene was obtained by concentrating it for an appropriate time. We added some pure patchoulol to it, and we
got our FUNCDYES(Fig.1)! We soaked the silk in FUNCDYES, and after 15 minutes of dyeing, we succeeded in getting
the red silk with fragrance!

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dowmstream-fig1-min.jpg)

##### Fig.1 The fragrant red silk we dyed.

Next, we experimented with varying the concentration of lycopene to obtain silks of different colors. Also, by
changing the ratio of patchouli alcohol to lycopene, we obtained silks with different aromatic intensity and
color shades.

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/igem-min.jpg)

##### Fig.2 We dye different colors of silk.

We observed the color and fragrance persistence of the silk obtained by dyeing, and we found that the color of
the silk lightened slightly over fifteen days, but no significant fading occurred, and the fragrance persisted.

Although we managed to get fragrant red silks, they are not very bright in color. In the following experiment,
we began to think about whether we could achieve better dyeing results by improving the staining conditions.

We changed the dyeing time, temperature, hoping to find the most suitable dyeing conditions. It was found that
prolonging the staining time had no significant effect on the dyeing results, so in the subsequent dyeing
experiments, we set the staining time at about 5 minutes. Moreover, the effect of temperature on the dyeing
effect is not significant, and considering the cost of temperature control in industry, we believe that dyeing
at room temperature is sufficient.

In addition, we have looked up the common composition of silk dyes on the market and found that most of them
are water soluble, while lycopene and patchoulol are hydrophobic substances. Therefore, we set different groups
to explore the more appropriate solvent.

|  |  |  |
| --- | --- | --- |
| Grouping of solvent selection experiments | | |
| Group Name | Solvent | Additional treatment |
| A | Ethyl acetate | / |
| B | Ethyl acetate | Add 0.5 g/mL of NaCl |
| C | Ethyl acetate | Add 1 g/mL of NaCl |
| D | Ethyl acetate: Water=1:1 | Use ultrasound during dyeing |
| E | Ethyl acetate: Water=1:1 | Add tween 80 |
| F | Ethyl acetate: Water=1:1 | Add 0.5 g/mL of NaCl |
| G | Ethyl acetate: Water=1:1 | Add 1 g/mL of NaCl |

##### Tbl.1 Grouping of solvent selection experiments.

It was found that the addition of sodium chloride did have a positive effect on the coloring of the dye, but
only in the solvent containing water. However, in solvents containing water, the phenomenon of uneven staining
is more likely to occur. The results of the rest of the experimental groups showed that none of them stained as
well as the pure ethyl acetate as a solvent. Therefore, we finally chose ethyl acetate as our solvent.

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dowmstream-fig4-min.jpg)

##### Fig.3 Experimental results of staining conditions

Since we did not get the desired results in our experiments with staining conditions, we came to the staining
plant hoping to talk to people with more experience and get some inspiration.

[Click this
link(part one about **Explore the
significance**) to see what we communicated~](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

After talking with other experienced people, we have suggested some possible experimental improvements in the
future. We proposed the idea of using cyclodextrins or liposomes to encapsulate the lycopene and patchoulol
molecules, and by modifying the encapsulants, they would chemically react with the amino acids in the silk, thus
giving the dyed silk superior color fastness and a longer lasting fragrance. It also occurred to us that it
might be possible to use a supercritical CO2 extractor to dye silk as a solution to the problem that
dye molecules are only soluble in organic reagents.

Although we did not obtain the best experimental results due to technical limitations, we believe that these
problems can be optimized in the process of industrial production.

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

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---

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Results

#### Ⅰ Overview

#### Ⅱ Promoter screening

#### Ⅲ PTS mutation

#### Ⅳ Dynamic regulation

#### Ⅴ Dowmstream-dyeing

In this section, we present all the experimental works performed during our iGEM year.
Through preliminary
design and engineering, we have obtained ideal experimental results. After engineering, we also demonstrated our
engineering success more completely. We start here with the result of promoter screening and *PTS*
mutation; dyeing and visualization are presented thereafter(Fig.1).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/overview-fig1-min.png)

##### Fig. 1 Experiment construction of FUNCDYES.

## 1. Characterization of Fluorescence Intensity

### (1) Purpose of the experiments

In order to screen according to the transcriptional intensity of the promoter, we first selected EGFP
(enhanced green fluorescent protein) as the reporter gene. By detecting the EGFP fluorescence and optical
density at 600 nm (OD600), the promoter activity can be preliminarily and intuitively judged.

### (2) Experiments

#### • Shaker Flask Cultivations

The engineered yeast 2OZPP which had the ability to express the lycopene synthetic genes from
*Corynebacterium glutamicum* ATCC13032 with the P*AOX1* promoter was first inoculated
into 10 mL of BMGY medium in a 50 mL Erlenmeyer flask. The cells were grown overnight at 30°C with shaking at
250 rpm. Then, the main cultures were inoculated into a 250 mL Erlenmeyer flask, containing 25 mL of BMMY
medium (yeast extract 1%, peptone 2%, YNB 1.34%, potassium phosphate (pH 6.0) 100 mM, and methanol 1%) with an
initial OD600 of 1 and cultured in a shaking incubator under the same conditions for 120 h. 1% (v/v) fresh
methanol was added each every 24 h. Samples were taken every 24h to measure the OD600 of the fermentation
broth and the fluorescence of the supernatant.

#### • Fluorescence Detection of EGFP

For the samples taken after each 24h of fermentation, we conducted the following experimental operations. Add
300 μL Tris-HCl buffer, wash the yeast strain by vortex shaking, centrifuge at 8000 R/min and 4℃ for 1 min to
remove the supernatant. Repeat this process three times. After the last washing, resuspend the yeast strain
with an appropriate amount of Tris-HCl buffer. Take the same volume of yeast suspension three times in
parallel to detect the fluorescence intensity (excitation wavelength 488 nm, emission wavelength 520 nm), and
determine the EGFP fluorescence and optical density at 600 nm (OD600).

### (3) Results and discussion

After quantifying fluorescence using the microplate reader and measuring od at 600nm, we preliminarily ranked
the promoter strength according to the fluorescence per OD (Fig.1). The order of the promoters from strong to
weak is: P*DAS2*, P*AOX1*, P*0547*,
P*GAP*, P*PET9*, P*ACC1*, P*PGK1*,
P*ERG9*. Overall, among the promoters we selected, the strength of methanol inducible
promoters is generally greater than that of constitutive promoters, among which P*DAS2* and
P*AOX1* are inducible promoters, and the others are constitutive promoters. To our delight,
*Pichia pastoris* strain with P*DAS2* has stronger transcription intensity. The above
results also paved the way for us to replace *EGFP* gene in *P.pastoris* genome with
*PTS* gene in the future.

![](https://static.igem.wiki/teams/4263/wiki/images/contribution-images/fluorescence.jpg)

##### Fig.1 Fluorescence per OD of different promoters within 120 hours.

## 2. The Analysis of Lycopene and Patchoulol

### (1) Purpose of the experiments

The strength of the promoter can be characterized by detecting the fluorescence of green fluorescent protein,
but ultimately, we need to analyze the terpenoid production of engineering yeast co-expressing patchoulol and
lycopene, and we hope to get the data that there is a trend of inverse relationship between the production of
these two terpenoids through analysis. Because patchoulol is volatile and can be extracted to the outside by
n-dodecane, we chose gas chromatography to quantify the yield of patchoulol. Lycopene will remain in cells after
synthesis and is not volatile, so we chose high performance liquid chromatography to quantify the yield of
lycopene.

### (2) Experiments

#### • Shaker Flask Cultivations

The engineered yeast cells were first inoculated into 10 mL of BMGY medium in a 50 mL Erlenmeyer flask. The
cells were grown overnight at 30°C with shaking at 250 rpm. Then, the main cultures were inoculated into a 250
mL Erlenmeyer flask, containing 25 mL of BMMY medium (yeast extract 1%, peptone 2%, YNB 1.34%, biotin 0.00004%,
potassium phosphate (pH 6.0) 100 mM, and methanol 1%) with an initial OD600 of 1 and cultured in a shaking
incubator under the same conditions for 120 h. 1% (v/v) fresh methanol was added each every 24 h[1].

#### • Analysis of Terpenoids

The quantification of lycopene was performed on a Waters 2695 series HPLC system (Waters Technologies, Milford,
MA, USA) equipped with Waters C18 column (4.6 mm×250 mm) and UV/VIS detector detected signals at 450 nm (Fig.2).
For separation, samples were eluted with an isocratic elution program acetonitrile-isopropanol-methanol
(50:20:30 v/v) at a flow rate of 1 mL/min at 40°C. The injection volume was set to 10 μL[1].

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-2-min.png)

##### Fig.2 High performance liquid chromatogram of lycopene.

The quantification of patchoulol was performed on a GC system (Agilent 7820 a, USA) equipped with HP-5 column
(30 m×0.25 mm, 0.25μM U.M film thickness) and a flame ionization detector (FID). The oven temperature shall be
kept at 90℃ for 0.5 min, then gradually increased to 120℃ at the speed of 20℃/ min, then increased to 160℃ at
the speed of 3℃/ min for 5 min, and finally increased to 250℃ at the speed of 20℃/ min for 10 min. Patchoulol
was quantified by external standard method (Fig.3).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-3-min.jpg)

##### Fig.3 Gas chromatogram of patchoulol.

#### • The standard curve

By extracting lycopene standard, we obtained the standard mother liquor with a concentration of 200 μg/ml,
respectively carry out a series of gradient dilutions on the standard mother liquor, and then determine the
relationship between the absorption peak of the standard and the concentration of the standard according to the
HPLC isocratic elution method[1]. Fit the linear regression equation y=71782x-587086, R2 =
0.9976, Y represents the peak area, X represents the concentration of the standard lycopene (Fig.4).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-4-min.png)

##### Fig.4 The standard curve of lycopene.

Weigh 0.5mg of patchoulol standard with an analytical balance and dissolve it in 5ml of n-dodecane. Volume it
to 5ml with a volumetric flask. The concentration of the standard mother liquor is 1g / L; Dilute the standard
mother liquor to 0.75g/l, 0.75g/l, 0.5g/l, 0.25g/l and 0.1g/l, and then determine the relationship between the
absorption peak of the standard and the concentration of the standard according to the GC method. Fit the linear
regression equation y=3×107-556362, R2 = 0.9995, Y represents the peak area, X represents
the concentration of the standard patchoulol (Fig.5).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-5-min.png)

##### Fig.5 The standard curve of patchoulol.

### (3) Results and discussion

#### • Production and Titers

Through quantitative analysis by GC and HPLC, we obtained the production and titer of patchoulol and lycopene
of the engineered yeast transformants corresponding to different promoters. It can be intuitively found that in
the inducible promoter, the production of lycopene and patchoulol of P*DAS2* is the highest,
and in the constitutive promoter, the production of lycopene and patchoulol of P*PET9* is the
highest, followed by P*GAP* (Fig.6&7). It is worth mentioning that because the data related to
the production and titer of patchoulol on the promoter P*DAS2* fluctuated greatly during the
previous experiment and there was data loss, the quality of the data of P*DAS2* was not good.

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/lpbar.jpg)![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-7-min.jpg)

##### Fig.6 The production of two terpenoids(left) Fig.7 The titer of two terpenoids(right)

#### • The Selection of Promoters

However, since our main goal is not only to achieve high production of lycopene and patchoulol at the same
time, we also hope to reduce the by-products in the synthesis of patchoulol, which requires us to mutate and
find suitable *PTS* genes using a quick screening method.

As mentioned in the project design, since the two terpenoids share the same precursor FPP, the promoters
corresponding to the strains that make the production of lycopene and patchoulol inversely proportional to each
other are particularly important in the subsequent visual screening experiments, and they can be used for the
preliminary screening of PTS mutants. And we found that the production of lycopene and patchoulol of the
engineering yeast strains corresponding to the promoters P*0547* and P*PGK1*
tended to be inversely proportional (Fig.8&9).

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/ppgk1.jpg)

##### Fig.8 Lycopene and patchoulol titer of yeast transformants corresponding to P*PGK1*. The lycopene production of P*PGK1*-1, P*PGK1*-2 and P*PGK1*-3 was 177 mg/L (15.63mg/g DCW), 194.75 mg/L (17.70mg/g DCW) and 199.95 mg/L (18.18 mg/g DCW), and the patchoulol production of P*PGK1*-1, P*PGK1*-2 and P*PGK1*-3 was 4.77mg/L (0.525mg/g DCW), 3.95 mg/L (0.448mg/g DCW) and 3.02 mg/L (0.343 mg/g DCW).

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/p0547.jpg)

##### Fig.9 Lycopene and patchoulol titer of yeast transformants corresponding to P*0547*. The lycopene production of P*0547*-2, P*0547*-3 and P*0547*-4 was 158.69 mg/L (14.43mg/g DCW), 208.58 mg/L (19.17mg/g DCW) and 204.3 mg/L (18.07 mg/g DCW), and the patchoulol production of P*0547*-2, P*0547*-3 and P*0547*-4 was 5.10mg/L (0.580mg/g DCW), 3.76 mg/L (0.437mg/g DCW) and 4.02 mg/L (0.427 mg/g DCW).

Previous studies have shown that color complementary analysis is a simple and effective method to identify
genes in the terpenoid biosynthesis pathway[2]. Therefore, we performed color complementation analysis on
P*0547*-2, P*0547*-3, P*0547*-4,
P*PGK1*-1, P*PGK1*-2 and P*PGK1*-3. 2OZPP yeast was used
as the control group, and six engineered 2OZPP yeast transformants containing PTS synthesis pathway and
different upstream promoters were used as the experimental group. The experimental results can be well
distinguished by the naked eye from the color complementation of different yeast transformants (Fig.10), which
has well proved that it is feasible to screen PTS mutants by visualized color complementation analysis.

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/experience.jpg)

##### Fig.10 Identification of two terpenoids in color complementation experiments. Blue represents the production of patchoulol, and red represents the production of lycopene

#### • Growth

FPP is a precursor for some other components of cell membrane in *P.pastoris*, such as ergosterol and
farnesyl. By comparing the growth curves of *P.pastoris* corresponding to the promoters, we speculate
that too strong promoter strength may affect the growth of the strains at the early stage of fermentation
(Fig.11).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-11-min.jpg)

##### Fig.11 Growth of *P.pastoris* corresponding to different promoters.

It is generally believed that PAOX1, the promoter of alcohol oxidase 1 in MUT pathway, is the strongest known
promoter of P. pastoris. We noticed that in our experimental results, the strength of the promoter PDAS2 was
stronger than that of the promoter PAOX1 (Fig.11). When we compared the growth curves of the two, we found that
the growth level of the yeast corresponding to PDAS2 during shake flask fermentation was generally lower than
that of PAOX1 (Fig.12). However, we had doubts about such data results. By comparing the fermentation data of
previous batches, we thought that the growth curve of different fermentation batches would have different
fluctuations due to the effects of experimental operation, environment and shaking table. Therefore, we could
not draw the conclusion that strong promoters would reduce the synthesis of squalene and affect the early growth
of the strain through this experiment.

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/promoter-fig-12-min.jpg)

##### Fig.12 Growth of *P.pastoris* corresponding to P*DAS2* and P*AOX1*.

### Reference

[1]Zhang, X., Wang, D., Duan, Y., Zheng, X., Lin, Y., & Liang, S. (2020). Production of lycopene by
metabolically engineered *Pichia pastoris*. Bioscience, biotechnology, and biochemistry, 84(3),
463-470. https://doi.org/10.1080/09168451.2019.1693250
[2]Araya-Garay, J. M., Feijoo-Siota, L.,
Veiga-Crespo, P., & Villa, T. G. (2011). cDNA cloning of a novel gene codifying for the enzyme lycopene
β-cyclase from *Ficus carica* and its expression in Escherichia coli. Applied microbiology and
biotechnology, 92(4), 769-777. https://doi.org/10.1007/s00253-011-3488-8

## 1. Construction of the mutation system

### (1) Purpose of the experiment

As depicted in Fig.1, there are at least 5 kinds of by-products during the patchoulol synthesis procedure.
Thus, we planned to do patchoulol synthase (PTS) mutation to increase the specificity of the enzyme, as well
as a supplement of static control system.

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/pts-fig1-min.png)

##### Fig.1 Postulated reaction pathways to account for the different products generated by the patchoulol synthase from farnesyl diphosphate (adapted from Croteau et al.).

### (2) Experiments

#### • Error-prone PCR

Generation of a maximally diverse gene library is particularly important when employing non-targeted
mutagenesis strategies. The method most often used to generate variants with random mutations is error-prone
PCR (epPCR). Here we use the basic method for using epPCR to generate gene variants that exhibit a relatively
balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of
those variants.

We chose manganese ions (Mn2+) as mutagen for patchouli alcohol synthase gene to produce random
mutations. The epPCR system are showing as follows. (Tbl.1)

|  |  |
| --- | --- |
| Fame | Volume |
| PCR Mix | 25μL |
| Top strand primers | 2μL |
| Bottom strand primers | 2μL |
| Mn2+ | - |
| dd water | ~50μL |

##### Tbl.1 System of epPCR.

### (3) Results and discussion

We chose different kinds of Mn2+ and tried different concertation and volume of it to construct
the best mutation system. Result are showing in the table below. (Tbl.2)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Cycle | Types of salt | Concentration | Volume | Result |
| 1 | MnSO4 | 2mM | 0.4μL | Agarose gel electrophoresis showed no bands or the wrong bands |
| 0.8μL |
| 1μL |
| 2μL |
| 2 | MnSO4 | 8mM | 1μL | The band are right through the agarose gel electrophoresis, but the sequencing results are all wrong. |
| 2μL |
| 3μL |
| 4μL |
| 3 | MnCl2 | 8mM | 2μL | There were much more mutations than we expected. |
| 4μL |
| 6μL | The band are right through the agarose gel electrophoresis, but the sequencing results show no sign. |
| 8μL |
| 4 | MnCl2 | 8mM | 0.5μL | There were less mutations than we expected. |
| 0.75μL | 3-5 bp mutation per 1000bp |
| 1.25μL | There were much more mutations |

##### Tbl.2 Result of PTS mutation.il

From the results above, we finally chose 0.75μL 8mM MnCl2 as our mutants to built our mutation
system.

## 2. Construction of a rapid selection system of mutants

### (1) Purpose of the experiment

As we know, traditional method to text the titer of terpenoids we use either GC or HPLC, which are not only
complex but time consuming. Thus, based on the relationship between lycopene and patchoulol production in
*P.pastoris* we have already mentioned, we developed a visual method to screen for efficient mutations in
*PTS*.

### (2) Experiments & Result

As mentioned before, we selected two promoters, P*0547* and P*PGK1*, from
the promoter library as the promoters of the mutant *PTS*, and transferred the mutant *PTS* into
*P.pastoris* strain 2OZPP by homologous recombination.

*P.pastoris* strain 2OZPP and strains containing unmutated *PTS* with P*0547*
and P*PGK1* as promoters were used as controls, and strains with mutated *PTS* were
inoculated on BMMY solid plates at 30℃ and given 1% methanol daily. After 96h of incubation, we observed
colonies of different colors (Fig.2).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/pts-fig2-min.jpg)

##### Fig.2 First round of *PTS* visualization screening.

We observed that colonies growing on the edges of the plates were generally lighter in color, and we believe
that this situation is due to the edge effect caused by nutrient limitation at the edges of the plates, which
can mislead the judgment of the *PTS* mutation effect. To reduce the interference of edge effects, we
replaced the smaller plates and re-cultured and re-screened for the lighter colored strains.

In the second round of screening, we found several strains that were lighter in color and might be good mutants
(Fig.3).

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/pts-fig3-min.png)

##### Fig.3 Second round of *PTS* visualization screening. The left one is the result of pPICZA-P*GAP*-mut PTS-His. The left one is the result of pPICZA-P*0547*-mut PTS-His.

Due to time constraints, we did not obtain enough positive strains, and we plan to continue to expand the
capacity of the *PTS* library to screen for more positive strains.

Considering that the issue of nutrient limitation may interfere with the judgment of strain color when cultured
on solid plates, we will select strains with lighter color and transfer them to BMMY liquid medium to be
cultured in 96-well plates for screening.

Strains that are lighter in color in the 96-well plates will be transferred to shake flasks for fermentation
and finally for quantitative validation of patchoulol yield. The best mutant *PTS* will thus be selected.

Screening in 96-well plates and shake flasks was not carried out due to time constraints, and we will continue
to do the screening in future experiments.

## Dynamic regulation with light-control system

### 1. Purpose of the experiment

The idea of constructing a light-control module in our host is derived from our previous wet lab results. As we
discovered that by applying mere static control, our function of interest can not be reach. We turn to seek a
dynamic control strategy to meet our need of changing the proportion of the two terpenoids at our will.

### 2. Experiments

In practice, we synthesize the parts, BBa\_K4263009 and BBa\_K4263010, and construct them on the same vector
pPICZA. Four groups of our host, three parallel tests each, are set with difference in the promoter which
control the expression of EL222 in 250ml shake flasks. All of the flasks are cultured for 48h, and methanol was
added every 24h.

As for hardware, we build up a LED device to spray blue light. LEDs (5W, 465 nm) are hang above the flasks to
create a blue light environment. The incubator was sealed with black cloth to block the blue light, preventing
it from doing harm to the people passing by and reducing the influence from the outside. (Fig.1)

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dynamic-1-1-min.png)![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dynamic-1-2-min.png)

##### Fig.1 The incubator with blue light device

### 3. Results and discussion

The titer from each parallel is examined by GC and HPLC. (Fig.2)

For the two control groups, we can see that lycopene and patchoulol are expressed normally, compared with our
previous results using static promoters. In group 3, we proved that the light-induced system is able to turn
some metabolic flux to the synthesis of patchoulol, meanwhile turning down that of lycopene. For group 4, in
comparison to group 3, we discovered that longer blue light induces more synthesis of patchoulol, which means
that the regulation of *PTS* is dose-dependent on light duration.

In conclusion, our blue light-induced system is proved to be effective in practice.

|  |  |  |  |
| --- | --- | --- | --- |
| Names | Dark | Blue light | Strain |
| Control-1 | 0 | 48h | 2OZPP |
| Control-2 | 0 | 48h | 2OZPP+PTS |
| Longer blue light duration | 0 | 48h | 2OZPP+PGAP-TF-C(120)5-PTS |
| Shorter blue light duration | 16h | 32h | 2OZPP+PGAP-TF-C(120)5-PTS |

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dynamic-2-2-min.jpg)![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/lightbar.jpg)

##### Fig.2 Titer of lycopene and patchoulol of each parallel. The lycopene production of Control-1, Control-2, Longer group and Shorter group was 141.58 mg/L (15.42 mg/g DCW), 141.39 mg/L (15.24 mg/g DCW), 60.34 mg/L (9.42 mg/g DCW) and 121.05 mg/L (12.33 mg/g DCW), and the patchoulol production of Control-1, Control-2, Longer group and Shorter group was 0 mg/L (0 mg/g DCW), 1.21 mg/L (1.64 mg/g DCW), 3.13 mg/L (5.96 mg/g DCW) and 1.36 mg/L (1.85 mg/g DCW).

Our next question is, naturally, whether the system efficient or not. How can we evaluate the performance and
what can we do to improve it. Thus, we use *EGFP* as the target gene to figure out certain parameters of
the system. However, limited time of wet lab disable us from continuing on our design, and we are not ready to
present our progress here. The characterization of the parts may be done in the future.

## 1. Purpose of the experiment

After our design, we got the great cell factory that produces FUNCDYES! Next, we performed experiments to
obtain FUNCDYES for dyeing to finish our downstream experiment.

## 2. Experiments & Result

In the industrial production scenario, we hope to use volatile solvent as the extractant to extract patchoulol
during fermentation and then concentrate it for dyeing. However, in the laboratory, we used n-dodecane as the
extraction agent in the fermentation process, which is not easy to concentrate. Therefore, in the subsequent
dyeing experiments, we used store-bought patchoulol for dyeing experiment.

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dowmstream-fig1-min.jpg)

##### Fig.1 The fragrant red silk we dyed.

At the end of fermentation, the fermentation broth was
collected and centrifuged to obtain lycopene containing
cells, which were then broken to obtain lycopene
dissolved in the extractant - ethyl acetate. The dye containing lycopene was obtained by concentrating it for
an
appropriate time. We added some pure patchoulol to it,
and we got our FUNCDYES! We soaked the silk in
FUNCDYES, and after 15 minutes of dyeing, we succeeded in getting the red silk with fragrance! (Fig.1)

Next, we experimented with varying the concentration of lycopene to obtain silks of different colors. Also, by
changing the ratio of patchouli alcohol to lycopene, we obtained silks with different aromatic intensity and
color shades. (Fig.2)

![](https://static.igem.wiki/teams/4263/wiki/images/proof-of-concept-images/igem-min.jpg)

##### Fig.2 The silk of different color we dyed

At the same time, we verified the dyeing effect of silk under different concentrations of lycopene. (Fig 3)

We observed the color and fragrance persistence of the silk obtained by dyeing, and we found that the color of
the silk lightened slightly over fifteen days, but no significant fading occurred, and the fragrance persisted.

Although we managed to get fragrant red silks, they are not very bright in color. In the following experiment,
we began to think about whether we could achieve better.

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dowmstream-fig3-min.jpg)

##### Figure 3 Silk dyed with different concentrations of lycopene

|  |  |
| --- | --- |
| Time | Temperature |
| 5min | 0℃ |
| 5min | Room temperature |
| 5min | 60℃ |
| 15min | 0℃ |
| 15min | Room temperature |
| 15min | 60℃ |
| 1h | 0℃ |
| 1h | Room temperature |
| 1h | 60℃ |

##### Tbl.1 Grouping of time and temperature selection

We changed the dyeing time, temperature, hoping to find the most suitable dyeing conditions. It was found
that prolonging the staining time had no significant effect on the dyeing results, so in the subsequent dyeing
experiments, we set the staining time at about 5 minutes. Moreover, the effect of temperature on the dyeing
effect is not significant, and considering the cost of temperature control in industry, we believe that dyeing
at room temperature is sufficient.

In addition, we have looked up the common composition of silk dyes on the market and found that most of them
are water soluble, while lycopene and patchoulol are hydrophobic substances. Therefore, we set different groups
to explore the more appropriate solvent.

|  |  |  |
| --- | --- | --- |
| Grouping of solvent selection experiments | | |
| Group Name | Solvent | Additional treatment |
| A | Ethyl acetate | / |
| B | Ethyl acetate | Add 0.5 g/mL of NaCl |
| C | Ethyl acetate | Add 1 g/mL of NaCl |
| D | Ethyl acetate: Water=1:1 | Use ultrasound during dyeing |
| E | Ethyl acetate: Water=1:1 | Add tween 80 |
| F | Ethyl acetate: Water=1:1 | Add 0.5 g/mL of NaCl |
| G | Ethyl acetate: Water=1:1 | Add 1 g/mL of NaCl |

##### Tbl.2 Grouping of solvent selection experiments

It was found that the addition of sodium chloride did have a positive effect on the coloring of the dye, but
only in the solvent containing water. However, in solvents containing water, the phenomenon of uneven staining
is more likely to occur. The results of the rest of the experimental groups showed that none of them stained as
well as the pure ethyl acetate as a solvent (Fig 4). Therefore, we finally chose ethyl acetate as our solvent.

![](https://static.igem.wiki/teams/4263/wiki/images/result-images/dowmstream-fig4-min.jpg)

##### Fig.4 Experimental results of solvent alteration

Since we did not get the desired results in our experiments with staining conditions, we came to the staining
plant hoping to talk to people with more experience and get some inspiration.

[Click this link(part one about
**Explore the
significance**) to see what we communicated~](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

After talking with other experienced people, we have suggested some possible experimental improvements in the
future. We proposed the idea of using cyclodextrins or liposomes to encapsulate the lycopene and patchoulol
molecules, and by modifying the encapsulants, they would chemically react with the amino acids in the silk, thus
giving the dyed silk superior color fastness and a longer lasting fragrance. It also occurred to us that it
might be possible to use a supercritical CO2 extractor to dye silk as a solution to the problem that
dye molecules are only soluble in organic reagents.

Although we did not obtain the best experimental results due to technical limitations, we believe that these
problems can be optimized in the process of industrial production.