iGEM 2021 — Team HUST-China

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# Team:HUST-China/Notebook

Notebook | iGEM HUST-China

[HUST-China](https://2021.igem.org/Team%3AHUST-China)

* Project

  [Description](https://2021.igem.org/Team%3AHUST-China/Description)[Design](https://2021.igem.org/Team%3AHUST-China/Design)[Proof of
  Concept](https://2021.igem.org/Team%3AHUST-China/Proof_Of_Concept)[Safety](https://2021.igem.org/Team%3AHUST-China/Safety)[Proposed
  Implementation](https://2021.igem.org/Team%3AHUST-China/Implementation)[Contribution](https://2021.igem.org/Team%3AHUST-China/Contribution)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHUST-China/Human_Practices)[Part
  1](https://2021.igem.org/Team%3AHUST-China/Human_Practices_01)[Part
  2](https://2021.igem.org/Team%3AHUST-China/Human_Practices_02)[Part
  3](https://2021.igem.org/Team%3AHUST-China/Collaborations)[Part 4](https://2021.igem.org/Team%3AHUST-China/Education)
* Modeling

  [Overview](https://2021.igem.org/Team%3AHUST-China/Model)[Gene
  Pathway](https://2021.igem.org/Team%3AHUST-China/Gene_Pathway)[Dye color
  forecast](https://2021.igem.org/Team%3AHUST-China/Dye_color_forecast)[Micronucleus
  Counter](https://2021.igem.org/Team%3AHUST-China/Micronucleus_Counter)
* Products

  [Overview](https://2021.igem.org/Team%3AHUST-China/Products)[Software](https://2021.igem.org/Team%3AHUST-China/Software)[Hardware](https://2021.igem.org/Team%3AHUST-China/Hardware)[Another
  Area](https://2021.igem.org/Team%3AHUST-China/Another_Area)
* Experiments

  [Overview](https://2021.igem.org/Team%3AHUST-China/Experiments)[Parts](https://2021.igem.org/Team%3AHUST-China/Parts)[Engineering
  Success](https://2021.igem.org/Team%3AHUST-China/Engineering)[Results](https://2021.igem.org/Team%3AHUST-China/Results)[Protocol](https://2021.igem.org/Team%3AHUST-China/Protocol)[Notebook](https://2021.igem.org/Team%3AHUST-China/Notebook)
* People

  [Team](https://2021.igem.org/Team%3AHUST-China/Team)[Partnership](https://2021.igem.org/Team%3AHUST-China/Partnership)[Attributions](https://2021.igem.org/Team%3AHUST-China/Attributions)
* Achievements

  [Award](https://2021.igem.org/Team%3AHUST-China/Award)[Promotion
  Video](https://video.igem.org/w/aMwNT6eBryGs8xG5avXD51)[Judging
  Form](https://igem.org/2021_Judging_Form?id=3711)

# Notebook

---

##### Contents

# NoteBook

CONTACT US: iGEMHUSTChina@163.com

Huazhong University of Sci. & Tech., Wuhan, China

1037# Luoyu Rd, Wuhan, P.R.China 430074

Copyright © HUST-China iGEM 2021

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# Team:HUST-China/Engineering

Engineering success | iGEM HUST-China

[HUST-China](https://2021.igem.org/Team%3AHUST-China)

* Project

  [Description](https://2021.igem.org/Team%3AHUST-China/Description)[Design](https://2021.igem.org/Team%3AHUST-China/Design)[Proof of
  Concept](https://2021.igem.org/Team%3AHUST-China/Proof_Of_Concept)[Safety](https://2021.igem.org/Team%3AHUST-China/Safety)[Proposed
  Implementation](https://2021.igem.org/Team%3AHUST-China/Implementation)[Contribution](https://2021.igem.org/Team%3AHUST-China/Contribution)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHUST-China/Human_Practices)[Part
  1](https://2021.igem.org/Team%3AHUST-China/Human_Practices_01)[Part
  2](https://2021.igem.org/Team%3AHUST-China/Human_Practices_02)[Part
  3](https://2021.igem.org/Team%3AHUST-China/Collaborations)[Part 4](https://2021.igem.org/Team%3AHUST-China/Education)
* Modeling

  [Overview](https://2021.igem.org/Team%3AHUST-China/Model)[Gene
  Pathway](https://2021.igem.org/Team%3AHUST-China/Gene_Pathway)[Dye color
  forecast](https://2021.igem.org/Team%3AHUST-China/Dye_color_forecast)[Micronucleus
  Counter](https://2021.igem.org/Team%3AHUST-China/Micronucleus_Counter)
* Products

  [Overview](https://2021.igem.org/Team%3AHUST-China/Products)[Software](https://2021.igem.org/Team%3AHUST-China/Software)[Hardware](https://2021.igem.org/Team%3AHUST-China/Hardware)[Another
  Area](https://2021.igem.org/Team%3AHUST-China/Another_Area)
* Experiments

  [Overview](https://2021.igem.org/Team%3AHUST-China/Experiments)[Parts](https://2021.igem.org/Team%3AHUST-China/Parts)[Engineering
  Success](https://2021.igem.org/Team%3AHUST-China/Engineering)[Results](https://2021.igem.org/Team%3AHUST-China/Results)[Protocol](https://2021.igem.org/Team%3AHUST-China/Protocol)[Notebook](https://2021.igem.org/Team%3AHUST-China/Notebook)
* People

  [Team](https://2021.igem.org/Team%3AHUST-China/Team)[Partnership](https://2021.igem.org/Team%3AHUST-China/Partnership)[Attributions](https://2021.igem.org/Team%3AHUST-China/Attributions)
* Achievements

  [Award](https://2021.igem.org/Team%3AHUST-China/Award)[Promotion
  Video](https://video.igem.org/w/aMwNT6eBryGs8xG5avXD51)[Judging
  Form](https://igem.org/2021_Judging_Form?id=3711)

# Engineering success

---

##### Contents

# Introduction:

We want to build an enzyme system that can produce pigments and degrade them according to our
needs. For the same idea, we need the second type of enzyme system to produce short peptides
for perm, and it can also degrade short peptides at any time. In order to achieve the goal,
we designed multiple devices and four systems. In the stage of pathway construction, we
tried a variety of methods, summed up the experience, learned from mistakes, and finally
achieved some results. Our exploration and work this year has also provided some valuable
experience for the follow-up teams.

# Cycle1:Construct by the homologous recombination method

Several homologous fragments are designed to link multiple genes altogether at one time,
using seamless cloning, to construct a system with complete function. Some success are
gained during construction, like the whole pathway of FMO dimer, while significant
difficulties occur as the same time. Lessons are learnt and settlement are designed, in
order to overcome the present reality of getting much shorter product than expected, after
comparing our onlu success to tons of failure.

DesignBuildTestLearn

##### ES1

A multi-functional system can achieve a more convenient effect, so that a
plasmid with multiple devices can individually complete tasks that
require plural mono-functional plasmids. According to this idea, we
connected devices with independent functions, forming systems with
complete functions.

In the process of synthesizing each device, the foundation for the
formation of the final system had been laid. The first homologous
segment was first added to the 3' end of the Panb1-crtE-AOX1 Terminator,
Panb1-α factor-4CL-AOX1 Terminator and to the 5' end of the
Panb1-crtB-AOX1 Terminator, Panb1-α factor-ACC-AOX1 Terminator as the
vector.

The second homologous segment was then added at the 5' end of
Panb1-crtB-AOX1 Terminator, Panb1-α factor-ACC-AOX1 Terminator and at
the 3' end of Panb1-crtI-AOX1 Terminator, Panb1-α factor-CUS-AOX1
Terminator.

Finally, the third segment homologous to the gene on the vector plasmid
was finally added at the back end of the Panb1-crtI-AOX1 Terminator,
Panb1-α factor-CUS-AOX1 Terminator.

Three specific homologous segments were designed for using different PCR
primers to add to different gene segments when constructing whole
systems, which, in principle, ensure the experimental feasibility.

Close

##### ES2

Using the most efficient in-fusion cloning principle, homologous
recombinase is selected to construct the pathway. We design primers
strictly in accordance with the instructions and implement the operating
procedures: first double-enzyme digestion of the plasmids as the
vectors, then PCR amplified gene fragments, and then gel electrophoresis
to verify the vectors and conduct gel extraction for purification.
Finally, the high-purity vectors and segments are connected by
homologous recombinase to construct a system, and the ligated products
are transformed into E.coli TOP10 F'.

Taking lycopene system as an example: firstly, the whole plasmid
containing Panb1-crtE-AOX1 Terminator was used as a vector through
double digestion, then the gene segments of Panb1-crtB-AOX1 Terminator
and Panb1-crtI-AOX1 Terminator was obtained through PCR. Finally, the
purified vector and the two purified segments were linked using
homologous recombinase. The ligated products were then transformed into
E.coli TOP10 F'. The cloned host was inoculated to LB plate containing
AMP 100mg/mL for screening. After a single colony grew, the spot was
picked for colony PCR verification. If the target band is observed, mix
the bacteria solution with a 1:1 mixture of glycerin and water, and keep
the seeds at -80℃.

Close

##### ES3

After many attempts, we have made some achievements, but some works has
not achieved the desired results, and most of the agarose gel
electrophoresis strips are shorter than the expected length.

It is gratifying that the FMO dimer we built has been successful.

![Fig1. Plasmid construction and colony PCR result of Pynr071c-α factor-FMO dimer-AOX1 Terminator transformed E.coli.The bands of FMO dimer (less than 5000bp) and is identical to the theoretical lengths of 4600bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.](https://static.igem.org/mediawiki/2021/e/eb/T--HUST-China--img--engineering--1.png)

Close

##### ES4

After many attempts and comparing the examples of success and failure, we
conclude that the reasons for failure may come from these two points: 1.
There are a large number of repeated segments on our system. Because all
the promoters and terminators in our device are the same, it means that
our target system will contain a large number of repeated fragments,
which may cause functional confusion in the homologous recombinase.
However, the successful FMO dimer does not contain multiple promoters
and terminators. 2. The principle of the enzyme we use is different from
the classic seamless cloning enzyme.

We came up with two solutions, 1. Replacement of method, using the enzyme
digestion method with a certain success rate to construct pathway. 2.
Replacement of the homologous recombinase.

Combining practical considerations, because we have successfully
constructed the devices using the enzyme digestion method, and
considering the impact of the epidemic on our progress, we chose to
change the construction method, the enzyme digestion method.

Close

# Cycle2:Construct by enzyme digestion and ligation

Classic, reliable but lenthy double-enzyme digestion and reconnection is used to construct
basic devices, along with systems with complete function. Eventually, good news is we have
succeeded in building all of our basic devices, but bad news is we have no choice but to put
off the construction of complete pathways after iGEM, due to the precious but limited time,
which undoubtly will be continuously carried forward.

DesignBuildTestLearn

##### ES5

We utilized the existing components, assemble each component, built the
device, and then connected the devices one by one, stably, got the
system, expecting to achieve our goal.

The first is to construct devices containing multiple single gene
fragments. We first transferred the basic plasmids containing single
gene which were synthesized by the company into E. coli for
amplification. Plasmids containing the promoter Panb1 or Pynr071c became
as vectors after double digestion; PCR was used to obtain the gene
segments, and then double digestion with the same enzyme. The vectors
that had undergone gel extraction and the purified segments are
connected. Finally, the ligated product was transformed into E.coli
TOP10F' and screened by ampicillin 100 ug/mL LB medium. If a single
colony grows, pick the spot and verify the colony by PCR. After
observing the target band, mix the bacterial solution with a 1:1 mixture
of glycerin and water, and keep the seeds at -80℃.

Expand the training of the verified devices, and obtain a large number of
devices needed to build the system.

Secondly, we constructed the system. The plasmid used as the vector is
firstly digested with double enzymes, the fragments were obtained from
previously built devices through PCR. Then the fragments were double
digested with the same enzymes, similarly, the vector needed to undergo
gel extraction. After the PCR fragments were purified, the vector and
the fragments are connected. Same as above, the ligated product was
transformed into E.coli TOP10 F', and then proceeded to screening, PCR
verification and seed preservation in sequence.

Close

##### ES6

First, double-enzyme digestion of the plasmid as the vector, then use PCR
to obtain the gene segments of each element, then use the same enzymes
to double-enzyme digest the gene segments obtained by PCR, and finally
use ligase to connect the fragments with the vector. Take the
construction of device: Panb1-α factor-crtE-AOX1 Terminator as an
example: firstly, double-enzyme digestion of the vector plasmid
containing the promoter Panb1. Then obtain the fragment from the AOX1-α
factor-crtE-AOX1 Terminator basic plasmid by PCR, and use the same
enzyme double enzyme digest the PCR fragments. After the fragment and
the vector are purified, use Ligation ligase to connect the fragment and
the vector to obtain the device: Panb1-α factor-crtE-AOX1 Terminator.
Then transfer the ligation product to E.coli TOP10 F', and culture it
into LB medium containing ampicillin 100 ug/mL for screening. After a
single colony grows, the spot is picked and verified by PCR. If the
target band is observed, mix the bacterial solution with a 1:1 mixture
of glycerin and water, and keep the seeds at -80℃. The remaining 12
devices are constructed using the same method.

After obtaining all the devices, we constructed the system.
simultaneously using both methods

The 1. multi-step connection.

Double-enzyme digestion of the plasmid as the vector, and then use PCR to
obtain the gene fragments of each device, and then use the same enzyme
to double-enzyme the gene fragments obtained by PCR. After the fragment
and the vector are purified, they are ligated with the vector by ligase.
Repeat the operation until the system is successfully built. Take the
lycopene pathway as an example: First, digest the entire plasmid
containing Panb1-crtE-AOX1 Terminator as a vector by double enzyme
digestion, exposing the sticky end at the back of its gene fragment.
Then obtain the gene fragment of Panb1-crtB-AOX1 Terminator by PCR,
double digesting the gene fragment. After the fragment and vector are
purified, ligase is used to connect with the vector to obtain a new
plasmid: Panb1-crtE-AOX1 Terminator-Panb1-crtI-AOX1 Terminator. Then
transfer the ligation product to E.coli TOP10 F' culture, and inoculate
it into LB medium containing AMP100mg/mL for selection. After a single
colony grows, the spot is picked, and the colony is verified by PCR.
After observing the target band, mix the bacterial solution with a 1:1
mixture of glycerin and water, and keep the seeds at -80℃. Then use the
new plasmid as a vector and Panb1-crtB-AOX1 Terminator as a fragment,
and repeat the operation.

2. One-step connetion

The vector and fragment are obtained in the same way. After the fragments
and the vector are purified, ligase is used to ligate multiple fragments
with the vector at once. Take the lycopene pathway as an example:
firstly digest the entire plasmid containing Panb1-crtE-AOX1 Terminator
as a vector, exposing the sticky end at the back of its gene fragment.
Then obtain Panb1-crtB-AOX1 Terminator and Panb1-crtI- by PCR, double
digesting the gene fragment. The purified fragment and vector are
ligated with the vector by ligase to obtain a new plasmid:
Panb1-Lycopene-AOX1 Terminator. The last step is the same
transformation, screening, verification and seed preservation.

Close

##### ES7

All the constructed devices passed the gel electrophoresis, and all the
respective bands were correct, indicating that the construction was
successful.

Due to time limit, when constructing the system, the Panb1-crtE-AOX1
Terminator-Panb1-crtI-AOX1 Terminator plasmid and FMO duplex were
finally successfully obtained through Method 1 only. The enzyme
digestion method is more complicated and has a long cycle, so it is
temporarily shelved. Method 2 failed after many attempts, and the band
was shorter than expected. It may be that the homologous fragment in the
connecting part of the two fragments is shorter, which affects the
connection.

![Fig2. Plasmid construction and colony PCR results of reconstructed plasmid with Panb1 and Pynr071C promoter.All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.](https://static.igem.org/mediawiki/2021/d/d2/T--HUST-China--img--engineering--2.png)

Figure 2: Fig2. Plasmid construction and colony PCR results of
reconstructed plasmid with Panb1 and Pynr071C promoter.All the bands
are identical to the theoretical lengths, which could demonstrate
that these plasmid are correctly constructed and successfully
transformed into E.coli, confirmed by sequencing.

![Fig3. Plasmid construction and colony PCR result of Panb1-α factor -crtE-AOX1 Terminator-Panb1-α factor -crtI-AOX1 Terminator transformed E.coli.The bands of Panb1-α factor -crtE-AOX1 Terminator-Panb1-α factor -crtI-AOX1 (less than 5000bp) and is identical to the theoretical lengths of 4500bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed](https://static.igem.org/mediawiki/2021/c/cd/T--HUST-China--img--engineering--3.png)

Figure 3: Fig3. Plasmid construction and colony PCR result of
Panb1-α factor -crtE-AOX1 Terminator-Panb1-α factor
-crtI-AOX1 Terminator transformed E.coli.The bands of Panb1-α
factor -crtE-AOX1 Terminator-Panb1-α factor -crtI-AOX1 (less
than 5000bp) and is identical to the theoretical lengths of 4500bp
estimated by the designed primer locations (promoter to terminator),
which could demonstrate that these target plasmid are successfully
constructed

Close

##### ES8

The partial success of Method 1 proved that our design is feasible in
principle, but due to the limited time and the impact of the epidemic,
we temporarily shelved the experiment but this does not mean that we
will give up. After the iGEM finishes, we will continue this project
until the complete system is successfully built. The failure of method 2
is also expected: inserting multiple fragments at once is "bite more
than one can chew", and its success rate is destined to be low. So
method 2 is only an attempt to accumulate experience and train team
members. Scientific research requires our persevering efforts. We insist
on our love and confidence in synthetic biology, and are willing to
contribute to the development of synthetic biology

Close

# Cycle3:Extracellular expression portion

In order to achieve our purpose of perming and dyeing hair directly, we originally designed
to express all enzymes and short peptides outside the cell. So after Electrotransforming the
plasmid into yeast, we directly tried to detect our target protein in the supernatant. After
centrifugation, we took the supernatant for SDS-PAGE detection. But after many tests, only
part of the target protein was detected. After analysis, we suspected that the target
protein could not be detected because the extracellular expression was low and unstable, or
due to the mismatch between the signal peptide and the spatial structure of the protein. So
we want to try express in the cell later.

DesignBuildTestLearn

##### ES9

In order to facilitate the synthesis of pigments and short peptides that
can be directly used for perm and dyeing, we initially chose to express
all enzymes and short peptides extracellularly so that we can use
related enzymes to synthesize pigments in the culture medium and can
directly obtain them for perm and straightening hair. So we added
α-factor as a signal peptide in front of all target genes, in order to
secrete all proteins. At the same time, in order to enhance expression,
we selected the strongest eukaryotic promoter that has been discovered:
AOX1, to initiate and enhance the expression of downstream genes. And in
order to achieve the stable existence of the target gene in yeast and
form a high copy for efficient expression, we selected a suitable
restriction site to linearize the plasmid to integrate into the yeast
genome to ensure that it can remain stable after passaging. And we
expect to achieve high copy through repeated integration. At the same
time, we added His-tag at the end of the gene for protein purification
and identification.

Close

##### ES10

We first transfer the synthesized plasmid into E. coli for amplification
in order to obtain enough target plasmid. We comprehensively considered
the hidden restriction sites of different target genes, and finally
selected two integration sites, BglII and SalI, to avoid the destruction
of all genes. We cut the plasmid with BglII or SalI to obtain a
linearized plasmid. Due to system limitations, we can only divide it
into multiple tubes for single digestion.

Initially, we considered the single enzyme digestion product to be
subjected to agarose gel electrophoresis, and concentration were
performed while verifying. However, in next experiments, we found that
the linear plasmid concentration we obtained was very low. It is
attributed to the lower efficiency of gel extraction and the operation
process: in the last step, repeating the experiment ddH2O elution will
cause a large amount of plasmid-containing solution to remain at the
bottom or wall of the ep tube; in the second elution , we cannot heat
the ddH2O again, and the linearized carrier with a larger molecular
weight is trapped by the purification column with lower elution
efficiency, causing waste. In the end, due to various reasons, the
plasmid concentration was not enough when we first performed the
electroporation. Although part of the linearized vector was successfully
electro-transformed in the follow-up test, and some of the yeast
integrated with the target gene was obtained, the target protein was
almost impossible to be detected in the follow-up identification due to
the low concentration and the failure to form a high copy.

After carefully reviewing the information and asking authoritative
sources, combined with the previous results, we found that at the
concentration of the plasmid we extracted, overnight single enzyme
digestion is completely enough to completely cut all the plasmids. This
means that there are very few unlinearized plasmids in the reaction
system, and there is no need to use electrophoresis to separate them.
Therefore, in the subsequent experiments, we chose the method of ethanol
precipitation to achieve the purification and concentration of the
single enzyme digestion system. After enzyme digestion, only a small
amount of products were taken for agarose gel electrophoresis
verification, and the remaining products were directly used for ethanol
precipitation and concentration, and finally a high concentration of
linearized plasmid was successfully obtained. After electroporation,
screening was performed on MD plates containing 500 mg/mL G418, and more
single yeast colonies integrated with the target gene were obtained, and
expression verification was performed.

Close

##### ES11

We use the colony PCR method to test whether the target gene is
successfully transformed into the yeast cells. The pipette tip of the
picked yeast single colony is lightly dipped in a PCR tube filled with
sterile water, and then filled with 1 mL of antibiotic YPD is cultured
in a 2mL ep tube to realize the amplification of bacterial species while
performing PCR verification, which saves time and reduces the risk of
infection by reducing openings.

![Figure 4 Results of PCR agarose gel electrophoresis of yeast colonies after electroporation.The bright band identified by colony PCR is consistent with the expection, which proves that the target gene was successfully transferred into yeast cells](https://static.igem.org/mediawiki/2021/9/9f/T--HUST-China--img--engineering--4.png)

Figure 4: Figure 4 Results of PCR agarose gel electrophoresis of
yeast colonies after electroporation.The bright band identified by
colony PCR is consistent with the expection, which proves that the
target gene was successfully transferred into yeast cells

After the PCR verification of the colony was correct, we expanded the
culture and induced expression. Since AOX1 is a methanol-inducible
promoter and is strictly inhibited by glycerol, we cultured it in the
initial glycerol-containing medium to expand the number of bacteria, and
then transferred all of its bacteria to a culture medium with methanol
as the sole carbon source. And add 0.5% of the fermentation volume of
methanol every day, and after sampling and centrifugation, the
supernatant was tested by SDS-PAGE. After multiple days of
identification, we successfully detected some target bands. At the same
time, because the molecular weight of PepACS is only 5.08 kDa, it is
almost impossible to separate from bromophenol blue, and its migration
is also greatly affected, so the position of the band is not accurate.
In order to judge whether it was expressed or not, we first filtered the
miscellaneous proteins through 10kDa ultrafiltration tube, then
concentrated it with 3kDa ultrafiltration tube, and then detected it by
SDS-PAGE. Judge whether it is expressed successfully by whether there is
coloring near the bottom line.

![Figure 5 for SDS-PAGE detection of the Laccase GS1154CL LOX2 ACC pepACS DsbC+pepACS supernatant](https://static.igem.org/mediawiki/2021/1/18/T--HUST-China--img--engineering--5.png)

Figure 5: Figure 5 for SDS-PAGE detection of the Laccase GS1154CL
LOX2 ACC pepACS DsbC+pepACS supernatant

Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS supernatant SDS-PAGE
detection.

Due to glycosylation modification during yeast expression, the apparent
molecular weight will be slightly larger than the molecular weight of
the actual protein. Preliminary detection of Laccase, 4CL, ACC bands
molecular weight is about 75kDa, LOX2 is above 100kDa, DsbC+pepACS is
about 40kDA, slightly larger than its actual molecular weight (Laccase:
57.01kDa, 4CL: 61.88kDa, ACC: 63.40kDa, LOX2: 102.88 kDa, DsbC+pepACS:
31.72 kDa), which are all within the acceptable range, can be considered
to be successfully expressed. At the same time, due to the small
molecular weight of 5-1, it is difficult to separate from bromophenol
blue completely, which means than its migration is to be greatly
affected. But according to the 10kDa lower part of the marker, it can be
seen that it is distinguished from the blank control: there is obvious
coloring, and the successful expression can be confirmed. There are
irregular staining bands on the bromophenol blue line in the PepACS
swimming lane. Although the molecular weight is not accurate, the
successful expression can be judged according to the detection method we
designed.

For the enzymes Laccase and LOX2 whose expression has been confirmed and
have standard enzyme activity determination methods, we tried to
identify their catalytic activity first.

For LOX2: use 27ul linoleic acid, 25ul tween 20, 8ml ddH20, 3M/L NaOH
183ul, and dilute to 50ml as a solution; take 200ul substrate, 1ml
supernatant, and 1ml pH6.0 buffer for measuring absorbance changes at
234nm. And every 0.001 increase in absorbance is regarded as an enzyme
activity unit. Its activity was successfully detected.

![Figure 6 Enzyme activity determination of LOX2](https://static.igem.org/mediawiki/2021/0/0f/T--HUST-China--img--engineering--6.png)

Figure 6: Figure 6 Enzyme activity determination of LOX2

On SDS-PAGE, Laccase showed a low concentration, so it is not ideal to
directly measure its catalytic activity. We were not sure about its
activity status, thus, we decided to purify it with a nickel column to
increase the concentration of Laccase so as to further detect the enzyme
activity. activity.

![Figure 7c Laccase nickel column purification SDS-PAGE verification](https://static.igem.org/mediawiki/2021/4/4c/T--HUST-China--img--engineering--7.png)

Figure 7: Figure 7c Laccase nickel column purification SDS-PAGE
verification

It can be seen that there is a clear band at 75kDa, which is consistent
with the band displayed in the supernatant, and the color of bands
darkens as the concentration of the eluent increases, which further
proves that the band we observed before is indeed the target protein
rather than a high-expressed hybrid protein

After obtaining a higher concentration of Laccase after purification, we
mixed 1mL of 1mmol/L ABTS solution with 1mL of supernatant after
centrifugation, and added CuSO4 with a final concentration of 10mmol/L.
The absorbance was measured at 420nm, and it was successfully detected
enzyme activity. On this basis, in order to explore the optimal
conditions for Laccase, we carried out further determinations.

Since the active center of Laccase is copper ion, we first studied the
relationship between the supplemental copper ion concentration and
enzyme activity. The CuSO4 concentration was adjusted to 9mmol/L and
10mmol/L respectively, and the enzyme activity of Laccase was measured
under the same conditions.

![Figure 8 Effects of concentration of CuSO4 on enzyme activity](https://static.igem.org/mediawiki/2021/9/9a/T--HUST-China--img--engineering--8.png)

Figure 8: Figure 8 Effects of concentration of CuSO4 on enzyme
activity

We found that the enzyme activity is the highest when the final
concentration is 10 mmol. This may be because when the concentration is
too low, it cannot meet the needs of the Laccase active center. And when
the concentration is too high, it affects the binding of Laccase to the
substrate.

We also explored the effect of pH and temperature on Laccase activity.

pH: The data showed that the optimal pH of Laccse was acidic, so we used
100mM citric acid and sodium citrate to configure pH=3, pH=4.8 and
pH=6.6 buffers respectively. Under the premise that the final
concentration of CuSO4 is still 10mmol/L, 1mL of buffer is added to
control the pH of the reaction system.

![Figure 9 Effects of different pH on enzyme activity](https://static.igem.org/mediawiki/2021/4/4f/T--HUST-China--img--engineering--9.png)

Figure 9: Figure 9 Effects of different pH on enzyme activity

After comparison, we found that among the three sets of data measured,
the activity of Laccase was the highest at pH=3.0. At pH=4.8, the
activity drops sharply, but at pH=6.6, it is completely inactivated. As
for the subsequent measurement value at pH 6.6 presenting lower than the
initial value, we speculated that it is caused by the inadequate mixing
of the solution at the beginning of the reaction.

Temperature: We separately kept the ABTS solution and supernatant at 10℃,
20℃, 37℃ in advance, and also kept the final concentration of CuSO4 at
10mmol/L, and mixed them before the measurement.

![Figure 10 Effects of different temperature on laccase activity](https://static.igem.org/mediawiki/2021/0/01/T--HUST-China--img--engineering--10.png)

Figure 10: Figure 10 Effects of different temperature on laccase
activity

The analysis and comparison of the three sets of data shows that under
the test conditions, the Laccase activity is highest at 20℃, and both
low temperature and high temperature will inhibit Laccase activity. As
for the decrease in absorbance at the late of the measurement at 10℃, it
is suspected that the low temperature has a certain effect on the ABTS
cation radicals, resulting in decrease in absorbance.

At the same time, during the detection process, we found that ROX1 has a
relatively vague band, and we cannot confirm its successful expression
based on the protein gel of supernatant. However, ROX1 is a key gene
involved in the regulation of all pathways in our entire design, whose
successful expression is very important to us, so we purified it through
a nickel column for further verification. Fortunately, we purified the
target band and confirmed that it can indeed be expressed.

![Figure 11 SDS-PAGE verification after ROX1 nickel column purification](https://static.igem.org/mediawiki/2021/0/0c/T--HUST-China--img--engineering--11.png)

Figure 11: Figure 11 SDS-PAGE verification after ROX1 nickel column
purification

SDS-PAGE verification after ROX1 nickel column purification

It can be seen that there is a band at 50-55kDa that is significantly
different from the sample after washing and flow-through treatment. Its
molecular weight is slightly larger than the theoretical value of
47.09kDa, but it can be confirmed within the range of the molecular
weight change caused by glycosylation. ROX1 was successfully expressed.
Although the gradient elution was not performed, there were many
contaminated bands when the target band appeared for the first time, but
according to the subsequent elution results, it can be judged that it is
indeed the target protein.

Close

##### ES12

However, after many times of SDS-PAGE tests, we found that there are
still some proteins that have not been detected in bands, but the
bacterial liquid PCR verification is correct. By analysis, we suspect
that there are two reasons: one is that the extracellular expression of
the protein is low, and it is unstable in the extracellular environment,
and is easily degraded by proteases, etc.; the other is that the signal
peptide we use is a universal signal peptide, absence of particular
design for the target protein, the two do not fit and the protein cannot
be successfully secreted to the outside of the cell.

Close

# Cycle4:Intracellular expression portion

After determining that under the existing system, the extracellular expression of some
proteins may not be achieved. We chose to reconstruct the plasmid, remove the signal peptide
and try to express it in the cell. So we amplified the target gene sequence without signal
peptide by PCR and inserted it behind the Panb1 promoter to construct a plasmid without
signal peptide. Also after electrotransformation, we extracted the total protein of yeast,
purified it with nickel column and examined it by SDS-PAGE. This time, we successfully
detected the rest of our protein and realized the synthesis of indigo and lycopene.

DesignBuildTestLearn

##### ES13

After determining that some proteins cannot be expressed extracellularly
through the existing system, we learned that the expression level of
intracellular expression is much higher than that of extracellular
expression. Intracellular proteins are more stable than they exist
outside the cell, and the problem that the expressed protein cannot be
detected due to signal peptide mismatch is avoided. Although it is more
difficult to detect the protein in the cell, after considering various
factors, we chose to rebuild the plasmid, remove the signal peptide, and
try to express protein in the cell.

Close

##### ES14

We designed new PCR primers on the basis of original sequence to ensure
that the signal peptide is eliminated when the gene is amplified.
However, because the signal peptide part of the plasmid with AOX1 as the
promoter did not have suitable restriction sites, we could not use
restriction enzymes to directly remove them, so we chose the method of
using restriction enzymes to connect the fragments and insert after the
constitutive promoter Panb1. Constitutive promoters can be used to
continuously synthesize the target protein without induction.

In order to ensure the successful construction of the target plasmid, we
first transformed the enzyme-linked product into E. coli for testing and
amplification. After confirming that the target plasmid was obtained, we
still used the same method as before, single enzyme digestion, ethanol
precipitation and concentration, electrotransfection of yeast and
expression.

Close

##### ES15

In order to confirm the successful construction of the target plasmid, we
first transformed the enzyme ligation product into E.coli, and spread it
on an LB plate containing Kan resistance. Only the E.coli successfully
transferred into the plasmid can grow normally. The colony PCR is used
to identify whether the transferred plasmid is correct, and further
sequence verification is performed to confirm that the plasmid is
successfully constructed without mutation.

![Fig12. Plasmid construction and colony PCR results of plasmid without signal peptides transformed E.coli](https://static.igem.org/mediawiki/2021/3/35/T--HUST-China--img--engineering--12.png)

Figure 12: Fig12. Plasmid construction and colony PCR results of
plasmid without signal peptides transformed E.coli

All the bands are identical to the theoretical lengths, which could
demonstrate that these plasmid are correctly constructed and
successfully transformed into E.coli, confirmed by sequencing.

Identical method is used in testing yeast after electroporation: colony
PCR, confirmation, bright bands, expand cultivation.

![Fig13. Colony PCR result of yeast after electroporation of reconstructed plasmid without the signal peptide](https://static.igem.org/mediawiki/2021/d/d6/T--HUST-China--img--engineering--13.png)

Figure 13: Fig13. Colony PCR result of yeast after electroporation of
reconstructed plasmid without the signal peptide

The bright bands are identical to the theoretical lengths, which could
demonstrate that this target plasmid had successfully transformed into
yeast. Target genes are confirmed exist in the yeast of multiple bands,
which could be the result of polluted electroporation cup.

The constitutive promoter doesn't need to be induced. To save time and
unnecessary steps to reduce the risk of bacterial infection, instead of
changing the culture medium, we culture our yeast continuously in the
same medium with daily supply of glycerol as carbon source.

Total yeast protein extraction kit is used to extract the whole proteome
of our yeast for further test because intracellular expression makes
direct test of target protein in the medium infeasible. Then, we go for
a purification through Nickel-affinity chromatography column, then apply
SDS-PAGE to separate target protein from the large amount and various
type of total protein to confirm the exist of our target protein.

![Fig14. SDS-PAGE result of FMO after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/8/88/T--HUST-China--img--engineering--14.png)

Figure 14: Fig14. SDS-PAGE result of FMO after purification of yeast
total protein extraction product through Nickel-affinity
chromatography column

Different from impure or permeate bands, the target protein located
around 60kDa, bigger than the theoretical 53.96kDa but still within
explainable and acceptable range of glycosylation modification. FMO
could be confirmed as successfully expressed. The concentration of yeast
total protein is so high that huge amount of impure protein is included
during elution. But due to difference from impure or permeate bands, its
dark color and consistency among several times of elution, this band
could be verified as our target FMO,

![Fig15. SDS-PAGE result of crtE after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/1/1d/T--HUST-China--img--engineering--15.png)

Figure 15: Fig15. SDS-PAGE result of crtE after purification of yeast
total protein extraction product through Nickel-affinity
chromatography column

Different from impure or permeate bands, the target protein located
around 50kDa, bigger than the theoretical 33.42kDa but still within
explainable and acceptable range of glycosylation modification. crtE
could be confirmed as successfully expressed.

![Fig16. SDS-PAGE result of crtB after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/8/80/T--HUST-China--img--engineering--16.png)

Figure 16: Fig16. SDS-PAGE result of crtB after purification of yeast
total protein extraction product through Nickel-affinity
chromatography column

Different from impure or permeate bands, the target protein located
around 50kDa, bigger than the theoretical 35.30kDa but still within
explainable and acceptable range of glycosylation modification. crtB
could be confirmed as successfully expressed.

![Fig17. SDS-PAGE result of crtI after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/2/2b/T--HUST-China--img--engineering--17.png)

Figure 17: Fig17. SDS-PAGE result of crtI after purification of yeast
total protein extraction product through Nickel-affinity
chromatography column

Different from impure or permeate bands, the target protein located
around 60kDa, bigger than the theoretical 55.86kDa but still within
explainable and acceptable range of glycosylation modification. crtI
could be confirmed as successfully expressed.

After confirmation of successful expression, we add indole as substrate
into culture medium of FMO and FPP into the mixture of crtE, crtB and
crtI to test whether the expressed enzyme is active in terms of color
change of the culture medium. After culturing for a period of time, blue
and red medium is occurred, which means we succeed in expression of
active enzymes used to synthesize indigo or lycopene. Although the
synthesis level of the pigments is too low to dye, still, it is one huge
step for us.

![Fig18 Medium for expression with substrates](https://static.igem.org/mediawiki/2021/3/3e/T--HUST-China--img--engineering--18.png)

Figure 18: Fig18 Medium for expression with substrates

From left to right:

GS115 medium with indole and FPP;

Panb1-FMO-AOX1 Terminator medium with indole;

Mixture of Panb1-crtE-AOX1 Terminator??Panb1-crtB-AOX1
Terminator??Panb1-crtI-AOX1 Terminator medium with FPP

Unfortunately, due to limited time, enzymes for synthesis and degradation
of curcumin aren't tested. Neither does the synthesis of curcumin

Close

##### ES16

Through intracellular experiment, we confirm that the suitability problem
between the signal peptide and the space structure of our protein do
exist, that is to say, even if the common signal peptide on the most
popular engineering plasmid couldn't fit protein with any kind of space
structure. This part of experiment rescues our experiment result by
expressing enzymes which we could not detect in extracellular expression
and realize the synthesis of pigments primarily.

It must be noted that the expression and synthesis level of our enzymes
and pigments are too low to be applied in actual dyeing due to the
shallow color of supernatant of intracellular expression after
certification. Across all our experiments, we would say it is because
the low copy number of our plasmid within the yeast. Due to limitation
of time and culture scale, we have to pick the relatively big colonies
which grow rapidly as yeast with high copy number. Without rounds of
screening of gradient concentration of antibiotics, our identification
of high copy number is only a suspect and not strict enough, since yeast
with higher copy number will express higher level of antibiotic
resistance product to grow faster in medium containing higher
concentration of antibiotics. So it is possible that the strain we
selected has too low the copy number to express enough amount of target
protein without gradie

Close

![](https://static.igem.org/mediawiki/2021/2/28/T--HUST-China--img--girl.png)

I think the methods in constructing engineering plasmids are quite valid, you? Well, I have a question here. What if you or I want to repeat their experiment? I want to know what [protocols](https://2021.igem.org/Team%3AHUST-China/Protocol) did they follow to get those result.

CONTACT US: iGEMHUSTChina@163.com

Huazhong University of Sci. & Tech., Wuhan, China

1037# Luoyu Rd, Wuhan, P.R.China 430074

Copyright © HUST-China iGEM 2021

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HUST-China/Experiments

Experiments | iGEM HUST-China

[HUST-China](https://2021.igem.org/Team%3AHUST-China)

* Project

  [Description](https://2021.igem.org/Team%3AHUST-China/Description)[Design](https://2021.igem.org/Team%3AHUST-China/Design)[Proof of
  Concept](https://2021.igem.org/Team%3AHUST-China/Proof_Of_Concept)[Safety](https://2021.igem.org/Team%3AHUST-China/Safety)[Proposed
  Implementation](https://2021.igem.org/Team%3AHUST-China/Implementation)[Contribution](https://2021.igem.org/Team%3AHUST-China/Contribution)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHUST-China/Human_Practices)[Part
  1](https://2021.igem.org/Team%3AHUST-China/Human_Practices_01)[Part
  2](https://2021.igem.org/Team%3AHUST-China/Human_Practices_02)[Part
  3](https://2021.igem.org/Team%3AHUST-China/Collaborations)[Part 4](https://2021.igem.org/Team%3AHUST-China/Education)
* Modeling

  [Overview](https://2021.igem.org/Team%3AHUST-China/Model)[Gene
  Pathway](https://2021.igem.org/Team%3AHUST-China/Gene_Pathway)[Dye color
  forecast](https://2021.igem.org/Team%3AHUST-China/Dye_color_forecast)[Micronucleus
  Counter](https://2021.igem.org/Team%3AHUST-China/Micronucleus_Counter)
* Products

  [Overview](https://2021.igem.org/Team%3AHUST-China/Products)[Software](https://2021.igem.org/Team%3AHUST-China/Software)[Hardware](https://2021.igem.org/Team%3AHUST-China/Hardware)[Another
  Area](https://2021.igem.org/Team%3AHUST-China/Another_Area)
* Experiments

  [Overview](https://2021.igem.org/Team%3AHUST-China/Experiments)[Parts](https://2021.igem.org/Team%3AHUST-China/Parts)[Engineering
  Success](https://2021.igem.org/Team%3AHUST-China/Engineering)[Results](https://2021.igem.org/Team%3AHUST-China/Results)[Protocol](https://2021.igem.org/Team%3AHUST-China/Protocol)[Notebook](https://2021.igem.org/Team%3AHUST-China/Notebook)
* People

  [Team](https://2021.igem.org/Team%3AHUST-China/Team)[Partnership](https://2021.igem.org/Team%3AHUST-China/Partnership)[Attributions](https://2021.igem.org/Team%3AHUST-China/Attributions)
* Achievements

  [Award](https://2021.igem.org/Team%3AHUST-China/Award)[Promotion
  Video](https://video.igem.org/w/aMwNT6eBryGs8xG5avXD51)[Judging
  Form](https://igem.org/2021_Judging_Form?id=3711)

# Experiments

---

##### Contents

# Overview

![](https://static.igem.org/mediawiki/2021/f/f6/T--HUST-China--img--Experiments-Overview.png)

CONTACT US: iGEMHUSTChina@163.com

Huazhong University of Sci. & Tech., Wuhan, China

1037# Luoyu Rd, Wuhan, P.R.China 430074

Copyright © HUST-China iGEM 2021

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HUST-China/Results

Results | iGEM HUST-China

[HUST-China](https://2021.igem.org/Team%3AHUST-China)

* Project

  [Description](https://2021.igem.org/Team%3AHUST-China/Description)[Design](https://2021.igem.org/Team%3AHUST-China/Design)[Proof of
  Concept](https://2021.igem.org/Team%3AHUST-China/Proof_Of_Concept)[Safety](https://2021.igem.org/Team%3AHUST-China/Safety)[Proposed
  Implementation](https://2021.igem.org/Team%3AHUST-China/Implementation)[Contribution](https://2021.igem.org/Team%3AHUST-China/Contribution)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHUST-China/Human_Practices)[Part
  1](https://2021.igem.org/Team%3AHUST-China/Human_Practices_01)[Part
  2](https://2021.igem.org/Team%3AHUST-China/Human_Practices_02)[Part
  3](https://2021.igem.org/Team%3AHUST-China/Collaborations)[Part 4](https://2021.igem.org/Team%3AHUST-China/Education)
* Modeling

  [Overview](https://2021.igem.org/Team%3AHUST-China/Model)[Gene
  Pathway](https://2021.igem.org/Team%3AHUST-China/Gene_Pathway)[Dye color
  forecast](https://2021.igem.org/Team%3AHUST-China/Dye_color_forecast)[Micronucleus
  Counter](https://2021.igem.org/Team%3AHUST-China/Micronucleus_Counter)
* Products

  [Overview](https://2021.igem.org/Team%3AHUST-China/Products)[Software](https://2021.igem.org/Team%3AHUST-China/Software)[Hardware](https://2021.igem.org/Team%3AHUST-China/Hardware)[Another
  Area](https://2021.igem.org/Team%3AHUST-China/Another_Area)
* Experiments

  [Overview](https://2021.igem.org/Team%3AHUST-China/Experiments)[Parts](https://2021.igem.org/Team%3AHUST-China/Parts)[Engineering
  Success](https://2021.igem.org/Team%3AHUST-China/Engineering)[Results](https://2021.igem.org/Team%3AHUST-China/Results)[Protocol](https://2021.igem.org/Team%3AHUST-China/Protocol)[Notebook](https://2021.igem.org/Team%3AHUST-China/Notebook)
* People

  [Team](https://2021.igem.org/Team%3AHUST-China/Team)[Partnership](https://2021.igem.org/Team%3AHUST-China/Partnership)[Attributions](https://2021.igem.org/Team%3AHUST-China/Attributions)
* Achievements

  [Award](https://2021.igem.org/Team%3AHUST-China/Award)[Promotion
  Video](https://video.igem.org/w/aMwNT6eBryGs8xG5avXD51)[Judging
  Form](https://igem.org/2021_Judging_Form?id=3711)

# Results

---

##### Contents

# Introduction

To realize hair dyeing & perming and decoloring & straightening as designed, we use
E.coli to construct all the needed plasmid and transfect them into yeast through
electroporation to express target enzymes and peptides. To confirm the validity of our
perming and dyeing theory and eventually, the whole project, real hair is used in lab
trails, which aim to estimate its color fastness, mechanical property, etc.

# Plasmid construction and amplification

## The transformation of plasmid with AOX1 as promoter

First of all, we need to amplificated all the commercially synthesized plasmid to acquire
enough amount for further study. After transformation, colony PCR is applied for
confirmation. Then we go for plasmid extraction.

![ Colony PCR confirmation of successful E.coli transfection](https://static.igem.org/mediawiki/2021/8/8e/T--HUST-China--img--result--1.png)

Bright bands of identical sizes from colony PCR result demonstrates that target plasmid had
successfully transformed into E.coli

Read More

##### result1

Plasmid with target gene is transformed into E.coli. From them, we
acquire large amount of target gene using as raw material for further
operation.

![Colony PCR result of AOX1-&alpha; factor-ROX1-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/9/9b/T--HUST-China--img--result--2.png)

Figure 2: Colony PCR result of AOX1-&alpha; factor-ROX1-AOX1
Terminator transformed E.coli

The band of AOX1-α factor-ROX1-AOX1 Terminator from colony PCR is about
3000bp, identical to the theoretical length of 3070bp estimated by the
designed primer location (promoter to terminator), which could
demonstrate that this target plasmid had successfully transformed into
E.coli

![Colony PCR result of AOX1-&alpha; factor-Laccase-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/5/50/T--HUST-China--img--result--3.png)

Figure 3: Colony PCR result of AOX1-&alpha; factor-Laccase-AOX1
Terminator transformed E.coli

The band of AOX1-α factor-Laccase-AOX1 Terminator from colony PCR is
about 3000bp, identical to the theoretical length of 3416bp estimated by
the designed primer location (promoter to terminator), which could
demonstrate that this target plasmid had successfully transformed into
E.coli

![Colony PCR results of AOX1-&alpha; factor-FMO-AOX1 Terminator, AOX1-&alpha; factor-crtE-AOX1 Terminator, AOX1-&alpha; factor-crtB-AOX1 Terminator and AOX1-&alpha; factor-crtI-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/5/55/T--HUST-China--img--result--4.png)

Figure 4: Colony PCR results of AOX1-&alpha; factor-FMO-AOX1
Terminator, AOX1-&alpha; factor-crtE-AOX1 Terminator,
AOX1-&alpha; factor-crtB-AOX1 Terminator and AOX1-&alpha;
factor-crtI-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-FMO-AOX1 Terminator (3000+bp), AOX1-α
factor-crtE-AOX1 Terminator (almost 3000bp), AOX1-α factor-crtB-AOX1
Terminator (less than 3000bp) and AOX1-α factor-crtI-AOX1 Terminator
(3000+bp) from colony PCR are identical to the theoretical lengths of
3214bp, 2746bp, 2767bp and 3316 bp estimated by the designed primer
locations (promoter to terminator), which could demonstrate that these
target plasmid had successfully transformed into E.coli

![Colony PCR results of AOX1-&alpha; factor-CUS-AOX1 Terminator, AOX1-&alpha; factor-ACC-AOX1 Terminator, AOX1-&alpha; factor-4CL-AOX1 Terminator and AOX1-&alpha; factor-LOX2-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/4/46/T--HUST-China--img--result--5.png)

Figure 5: Colony PCR results of AOX1-&alpha; factor-CUS-AOX1
Terminator, AOX1-&alpha; factor-ACC-AOX1 Terminator,
AOX1-&alpha; factor-4CL-AOX1 Terminator and AOX1-&alpha;
factor-LOX2-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-CUS-AOX1 Terminator (3000bp) , AOX1-α
factor-ACC-AOX1 Terminator (3000+bp), AOX1-α factor-4CL-AOX1 Terminator
(3000+bp) and AOX1-α factor-LOX2-AOX1 Terminator (almost 5000bp) from
colony PCR are identical to the theoretical lengths of 3046bp, 3619bp,
3523bp and 4528bp estimated by the designed primer locations (promoter
to terminator), which could demonstrate that these target plasmid had
successfully transformed into E.coli

![Colony PCR results of AOX1-&alpha; factor-curA-AOX1 Terminator, AOX1-&alpha; factor-pepACS-AOX1 Terminator and AOX1-&alpha; factor-DsbC-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/2/2a/T--HUST-China--img--result--6.png)

Figure 6: Colony PCR results of AOX1-&alpha; factor-curA-AOX1
Terminator, AOX1-&alpha; factor-pepACS-AOX1 Terminator and
AOX1-&alpha; factor-DsbC-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-curA-AOX1 Terminator (almost 3000bp), AOX1-α
factor-pepACS-AOX1 Terminator (almost 2000bp) and AOX1-α
factor-DsbC-AOX1 Terminator (almost 3000bp) from colony PCR are
identical to the theoretical lengths of 2875bp, 1987bp and 2722bp
estimated by the designed primer locations (promoter to terminator),
which could demonstrate that these target plasmid had successfully
transformed into E.coli

Close

## The construction of plasmid with Panb1 or Pynr071c as promoter

AOX1 promoter is the strongest eukaryotic promoter currently known in yeast expression
system. So we choose AOX1 as the primary promoter when we synthesized all these plasmid for
the sake of more convenient expression. But noticing that methanol is hazardous, flammable,
combustible and therefore, inappropriate to have direct contact with the hair, we need to
substrate AOX1 for constitutive promoter Panb1 and xylose induced promoter Pynr071C to
realize the projected regulation function as designed. Double-enzyme cleavage and rejointing
is used to achieve this. We amplify the target gene located in the primarily synthesized
plasmid without AOX1 promoter, and digest the acquired fragments and two kinds of plasmid,
containing promoter only, with EcoR I and BamH I, then transform them into E.coli after
linking the product together. Through this, we successfully substrate the primary AOX1 for
Panb1 and Pynr071C.

![Plasmid construction and colony PCR results of reconstructed plasmid with Panb1 and Pynr071C promoter](https://static.igem.org/mediawiki/2021/b/b7/T--HUST-China--img--result--7.png)

Figure 7: Plasmid construction and colony PCR results of reconstructed plasmid with Panb1
and Pynr071C promoter

All the bands are identical to the theoretical lengths, which could demonstrate that these
plasmid are correctly constructed and successfully transformed into E.coli, confirmed by
sequencing.

Read More

##### result2

AOX1 promoter is the strongest eukaryotic promoter currently known in
yeast expression system. So we choose AOX1 as the primary promoter when
we synthesized all these plasmid for the sake of more convenient
expression. But noticing that methanol is hazardous, flammable,
combustible and therefore, inappropriate to have direct contact with the
hair, we need to substrate AOX1 for constitutive promoter Panb1 and
xylose induced promoter Pynr071C to realize the projected regulation
function as designed. Another series of plasmid with target gene and new
promoter are constructed.

![Plasmid construction and colony PCR results of Panb1-&alpha; factor-FMO-AOX1 Terminator and Pynr071c-&alpha; factor-ROX1-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/d/d8/T--HUST-China--img--result--8.png)

Figure 8: Plasmid construction and colony PCR results of
Panb1-&alpha; factor-FMO-AOX1 Terminator and
Pynr071c-&alpha; factor-ROX1-AOX1 Terminator transformed E.coli

The bands of Panb1-α factor-FMO-AOX1 Terminator (less than 3000bp) and
Pynr071c-α factor-ROX1-AOX1 Terminator (3000+bp) from colony PCR are
identical to the theoretical lengths of 2676bp and 3321bp estimated by
the designed primer locations (promoter to terminator), which could
demonstrate that these target plasmid are successfully constructed.

![Plasmid construction and colony PCR results of Panb1-&alpha; factor-4CL-AOX1 Terminator, Panb1-&alpha; factor-crtI-AOX1 Terminator, Panb1-&alpha; factor-crtB-AOX1 Terminator and Panb1-&alpha; factor-crtE-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/9/92/T--HUST-China--img--result--9.png)

Figure 9: Plasmid construction and colony PCR results of
Panb1-&alpha; factor-4CL-AOX1 Terminator, Panb1-&alpha;
factor-crtI-AOX1 Terminator, Panb1-&alpha; factor-crtB-AOX1
Terminator and Panb1-&alpha; factor-crtE-AOX1 Terminator
transformed E.coli

The bands of Panb1-α factor-4CL-AOX1 Terminator (3000+bp), Panb1-α
factor-crtI-AOX1 Terminator (3000bp), Panb1-α factor-crtB-AOX1
Terminator (2000+bp) and Panb1-α factor-crtE-AOX1 Terminator (2000+bp)
from colony PCR are identical to the theoretical lengths of 3185bp,
3046bp, 2198bp and 2177bp estimated by the designed primer locations
(promoter to terminator), which could demonstrate that these target
plasmid are successfully constructed.

![Plasmid construction and colony PCR results of Panb1-&alpha; factor-CUS-AOX1 Terminator, Panb1-&alpha; factor-ACC-AOX1 Terminator and Panb1-&alpha; factor-PepACS-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/6/6f/T--HUST-China--img--result--10.png)

Figure 10: Plasmid construction and colony PCR results of
Panb1-&alpha; factor-CUS-AOX1 Terminator, Panb1-&alpha;
factor-ACC-AOX1 Terminator and Panb1-&alpha; factor-PepACS-AOX1
Terminator transformed E.coli

The bands of Panb1-α factor-CUS-AOX1 Terminator (2500bp), Panb1-α
factor-ACC-AOX1 Terminator (3000bp) and Panb1-α factor-PepACS-AOX1
Terminator(less than 1500bp) from colony PCR are identical to the
theoretical lengths of 2508bp, 3081bp and 1312bp estimated by the
designed primer locations (promoter to terminator), which could
demonstrate that these target plasmid are successfully constructed.

Close

## The construction of plasmid without signal peptide

During the experiment, we find that not all the protein we want could be secreted
extracellularly. Target protein is only detected in the yeast but not in the culture. while
no signal appears in the supernatant without them, which indicates that not all of our
signal peptides works. We reckon that as the α-factor on the plasmid we used is only a
common basic signal peptide, which has some degree of universality but couldn't fit all the
protein due to unseen matters like space structure of the protein, our target protein
couldn't be induced to secret into the extracellular space by α-factor, or the low
expression level coupled with degradation from protease existing in the extracellular
environment could do the same. So, to get the rest of our enzymes which don't want to be
outside of the cell with α-factor, and whether or not we could raise the expression level,
we design new primers for PCR, which eliminate the signal peptide part off the whole target
genes, and reconstruct the plasmid without the α-factor to verify if they prefer to be
expressed inside of the cell.

![Plasmid construction and colony PCR results of plasmid without signal peptides transformed E.coli](https://static.igem.org/mediawiki/2021/d/de/T--HUST-China--img--result--11.png)

Figure 11: Plasmid construction and colony PCR results of plasmid without signal peptides
transformed E.coli

All the bands are identical to the theoretical lengths, which could demonstrate that these
plasmid are correctly constructed and successfully transformed into E.coli, confirmed by
sequencing.

Read More

##### result3

Not quite to what we expect, after repeated transfection to the yeast,
only a few products are expressed inside of eukaryotic system. Because
of the large molecular weight and various types of some of our protein,
we suspect that the common signal peptide we use, α-factor, is not
enough to bring our protein out of the cell. While there is some of the
genes without detectable products and we are hoping to get higher
expression level, new primers for PCR are designed to ignore α-factor
from our target gene in PCR. Then, likewise, we reconstruct this series
of plasmid without α-factor through similar double-enzyme digestion and
reconnection which insert our target genes right behind Panb1 promoter.

![Plasmid construction and colony PCR results of Panb1-crtB-AOX1 Terminator, Panb1-crtE-AOX1 Terminator and Panb1-FMO-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/5/58/T--HUST-China--img--result--12.png)

Figure 12: Plasmid construction and colony PCR results of
Panb1-crtB-AOX1 Terminator, Panb1-crtE-AOX1 Terminator and
Panb1-FMO-AOX1 Terminator transformed E.coli

The bands of Panb1-crtB-AOX1 Terminator (less than 2000bp),
Panb1-crtE-AOX1 Terminator (less than 2000bp) and Panb1-FMO-AOX1
Terminator (2500bp) from colony PCR are identical to the theoretical
lengths of 1859bp, 1838bp and 2437bp estimated by the designed primer
locations (promoter to terminator), which could demonstrate that these
target plasmid are successfully constructed.

![Plasmid construction and colony PCR results of Panb1-CUS-AOX1 Terminator, Panb1-ACC-AOX1 Terminator, Panb1-4CL-AOX1 Terminator and Panb1-crtI-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/8/8a/T--HUST-China--img--result--13.png)

Figure 13: Plasmid construction and colony PCR results of
Panb1-CUS-AOX1 Terminator, Panb1-ACC-AOX1 Terminator, Panb1-4CL-AOX1
Terminator and Panb1-crtI-AOX1 Terminator transformed E.coli

The bands of Panb1-CUS-AOX1 Terminator (2000+bp), Panb1-ACC-AOX1
Terminator (3000bp), Panb1-4CL-AOX1 Terminator (2500+bp) and
Panb1-crtI-AOX1 Terminator (2500bp) from colony PCR are identical to the
theoretical lengths of 2158bp, 2832bp, 2688bp and 2437bp estimated by
the designed primer locations (promoter to terminator), which could
demonstrate that these target plasmid are successfully constructed.

Close

## Exploring the construction of plasmid for pigment synthesis

After all the expression product is detected, to explore the molecular mechanism laid behind
and to save more experience for future teams, we tried to construct the complete whole
pathways. Using seamless cloning, tentative experiments are performed by new primer for
homologous recombination.

Unfortunately, no plasmid with multiple target genes are constructed. Good news is
double-target-gene plasmid obtained. It seems that the identical promoters and terminators
lead to the much shorter product of seamless cloning than theoretical, which means the
enzymes used to join the designed homologous arms by primers falsely take some of the shared
promoters and terminators as substrates as well. Although FMO dimers were successfully
obtained, other plasmids were always shorter than expected .Then we give double-enzyme
digestion and reconnection a try but limited to the restriction sites existed in our target
genes and more fragments less odds and, well, time, no complete pathway is constructed. What
we do have now is the precious experimental experience for all of our team members, and
useful information for future teams.

Read More

##### result4

![Plasmid construction and colony PCR result of Panb1-&alpha; factor -crtE-AOX1 Terminator-Panb1-&alpha; factor -crtI-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/4/40/T--HUST-China--img--result--14.png)

Figure 14: Plasmid construction and colony PCR result of
Panb1-&alpha; factor -crtE-AOX1 Terminator-Panb1-&alpha;
factor -crtI-AOX1 Terminator transformed E.coli

The bands of Panb1-α factor-crtE-AOX1 Terminator-Panb1-α factor
-crtI-AOX1 (less than 5000bp) and is identical to the theoretical
lengths of 4574bp estimated by the designed primer locations (promoter
to terminator), which could demonstrate that these target plasmid are
successfully constructed.

![Plasmid construction and colony PCR result of Pynr071c-&alpha; factor-FMO dimer-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/0/0c/T--HUST-China--img--result--15.png)

Figure 15: Plasmid construction and colony PCR result of
Pynr071c-&alpha; factor-FMO dimer-AOX1 Terminator transformed
E.coli

The bands of FMO dimer (less than 5000bp) and is identical to the
theoretical lengths of 4740bp estimated by the designed primer locations
(promoter to terminator), which could demonstrate that these target
plasmid are successfully constructed. After serious of trails, it occurs
that when multiple repeated fragments existed, seamless cloning may
cause reduction of intended genes and of construction failure. We won't
be able to finish the construction of complete pathway when going back
for double-enzyme digestion and reconnection, which is much more
difficult and complex.

Close

# Electroporation and expression of yeast

To verify the expression of our genes and to acquire corresponding enzymes, we choose Pichia
Pastoris GS115 as chassis and using electroporation to blend our plasmids into them. Large
amount of target gene containing plasmid is extracted, then we digest them with Bgl II or
Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting
lost while being frozen. Concentration is also applied to achieve higher copy number and
higher expression level.

After electroporation of plasmid with AOX1 as promoter and those without signal peptide, we
acquire identical bands through Nickel-affinity chromatography column and SDS-PAGE.

For some of our enzymes don't have standard protocol to estimate their activity at present,
we add substrates into culturing medium accordingly to find out whether there exists active
target enzymes and do get our indigo and lycopene synthesized.

![Medium for expression with substrates From left to right: GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole;  mixture of Panb1-crtE-AOX1 Terminator, Panb1-crtB-AOX1 Terminator, Panb1-crtI-AOX1 Terminator medium with FPP](https://static.igem.org/mediawiki/2021/7/79/T--HUST-China--img--result--16.png)

Figure 16: Medium for expression with substrates From left to right: GS115 medium with
indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole; mixture of
Panb1-crtE-AOX1 Terminator, Panb1-crtB-AOX1 Terminator, Panb1-crtI-AOX1 Terminator
medium with FPP

From left to right: GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator
medium with indole; mixture of Panb1-crtE-AOX1 Terminator, Panb1-crtB-AOX1 Terminator,
Panb1-crtI-AOX1 Terminator medium with FPP

Read More

##### result5

Using E.coli for amplification, we extract and digest them with Bgl II or
Sal I to get linear plasmid, which could be integrated into yeast genome
to avoid getting lost while being frozen. Then, concentration of linear
plasmid is also applied to achieve higher copy number and higher
expression level. Several rounds of electroporation later, we
successfully get all the plasmid with AOX1 as promoter into yeast.

![Colony PCR result of yeast after electroporation](https://static.igem.org/mediawiki/2021/f/f3/T--HUST-China--img--result--17.png)

Figure 17: Colony PCR result of yeast after electroporation

The bright bands are identical to the theoretical lengths, which could
demonstrate that this target plasmid had successfully transformed into
yeast.

After confirmation from colony PCR and sequencing, we using the
successfully integrated yeast for expression. At first, we try to detect
our target protein in the supernatant since there is signal peptide. At
the same time, because the molecular weight of PepACS is only 5.08 kDa,
it is almost impossible to separate from bromophenol blue, and its
migration is also greatly affected, so the position of the band is not
accurate. In order to judge whether it was expressed or not, we first
filtered the miscellaneous proteins through 10kDa ultrafiltration tube,
then concentrated it with 3kDa ultrafiltration tube, and then detected
it by SDS-PAGE. Judge whether it is expressed successfully by whether
there is coloring near the bottom line.

![SDS-PAGE result of Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS detecetion in the supernatant](https://static.igem.org/mediawiki/2021/9/9a/T--HUST-China--img--result--18.png)

Figure 18: SDS-PAGE result of Laccase GS115 4CL LOX2 ACC pepACS
DsbC+pepACS detecetion in the supernatant

Due to glycosylation modification of yeast expression, the molecular
weight exhibited on SDS-PAGE will be larger than theoretical. Primary
detection shows that we have laccase, 4CL and ACC bands of about 75kDa,
LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a
bit larger(Laccase: 57.01 kDa; 4CL: 61.88 kDa; ACC: 63.40 kDa; LOX2:
102.88 kDa; DsbC+pepACS: 31.72 kDa) but still within explainable and
acceptable range, which could be evidence of successful expression.
There are irregular staining bands on the bromophenol blue line in the
PepACS swimming lane. Although the molecular weight is not accurate, the
successful expression can be judged according to the detection method we
designed.

After confirmation of successful secret of some of the protein, we test
the enzymatic activities of laccase and LOX2, which have standard
activity testing protocol. Because of the thin band of laccase from
SDS-PAGE, we can't be entirely sure whether it's inactive or active but
too low the concentration to be detected. So, purification through
Nickel-affinity chromatography column is used to raise its concentration
for further test of enzymatic activity.

![SDS-PAGE result of laccase after purification through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/c/c7/T--HUST-China--img--result--19.png)

Figure 19: SDS-PAGE result of laccase after purification through
Nickel-affinity chromatography column

Clear and thick band of about 75kDa is consistent to the result in the
supernatant. Higher the eluent concentration, thicker the band. This
indicates that the band of laccase in Fig18 is our target protein
instead of other impurity with a high expression level.

During the test, we find a vague band of ROX1 which couldn't be verified
as successfully expressed according to its SDS-PAGE result in the
supernatant. As the key component of our xylose responding system, the
successful expression of ROX1 is most vital to our project, so we apply
purification through Nickel-affinity chromatography column to ROX1 to
further identify its expression. Lucky us, target band is acquired and
confirmed. It feels so good to announce that our ROX1 can be expressed
like the others.

![SDS-PAGE result of ROX1 after purification through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/2/21/T--HUST-China--img--result--20.png)

Figure 20: SDS-PAGE result of ROX1 after purification through
Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located
around 55-55kDa, bigger than the theoretical 47.09kDa but still within
explainable and acceptable range of glycosylation modification. ROX1
could be confirmed as successfully expressed. Although no gradient
elution is applied, following elution result could verify this is ROX1,
with some impurity bands occurring in the first ROX1 elution.

At the same time, after several SDS-PAGE confirmation, some of our
proteins still don't want to show themselves. No band detected. We infer
that this is due to A. the low level of extracellular expression and
degradation by protease existing in the extracellular environment and B.
the common signal peptide we use-α-factor-isn't specialized according to
the unique space structure of our proteins so the mismatch causes
failure of extracellular expression. To solve this, we reconstruct
plasmids without the signal peptide and try to do intracellular
expression. This is aim at all the undetectable or low-expressed genes.

![Colony PCR result of yeast after electroporation of reconstructed plasmid without the signal peptide](https://static.igem.org/mediawiki/2021/d/db/T--HUST-China--img--result--21.png)

Figure 21: Colony PCR result of yeast after electroporation of
reconstructed plasmid without the signal peptide

The bright bands are identical to the theoretical lengths, which could
demonstrate that this target plasmid had successfully transformed into
yeast. Target genes are confirmed exist in the yeast of multiple bands,
which could be the result of polluted electroporation cup.

After verification of successful transfection, we can't test the protein
directly due to intracellular expression. So, we extract the total
protein in yeast and go for a purification through Nickel-affinity
chromatography column, then apply SDS-PAGE to separate target protein
from the large amount and various type of total protein to confirm
whether our target protein could be expressed and value its expression
level quantitatively.

![SDS-PAGE result of FMO after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/e/ed/T--HUST-China--img--result--22.png)

Figure 22: SDS-PAGE result of FMO after purification of yeast total
protein extraction product through Nickel-affinity chromatography
column

Different from impure or permeate bands, the target protein located
around 60kDa, bigger than the theoretical 53.96kDa but still within
explainable and acceptable range of glycosylation modification. FMO
could be confirmed as successfully expressed. The concentration of yeast
total protein is so high that huge amount of impure protein is included
during elution. But due to difference from impure or permeate bands, its
dark color and consistency among several times of elution, this band
could be verified as our target FMO,

![SDS-PAGE result of crtE after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/1/16/T--HUST-China--img--result--23.png)

Figure 23: SDS-PAGE result of crtE after purification of yeast total
protein extraction product through Nickel-affinity chromatography
column

Different from impure or permeate bands, the target protein located
around 50kDa, bigger than the theoretical 33.42kDa but still within
explainable and acceptable range of glycosylation modification. crtE
could be confirmed as successfully expressed.

![SDS-PAGE result of crtB after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/f/fc/T--HUST-China--img--result--24.png)

Figure 24: SDS-PAGE result of crtB after purification of yeast total
protein extraction product through Nickel-affinity chromatography
column

Different from impure or permeate bands, the target protein located
around 50kDa, bigger than the theoretical 35.30kDa but still within
explainable and acceptable range of glycosylation modification. crtB
could be confirmed as successfully expressed.

![SDS-PAGE result of crtI after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/6/6f/T--HUST-China--img--result--25.png)

Figure 25: SDS-PAGE result of crtI after purification of yeast total
protein extraction product through Nickel-affinity chromatography
column

Different from impure or permeate bands, the target protein located
around 60kDa, bigger than the theoretical 55.86kDa but still within
explainable and acceptable range of glycosylation modification. crtI
could be confirmed as successfully expressed. After confirmation of
successful expression, we add indole as substrate into culture medium of
FMO and FPP into the mixture of crtE, crtB and crtI to test whether the
expressed enzyme is active in terms of color change of the culture
medium. As for the result, we succeed in expression of enzymes and
synthesis of indigo and lycopene.

![Medium for expression with substrates From left to right: GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole;  mixture of Panb1-crtE-AOX1 Terminator, Panb1-crtB-AOX1 Terminator, Panb1-crtI-AOX1 Terminator medium with FPP](https://static.igem.org/mediawiki/2021/0/03/T--HUST-China--img--result--26.png)

Figure 26: Medium for expression with substrates From left to right:
GS115 medium with indole and FPP as control; Panb1-FMO-AOX1
Terminator medium with indole; mixture of Panb1-crtE-AOX1
Terminator, Panb1-crtB-AOX1 Terminator, Panb1-crtI-AOX1 Terminator
medium with FPP

From left to right:

GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator
medium with indole; mixture of Panb1-crtE-AOX1
Terminator,Panb1-crtB-AOX1 Terminator,Panb1-crtI-AOX1 Terminator medium
with FPP

Close

Also, In order to verify whether the catalytic effect of FMO dimer is better than FMO. We
electrotransformed the FMO dimer into yeast and tried to detect its expression and compare
it with FMO.

The color of the medium and the cell were significantly darker than FMO under the same
culture conditions. This shows that the catalytic activity of FMO dimer is stronger than
that of FMO.

![FMO dimer medium with indole after centrifugation.It is obvious that the culture medium and bacteria are dark blue.](https://static.igem.org/mediawiki/2021/3/3a/T--HUST-China--img--result--27.png)

Figure 27: FMO dimer medium with indole after centrifugation.It is obvious that the
culture medium and bacteria are dark blue.

Read More

##### result6

After electrotransformed the FMO dimer into yeast, we still used Colony
PCR to confirm the target gene is successfully transformed into the
yeast cells.

![Colony PCR result of yeast after electroporation of FMO dimer.](https://static.igem.org/mediawiki/2021/e/ef/T--HUST-China--img--result--28.png)

Figure 28: Colony PCR result of yeast after electroporation of FMO
dimer.

The bands of FMO dimer (less than 5000bp) and is identical to the
theoretical lengths of 4600bp estimated by the designed primer locations
(promoter to terminator), which could demonstrate that this target
plasmid had successfully transformed into yeast.

At first, we tried to detect the protein in the supernatant, but no
results were obtained. Because we have already experienced similar
problems. We extracted the total protein directly and go for a
purification to tested whether it was in the cell. Actually we detected
the protein this time but it was smaller than expected.

![SDS-PAGE result of FMO dimer after purification of yeast total protein extraction product through Nickel-affinity chromatography column](https://static.igem.org/mediawiki/2021/e/e6/T--HUST-China--img--result--29.png)

Figure 29: SDS-PAGE result of FMO dimer after purification of yeast
total protein extraction product through Nickel-affinity
chromatography column

Different from impure or permeate bands, the target protein located
around 60kDa, smaller than the theoretical 107.52kDa, but similar to the
theoretical molecular weight of FMO (53.96kDa).

This indicated that the FMO dimer was broken in the process of expression
or extraction. In order to confirm whether the catalytic effect is
better, we also added indole to its medium to observe its effect on
indigo synthesis.

![FMOdimer medium with indole after centrifugation](https://static.igem.org/mediawiki/2021/e/ef/T--HUST-China--img--result--28.png)

Figure 30: FMOdimer medium with indole after centrifugation

It is obvious that the culture medium and bacteria are dark blue.

The color of the medium and the cell were significantly darker than FMO
under the same culture conditions. This shows that the catalytic
activity of FMO dimer is stronger than that of FMO. So we can confirm
that the expression is FMO dimer, and its effect is better. As for the
problem of suspected fracture, we suspect that it is due to the short
connection. It will withstand a large torsional strain when folding,
resulting in its fragility, and fracture occurs in the process of
extraction or sample preparation.

Close

# Determination of enzyme avtivity

After the target strips appeared on SDS-PAGE, for the enzymes with standard enzyme activity
assay such as Laccase and LOX2, we measured their enzyme activity according to the standard
measurement methods to detect whether our enzymes are catalytically active. Laccase and LOX2
show high activity after adjusting the relevant conditions such as pH, temperature and ion
concentration

![Enzyme activity determination of Laccase](https://static.igem.org/mediawiki/2021/f/f2/T--HUST-China--img--result--31.png)

Figure 31: Enzyme activity determination of Laccase

![Enzyme activity determination of LOX2](https://static.igem.org/mediawiki/2021/0/02/T--HUST-China--img--result--32.png)

Figure 32: Enzyme activity determination of LOX2

Read More

##### result7

After target strips been detected, to measure the activity of Laccase, we
mixed 1ml of 1mmol / L ABTS solution with 1ml of centrifuged supernatant
and adjusted the final concentration of Cu2+ to 10mmol/L, and measured
its absorbance at 420nm. On this basis, we carried out determination
experiments to further explore the optimal conditions for inducing the
highest activity of Laccase.

Since the active center of Laccase is copper ion, we first explored the
copper ion concentration to be added into the culture media. Adjusted
the concentration of supplementary CuSO4 solution to 9mmol / L and
10mmol / L respectively, and measured the enzyme activity of Laccase
under the same conditions

![Effects of different concentrations of CuSO4 on enzyme activity](https://static.igem.org/mediawiki/2021/7/79/T--HUST-China--img--result--33.png)

Figure 33: Effects of different concentrations of CuSO4 on enzyme
activity

We found that the enzyme activity reaches its zenith when the
concentration of copper ion is 10mmol/L. It is probably because the
concentration of Cu2+ can't meet the need of Laccase active center when
it is too low. When the concentration is too high, it will affect the
binding of Laccase and substrate due to the existence of too much copper
ions.

We also explored the effects of pH and temperature on activity of Laccase
at the same time.

The optimum pH of Laccase was found to be acidic, therefore we prepared
buffers with pH = 3, pH = 4.8 and pH = 6.6 respectively, with 100 mM
citric acid and sodium citrate and added 1 ml buffer to control the pH
of the reaction system while maintaining the final concentration of
CuSO4 at 10 mmol / L

![Effects of different pH on enzyme activity](https://static.igem.org/mediawiki/2021/a/a0/T--HUST-China--img--result--34.png)

Figure 34: Effects of different pH on enzyme activity

After comparison, we found that among the three groups of data measured,
Laccase activity is highest at pH = 3.0, decreasing to nadir at pH =
4.8, and completely inactivated at pH = 6.6. As for the reason why the
subsequent measured value was lower than that at the beginning at pH
6.6, we speculated that it was caused by undermixing of solution when
started reading.

For the effect of temperature, we kept ABTS solution and supernatant at
10℃, 20℃ and 37℃ respectively in advance, and kept the final
concentration of CuSO4 at 10mmol / L.

![](https://static.igem.org/mediawiki/2021/6/6a/T--HUST-China--img--result--35.png)

Figure 35:

Through the analysis and comparison of the three groups of data, it is
found that under testing conditions, the activity of Laccase is the
highest when the temperature is 20℃, and the Laccase activity is
inhibited by both low and high temperatures. As for the decrease of
absorbance at the final phase of measuring enzyme activity at 10℃, it is
suspected that the low temperature has a certain impact on ABTS cationic
free radical, resulting in the decrease in absorbance

Close

# Verification of promoters

As we had successfully used Panb1 as promoter to express our protein, which can confirm that
the Panb1 promoter can work smoothly. So to verify whether our Pynr071c promoter could
initiate the expression of downstream genes, we linked GFP it, then transfect it into yeast
and test the exist of fluorescence.

![Fluorescence result of Pynr071c-GFP-AOX1 Terminator](https://static.igem.org/mediawiki/2021/d/d5/T--HUST-China--img--result--36.png)

Figure 36: Fluorescence result of Pynr071c-GFP-AOX1 Terminator

Read More

##### result8

To verify whether our Pynr071c promoter could initiate the expression of
downstream genes, we linked GFP it, then transfect it into yeast and
test the exist of fluorescence.

![Plasmid construction and colony PCR results of Pynr071c-GFP-AOX1 Terminator transformed E.coli](https://static.igem.org/mediawiki/2021/8/88/T--HUST-China--img--result--37.png)

Figure 37: Plasmid construction and colony PCR results of
Pynr071c-GFP-AOX1 Terminator transformed E.coli

The bands of Pynr071c-GFP-AOX1 Terminator from colony PCR are identical
to the theoretical lengths of 2266bp estimated by the designed primer
locations (promoter to terminator), which could demonstrate that these
target plasmid are successfully constructed.

We successfully construct the plasmid with the GFP and sequencing of
which is correct. E.coli is used for amplification as well. Extract
plasmid from E.coli, digest with Bgl II to get linear plasmid and
concentrate for electroporation of yeast.

![Colony PCR result of yeast after electroporation. The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.](https://static.igem.org/mediawiki/2021/3/34/T--HUST-China--img--result--38.png)

Figure 38: Colony PCR result of yeast after electroporation. The
bright bands are identical to the theoretical lengths, which could
demonstrate that this target plasmid had successfully transformed
into yeast.

We induce the plasmid with confirmation of colony PCR to express. Adding
xylose everyday for xylose induced promoter Pynr071C to induce synthesis
of GFP. Using fluorescence microscopy to detect the exist of green
fluorescence, which indicates whether our promoters function regularly.

![Fluorescence result of Pynr071c-GFP-AOX1 Terminator](https://static.igem.org/mediawiki/2021/4/42/T--HUST-China--img--result--39.png)

Figure 39: Fluorescence result of Pynr071c-GFP-AOX1 Terminator

Close

# The Experiment Standard

We measured the standard curves of three pigments before using them for hair dyeing
experiment. We also found that the amount of melanin contained in hair can have a
significant effect on hair dyeing outcomes. Therefore, we define different colors of hair
based on bleaching.

Read More

##### result9

![Curcumin concentration standard curve](https://static.igem.org/mediawiki/2021/8/89/T--HUST-China--img--result--result-curcumin_concentration_sc.png)

Figure 40: Curcumin concentration standard curve

![Indigo concentration standard curve](https://static.igem.org/mediawiki/2021/4/42/T--HUST-China--img--result--result-Indigo_concentration_sc.png)

Figure 41: Indigo concentration standard curve

![sLycopene concentration standard curve](https://static.igem.org/mediawiki/2021/5/55/T--HUST-China--img--result--result-Lycopene_concentration_sc.png)

Figure 42: sLycopene concentration standard curve

![Series of bleached hair](https://static.igem.org/mediawiki/2021/e/e9/T--HUST-China--img--result--result-the_sample_from_shop.png)

Figure 43: Series of bleached hair

For the convenience of dyeing, we buy a series of bleached hair. The
lightest color is the 9°hair, and the darkest is the 4 ° hair.

Close

# The Best Condition of hair dye

We have gained the best dye conditions of three kinds of hair dye(indigo, curcumin and
lycopene) at a certain concentration. Under optimal conditions, we dyed 4-9 degrees of hair
to get a series of dyeing discs. And we found that as for the three colors selected for the
experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.

Read More

##### result10

Chart of the best condition of hair dye

| Dye/Condition | temperature | Dyeing aid ingredients | concentration(g/L) | comment |
| --- | --- | --- | --- | --- |
| indigo 2min | Room temperature | none | 2 | The color deepens significantly while dyeing for multiple times |
| curcumin 30min | 50℃ | Ethyl alcohol | 0.5 |  |
| lycopene 30min | Room temperature | alum | 2 |  |

*The best dyeing condition*

The exhibition of hair dyed on the best condition

Under the best conditions, we dyed the hair from 4 degree to 9 degree,
and got a series of colors. It is found that it only needed to be
bleached to 8 degree so that the hair would show a bright color for all
three kinds of dye.

As to lycopene hair, 8 or 9 degree hair was red, 7 degree(or below) hair
was brownish, and the longer the hair was dyed, the redder it would
become.

As to indigo hair, 7 to 9 degree hair would become blue. As the dyeing
time goes, the color would turn blue from an indigo color; 5 to 6 degree
hair would be dyed to celadon, and 4 degree hair was still brown.

![The dyeing results of lycopene(room temperature, adding alum, 2g/L). From left to right: 9°;(5,10,30min), 8°(5,10,30min), 7°(5,10,30min), 6°(5,10,30min), 5°(10,30min), 4°(5,10,30min)](https://static.igem.org/mediawiki/2021/9/92/T--HUST-China--img--result--result-the_best_condition_for_lycopene_to_dye_.png)

Figure 44: The dyeing results of lycopene(room temperature, adding
alum, 2g/L). From left to right: 9°(5,10,30min),
8°(5,10,30min), 7°(5,10,30min),
6°(5,10,30min), 5°(10,30min),
4°(5,10,30min)

![The dyeing results of indigo (room temperature, 2g/L). From left to right: 9°(0.5,2,6min), 8°(0.5,2,6min), 7°(0.5,2,6min), 6°(0.5,2,6min), 5°(0.5,2,6min), 4°(0.5,2,6min)](https://static.igem.org/mediawiki/2021/8/89/T--HUST-China--img--result--result-the_best_condition_for_indigo_to_dye.png)

Figure 45: The dyeing results of indigo (room temperature, 2g/L).
From left to right: 9°(0.5,2,6min),
8°(0.5,2,6min), 7°(0.5,2,6min),
6°(0.5,2,6min), 5°(0.5,2,6min),
4°(0.5,2,6min)

![The dyeing results of indigo(room temperature, ethanol added, 0.5g/L). From left to right: 10min(9-4°), 30min(9-4°)8°](https://static.igem.org/mediawiki/2021/e/e4/T--HUST-China--img--result--result-the_best_condition_for_turmeric_to_dye.png)

Figure 46: The dyeing results of indigo(room temperature, ethanol
added, 0.5g/L). From left to right: 10min(9-4°),
30min(9-4°)8°

Problems occurred during the research:

**Lycopene**

Problem: No literature on coloring fabrics or hair with lycopene

Solution: We conducted a gradient experiment (0.5, 1, 2, 5 g/L) to
explore the effective concentration of lycopene for hair dyeing.
Finally, 2g/L of lycopene is selected, at which concentration the dye
fluid will not be too viscous, and has a better dyeing effect as the
picture below shows.

![(From left to right: 0.5(30 min), 1(5, 10, 30 min), 2(5, 10, 30min), 5(5, 10, 30min)g/L of lycopene)](https://static.igem.org/mediawiki/2021/3/35/T--HUST-China--img--result--result-the_effort_to_confirm_the_best_concentration_for_lycopene_dying.png)

Figure 47: (From left to right: 0.5(30 min), 1(5, 10, 30 min), 2(5,
10, 30min), 5(5, 10, 30min)g/L of lycopene)

Problem: Lycopene dye the hair with a low efficiency, a low color
fastness, and a constantly discoloring process when the hair is showered
by water.

Solution: We looked up the data and selected three eco-friendly color
aids (alum, potassium tartrate, citric acid). Through direct color
comparison and elution experiments, we found that alum can significantly
improve the coloration rate and color fastness of lycopene.

![(From left to right, 1st-4th groups: alum(30min), potassium tartrate(40min), citric acid(40 min), no color aids(40min); 5th-8th groups: the 1st-4th hair after washing 7 times)](https://static.igem.org/mediawiki/2021/6/6d/T--HUST-China--img--result--result-the_effort_for_confirming_the_maintaining_degree_of_lycopene.png)

Figure 48: (From left to right, 1st-4th groups: alum(30min),
potassium tartrate(40min), citric acid(40 min), no color
aids(40min); 5th-8th groups: the 1st-4th hair after washing 7 times)

![Color fastness test of lycopene](https://static.igem.org/mediawiki/2021/c/ca/T--HUST-China--img--result--result-Color_fastness_test_of_lycopene.png)

Figure 49: Color fastness test of lycopene

**Curcumin**

Problem: The coloration rate of the curcumin aqueduct solution is low

Solution: We carried out the same dye addition and elution experiments as
lycopene, and found that alum, potassium tartarate and citric acid
cannot improve the coloring effect of curcumin. The data showed that
curcumin is soluble in ethanol, and the optimal coloring temperature is
50 degree, so we adjusted the solvent to 33% ethanol solution. The
process of coloring was conducted in the oven at 50 degrees, and the
results showed that the coloring effect significantly improved.

![Dyeing result of curcumin made in different solvents(30 min, 50°C). Left: water solution, Right: 33% ethanol alcohol solution](https://static.igem.org/mediawiki/2021/c/c6/T--HUST-China--img--result--result-Dyeing_result_of_curcumin_made_in_different_solvents.png)

Figure 50: Dyeing result of curcumin made in different solvents(30
min, 50°C). Left: water solution, Right: 33% ethanol
alcohol solution

**Indigo** Problem: At the beginning of the experiment, we
found that indigo could not be successfully adsorbed by the hair.

Solution: We looked up the data and found that in the printing and dyeing
industry, it is necessary to restore the low solubility of indigo to
water-soluble leucoindigo, which is used to color. We used the textile
reducing agent - sodium sulfate to reduce indigo dye, and it came out to
be successful.

Considering the safety of hair dyeing, we tried to use glucose as a
reducing agent, by which we also obtained effective dyeing fluids. The
method of using is indigo: glucose: caustic soda: 1:6:1, react for 15
min under 50°C. In the final product, we expected that yeast could
conduct oxygen-free fermentation to reduce indigo with ethanol.

Problem: After dyeing your hair indigo, your hair becomes noticeably
brittle.

Solution: Testing the average pull limit of a single hair, we found that
the hair pull limit for indigo treatment was indeed significantly lower
than that of normal hair. We assumed that this is related to the
destruction of hair by alkaline substances. Therefore, we shorten the
processing time of indigo dye, and finally designed to dye 2min at a
time, repeated the procedure five times to achieve a saturation effect.

Close

# Hair stress experiment

We used a micro scale experimental instrument to quantitatively evaluate the changes in
mechanical properties of hair after hair dyeing. The results show that Indigo and lycopene
as dyes have no obvious damage to hair. Curcumin has a repairing effect on hair.

![Tensile Stress Strain Curves](https://static.igem.org/mediawiki/2021/e/ec/T--HUST-China--img--result--result-Tensile_Stress_Strain_Curves.png)

Figure 51: Tensile Stress Strain Curves

Read More

##### result11

After hair dyeing, the mechanical properties of hair in each group
changed to varying degrees. For example, the hair softens after a long
treatment of the indigo solution becomes soft, elastic but easy to
break. So we borrowed the micro scale experimental instrument
independently developed by Professor Liu.

During the pre-experiment, we found there is no significant difference in
ultimate stress between traditional dyeing hair and the untreated one,
and we believe it's the added hair repair component causes the slug.
Indigo and lycopene as dyes also have no obvious damage to hair. What's
more, the yang's modulus of curcumin treatment hair is significantly
higher. So we believe it keeps hair tough.

![Indigo dyed hair being treated with micro tensile / micro torsional mechanical tester](https://static.igem.org/mediawiki/2021/b/b5/T--HUST-China--img--result--result-Indigo_dyed_hair_being_treated_with_micro_tensile_micro_torsional_mechanical_tester.png)

Figure 52: Indigo dyed hair being treated with micro tensile / micro
torsional mechanical tester

Close

# Compound hair dye paste

After finishing the solution experiment, we try to mix the natural pigment into a dye that
can be applied directly to the hair. At present, lycopene dye and curcumin dye with NO.1
cream matrix as carrier are obtained, and natural essence is added to improve the odor of
dye paste. Indigo is an oxidizing dye with special properties, so we designed a timely
fermenter. In this way, we can use our product right now when indigo is produced and reduced
to indigo white.

![The expected product](https://static.igem.org/mediawiki/2021/5/55/T--HUST-China--img--result--result-the_expected_product.png)

Figure 53: The expected product

Read More

##### result12

**Lycopene**

Difficulty: when we use lycopene paste to dye hair, it is not red but
orange

Solution: by analyzing the cream formula, we think what is causing this
problem is sodium sulfite. We add sodium sulfite to prevent further
oxidation of the pigment, but it may also reduce and fade the pigment.
The solution experiment proved our conjecture. A new lycopene dye that
doesn't contain sodium sulfite found its way to red hair.

**Lycopene dying cream**

| Ingredient | Content |
| --- | --- |
| Cream matrix | 100g |
| Sodium sulfite | 0.2g |
| Absolute ethanol | 1mL |
| pH 6.8 phosphate buffer | 1mL |
| Solid paraffin | 1 drop or not |
| Essence | 1 drop |
| 20% Lycopene | 5g |
| Alum | 0.6g |

**Curcumin** EDTA is added to the curcumin paste to prevent
metal ions from interfering with the effect -- for example, Fe3 +
producing a reddish-brown tint.

**Curcumin dying cream**

| Ingredient | Content |
| --- | --- |
| Cream matrix | 100g |
| EDTA | 0.2g |
| Sodium sulfite | 0.1g |
| Absolute ethanol | 1mL |
| pH 6.8 phosphate buffer | 1mL |
| Isopropyl alcohol | 2mL |
| Solid paraffin | 1 drop or not |
| Essence | 1 drop |
| curcumin | 0.5g |

![The result of curcumin staining cream. 9 ° from left to right. Hair was dyed at room temperature for 30min, 60min and 0min; Dyeing at 50 &#8451; for 10min and 30min](https://static.igem.org/mediawiki/2021/a/a0/T--HUST-China--img--result--result-the_different_effort_of_termeric_dying_at_changing_tep.%26time.png)

Figure 54: The result of curcumin staining cream. 9 ° from
left to right. Hair was dyed at room temperature for 30min, 60min
and 0min; Dyeing at 50 &#8451; for 10min and 30min

The result of curcumin staining cream. 9 ° from left to right. Hair was
dyed at room temperature for 30min, 60min and 0min; Dyeing at 50 ℃ for
10min and 30min

**Indigo:**

Difficult: we can make indigo paste, but the hair does not dye well.

Solution: Indigo is a water-soluble component, and need to be oxidized to
indigo after fixing to the hair. With water-in-oil paste as the matrix,
indigo white can not fully enter the interior of the hair, and the oily
substances in the matrix and excessive reductant prevent indigo white
from oxidation in the hair, resulting in no effective coloring.

So we decided to design a timely manner in which indigo could be produced
and used at the same time. Therefore, we consider that indigo dye can be
produced and used in time -- the direct production of indigo by yeast,
and the production of indigo solution as a dye in time.

For this idea, we dye indigo solution directly on the hair and find that
it can be painted, but it can not color the hair evenly. So we designed
a hair dye comb to make it possible to evenly smear indigo cryptosomes
on the hair. The matching device is a timely fermentation tank, which
can meet the needs of users with our engineering bacteria as raw
materials, timely production and timely use of indigo white. For
detailed information, please refer to [Hardware](https://2021.igem.org/Team%3AHUST-China/Results/Hardware)
part.

![the different effort of indigo](https://static.igem.org/mediawiki/2021/5/5b/T--HUST-China--img--result--result-the_different_effort_of_indigo_dying_at_changing_tep.%26time.png)

Figure 55: the different effort of indigo

Close

# Color fastness test

Color fastness is an important aspect to measure the effect of dye, so we design a set of
elution scheme and test the color fastness of three kinds of natural pigment dye products
and the same color traditional dye paste. The results showed that the color fastness of the
natural pigment dyes was better than that of the traditional dyes.

![The comparison of biological dying with traditional dying](https://static.igem.org/mediawiki/2021/e/ee/T--HUST-China--img--result--result-the_comparison_of_biological_dying_with_traditional_dying.png)

Figure 56: The comparison of biological dying with traditional dying

Read More

##### result13

The experiment of color fastness:

Compound 25g/L shampoo solution. Add 10 ML shampoo solution and a bunch
of hair in a 15 ML centrifuge tube for 5 min at 100 r/min. The
experiment was repeated seven times. The absorbance of the eluent at the
maximum absorption band of the corresponding color was measured and the
curve was drawn. Results as shown in figure, the single elution amount
of three kinds of natural pigment was less than that of the same color
traditional dyes, and the color change after 7 times elution was also
less than that of the traditional dyes. Furthermore, curcumin dyeing
cream treatment of hair is almost non-decolorization.

Close

# Toxicological experiment of broad bean root tip

In order to test the safety of our dye paste, we carried out a toxicology experiment of broad
bean root tip. The rate of micronucleus in root tip cells on broad bean was measured. The
higher the micronucleus rate was, the higher the teratogenic rate was. The results showed
that the teratogenicity of our product was significantly lower than that of the two
traditional hair dyes on the market, indicating that our product was less toxic.

![Micronucleus Frequency](https://static.igem.org/mediawiki/2021/0/0a/T--HUST-China--img--result--result-micronucleus_frequency.png)

Figure 57: Micronucleus Frequency

Read More

##### result14

Micronucleus (MCN) is an abnormal structure in eukaryotic cells, which is
often produced by the action of radiation or chemical drugs. In the
intercellular phase, the Micronuclei were round or oval, free from the
main nucleus, the size should be less than 1/3 of the main nucleus. Only
the eucaryotic test system can directly predict the genetic damage of
mutagens to human beings and other higher organisms. In this respect,
the micronucleus test is an ideal method. And some studies show that the
plant micronucleus test and the animal test have a coincidence rate of
up to 99%. Micronucleus test using broad bean root tip as experimental
material can accurately show the effect of various treatments on induced
distortion.

![Patricles](https://static.igem.org/mediawiki/2021/d/d7/T--HUST-China--img--result--result-patricles.png)

Figure 58: Patricles

Specific experimental process:

1. We bought 2 catties of sun-dried broad bean seeds, put them in a wet
   porcelain tray after soaking in water overnight. After 2 days, about
   95% of the bean seeds grew roots about 1-2 cm long.
2. We divided 10 broad beans into 1 group. Each group of root tips were
   immersed in the following concentrations of dye paste solution for
   24 hours.
3. We took the broad beans soaked in the dye solution, washed them with
   distilled water, then put them into distilled water and cultured for
   24 hours.
4. The root tips of Vicia Faba were cut off and fixed in 15-fold Kano
   Solution (anhydrous ethanol: Glacial Acetic acid = 3:1) for 24
   hours.
5. We took out the root tip, washed it with distilled water, put it
   into 1 m hydrochloric acid solution, and reacted at 60 °C for 20
   minutes to soften the root tip. The root tip was cut 1 mm, smashed
   on the slide, stained with the modified carbolic acid red dye for 15
   min, covered with the slide, then flattened and observed under the
   microscope.
6. We first find the meristematic cells, which are closely arranged and
   square, look at 1000 cells, and record the number of cells with
   micronucleus. Micronucleus rate = micronucleus cell number/1000 ??
   1000%

| Indigo | Traditional blue | Lycopene | Traditional red | curcumin | Negative control:Distilled water | Positive control:1g/L CuSO4 |
| --- | --- | --- | --- | --- | --- | --- |
| 9g/L | 9g/L | 9g/L | 9g/L | 9g/L |  |  |
| 6g/L | 6g/L | 6g/L | 6g/L | 6g/L |  |  |
| 4.5g/L | 4.5g/L | 4.5g/L | 4.5g/L | 4.5g/L |  |  |
| 3g/L | 3g/L | 3g/L | 3g/L | 3g/L |  |  |
| 1.5g/L | 1.5g/L | 1.5g/L | 1.5g/L | 1.5g/L |  |  |

![Broad bean dyed with pigment](https://static.igem.org/mediawiki/2021/7/70/T--HUST-China--img--result--result-broad_bean_dyed_with_pigment.png)

Figure 59: Broad bean dyed with pigment

Due to the huge workload of counting micronucleus, we tried to develop counting algorithm. We
use the algorithm framework of Yolov5 to implement what is available on the author's Github
or Model section, which is not covered here. We selected the typical normal nuclei and
Micronuclei from the frame of the microscope. There were about 200 chromatin images and 50
micronuclei images in the normal nuclei. We trained a recognition model by using these
images as training set. With this model, a large number of imaging results can be analyzed
in a short time. We use this model to count micronuclei and measure the cytotoxicity of
various substances. The results show that this model is in good agreement with the commonly
used counting standards under the experimental conditions. It can be used to compare the
difference in toxicity between different substances. For detailed information, please refer
to [Model](https://2021.igem.org/Team%3AHUST-China/Results/Model) part.

Close

# Color stability testing

We would expose our hair dyed with our dye paste and two chemical dyes
(red and blue) to constant bright light, take pictures before and after lighting, and
calculate the degree of discoloration. The result shows that the light stability of our dye
paste is slightly less than that of the chemical dye paste, but the difference is invisible
to the naked eye in the actual application scene, so the color of our dye paste can exist
stably in the natural light, it's still very practical.

![Light exposure's influence on dying](https://static.igem.org/mediawiki/2021/f/ff/T--HUST-China--img--result--result-light_exposure%27s_influence_on_dying_.png)

Figure 60: Light exposure's influence on dying

Read More

##### result15

What we did: We cut a bunch of hair in half, pasted half of it onto a
piece of cardboard, and exposed it to a constant, bright light for 11
days. Our lights are LED lights and sodium lamps, cycle for 22 hours of
light and 2 hours of darkness alternately. At the end of the
illumination, half of the hair without illumination was pasted on, and
the fade index was analyzed by the algorithm. Considering the change of
RGB values, it is difficult to determine the model parameters, and there
may be multi-value based on the prediction results given by the neural
network. We choose the linear combination of RGB to calculate the gray
value, and take the gray value as the index of the concentration. It can
be used as an index to evaluate the degree of fading by calculating the
change of gray difference between the two groups. This indicator is not
linear and therefore should not be understood as an indicator in a
percentage sense. On a scale of one to one, the higher the score, the
deeper the discoloration. There was no significant difference between
indigo and chemical blue, the scores of indigo were 0.178 and 0.161,
respectively. Chemical red faded more slowly than lycopene, with a score
of 0.186 and 0.313, respectively.

The process of exploration: When we first carried out the strong light
experiment, we irradiated single hair and only treated it for 2 days.
The results showed that the dyeing of each hair in the same strand was
very different due to the uneven dyeing, and the treatment time is so
short that almost all the hair remains the same. In the second
experiment, we took a photo of the hair in front of the light and
compared it with the photo after the light exposure. The light exposure
was extended to 7 days. Although the discoloration was detectable this
time, it varied widely between the groups, suggesting that one hair's
discoloration was highly accidental. So on the third occasion, we
decided to reduce the chance by increasing the amount of hair, using a
single strand of hair, and extending the light again to get the
difference.

Close

# Color matching experiment and Sumu experiment

By adding color aids, mixing two dyes or secondary coloring, we get more hair colors. Now, we
have different brightness of the rainbow seven colors and gray, red, brown, tawny and other
colors. At the same time, we also used hematoxylin as a dye to explore a series of dyeing.
Hematoxylin itself can be brown, add alum can be red, add alkali can be purple. Hematoxylin
's synthetic biology production will be a future extension project.

![Olympic rings and Cartoon kid](https://static.igem.org/mediawiki/2021/0/04/T--HUST-China--img--result--result-Olympic_rings_and_Cartoon_kid.png)

Figure 61: Olympic rings and Cartoon kid

Read More

##### result16

A coloring list of the three pigments

| Picture | Color | Coloring method |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2021/d/d9/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_red.png) | Red | 8 ° hair in the room temperature treated with lycopene for 30 minutes |
| ![](https://static.igem.org/mediawiki/2021/e/e9/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_orange.png) | Orange | 9 ° hair in the room temperature treated with curcumin for 30 minutes then treated with lycopene for another 30 minutes |
| ![](https://static.igem.org/mediawiki/2021/6/67/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_light_orange.png) | Light orange | 9 ° hair in the room temperature treated with curcumin mixed with lycopene (2:1) for 30 minutes |
| ![](https://static.igem.org/mediawiki/2021/1/17/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_yellow.png) | Yellow | 9 ° hair in 50 ℃ treated with curcumin for 30 minutes |
| ![](https://static.igem.org/mediawiki/2021/f/fc/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_green.png) | Green | 7 ° hair in the room temperature treated with indigo for 2 minutes |
| ![](https://static.igem.org/mediawiki/2021/2/29/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_cyan.png) | Cyan | 9 ° hair in the room temperature treated with indigo for 30 seconds |
| ![](https://static.igem.org/mediawiki/2021/8/89/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_blue.png) | Blue | 9 ° hair in the room temperature treated with indigo for 2 minutes |
| ![](https://static.igem.org/mediawiki/2021/7/78/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_purple.png) | Purple | 9 ° hair in the room temperature treated with indigo for 5 minutes then treated with lycopene for another 10 minutes |
| ![](https://static.igem.org/mediawiki/2021/0/0b/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_grey.png) | Gray | 9 ° hair in the room temperature treated with lycopene for 5 minutes then treated with indigo for another 30 seconds |
| ![](https://static.igem.org/mediawiki/2021/5/5b/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_reddish_brown.png) | Reddish brown | 6 ° hair in the room temperature treated with lycopene for 10 minutes |
| ![](https://static.igem.org/mediawiki/2021/8/8b/T--HUST-China--img--result--result-the_teaching_of_dying_exmples_tan.png) | Tan | 9 ° hair in the room temperature added tannic acid treated with curcumin for 10 minutes |

Close

![the coloring effect of hematoxylin under various conditions](https://static.igem.org/mediawiki/2021/7/7b/T--HUST-China--img--result--result-the_overview_of_all_possible_colors.png)

Figure 62: the coloring effect of hematoxylin under various conditions

# Experiment of short peptide perm

## Short peptides perming hair experiment

![comparison of short peptides and water](https://static.igem.org/mediawiki/2021/6/61/T--HUST-China--img--result--result-comparison_of_short_peptides_and_water.png)

Figure 63: comparison of short peptides and water

We used 0.1% and 0.01% short peptides for perm experiment, and set chemical perm and up water
perm as control group. The perming effect of short peptide is more obvious than that of
ultra-pure water, which shows that in alkaline and 50° conditions, the hair cuticle is
opened and the longer react time it takes, the better perming result we get. And after a
series experiment, we found that hair dealt with SDS can have more obvious curl effect.

![comparison of short peptides with different mixture with other chemicals](https://static.igem.org/mediawiki/2021/7/78/T--HUST-China--img--result--result-comparison_of_short_peptides_with_different_mixture_with_other_chemicals.png)

Figure 64: comparison of short peptides with different mixture with other chemicals

Read More

##### result17

| Chemical perm agent(bought online) | Ultra-pure water | 0.001% short peptide solution | 0.1% short peptide solution |
| --- | --- | --- | --- |
| 500ul | | | |
| Immerse hair(washed and dried) 20min | | | |

The experimental results showed that the Chemical perm agent had the most
obvious effect, while others didn't have any obvious perming effect.
We've considered 3 reasons why short peptides have no obvious effect:

(1) Maybe the peptides didn't get into hair cuticle

(2) The time for short peptides to form disulfide bonds wasn't enough

(3) The reaction temperature is not enough.

So, we get another exploring experiment.

Time and Temperature:

(1)Drop a drop of 3M NaOH into 0.01% short peptides solution. [PH test
paper: PH=10]

(2)Control experiment: drop a drop of 3M NaOH into UP water

(3)Both react at 50° for 1h.

Re-explore a better condition for short peptides'perming hair

1)immerse 10 hairs in SDS 3 times to remove charges on hair surface.

2)Wash and put them in 0.01% short peptides solution

3)Control experiment: Up water, the subsequent processing steps are the
same as 1)2)

4)both:50°, 1h

Close

# Perming hair stress experiment-9

We used different kinds of perms for hairs with 2 groups of different hair, and the hair
strains will be damaged after perming. So we designed and conducted stress experiments to
test the mechanics of the hair after perming. The figure below shows the experiment data, by
which we can know that perming by short peptides will have very little damage to the hair,
and short peptide perm damage is less than chemical perm. Moreover, the results of the
damaged hair groups showed that the damage of short peptide perm is much smaller than that
of chemical perm. Hence, we speculated that perming procedure would cause some damage to
hair, but the use of short peptide perm could be used to repair the damaged hair.

### Healthy hair stress test results (Horizontal: the deformation length; Ordinate: pull force

![Healthy hair stress test results (Horizontal:  the deformation length; Ordinate: pull force)](https://static.igem.org/mediawiki/2021/4/4b/T--HUST-China--img--result--result-healthy_hair_stress_test_result.png)

Figure 65: Healthy hair stress test results (Horizontal: the deformation length;
Ordinate: pull force)

It is shown that the hair of the short peptide perm group changes in shape under constantly
increasing pull force, and most hair strains can withstand more pull than hair permed by
chemical perms. This suggests that short peptide perms do less damage to hair performance
than chemical perms. According to the experimental results, we judge that the concentration
of short peptides has little effect on the results.

### Damaged hair stress test results (Horizontal: the maximum length; Ordinate: pull force)

![Damaged hair stress test results (Horizontal: the maximum length; Ordinate: pull force](https://static.igem.org/mediawiki/2021/e/e9/T--HUST-China--img--result--result-damaged_hair_stress_test_result.png)

Figure 66: Damaged hair stress test results (Horizontal: the maximum length; Ordinate:
pull force

This set of data comes from a pulling test of damaged hair. It is shown in this figure that
both sets of hairs that use short peptides for perm can withstand more pull than the hairs
after chemical perm. And there isn't much difference between the results of short peptide
group and that of the water group. We speculate that short peptides can by some means repair
the damaged hair, which also portrays the excellent performance of short peptide perm that
it can protect the hair and maintain the hair quality.

# Microscopic effects of PepACS on hair

We used scanning electron microscopy to look at healthy hair, bleached hair and bleached hair
treated with the PepACS. The results showed that compared with normal hair, the surface of
bleached hair was obviously damaged, but no obvious damage was observed after treatment with
PepACS, indicating that PepACS may have a certain repair effect, which can be further
studied in the future.

Read More

##### result18

We soaked the damaged hair with 0.1% PepACS solution and reacted at 50℃
for 3 hours to obtain the damaged hair samples treated with PepACS. No
surface damage was clearly observed under scanning electron microscopy
after the PepACS treatment, but given the differences between the same
batch of damaged hair, we cannot say that the PepACS has a repairing
effect. We expect to continue to carry out relevant experiments in the
future.

![From left to right: healthy hair,bleached hair,bleached hair treated with the PepACS](https://static.igem.org/mediawiki/2021/4/4b/T--HUST-China--img--result--result-elect.png)

Figure 67: From left to right: healthy hair,bleached hair,bleached
hair treated with the PepACS

Close

![](https://static.igem.org/mediawiki/2021/2/28/T--HUST-China--img--girl.png)

Wow! That’s eye-opening! I guess the old saying that “Rome was not build in a
day” does make sense. All those results require accumulation, which is recorded
in the [notebook](https://2021.igem.org/Team%3AHUST-China/Notebook).

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