iGEM 2023 — Team um-macau

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering{% endblock %}
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{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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* Engineering
* [Primary design](#P1)
* [Secondary design](#P2)
* [Further study](#P3)

# Primary design

Adhesion module

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-1.png)

Figure 2.2.1 Biobircks of adhesion primary design, ① pSB-Pasr-cp19k-mSandy; ② pSB-J23119-cp19k-mSandy; ③
pSB-Pasr-mSandy-Control; ④ pSB-J23119-mSandy-Control. Our key adhesion protein is cp19k (BBa\_ 4716014), the 19 kDa
cement protein, was extracted from Pollicipes pollicipes and have an adhesion effect. We put its genetic sequence on the
cell membrane of E. coli in the hope that it will help the bacteria adhere; It's like glue, sticking bacteria to the
intestines.

When choosing our key protein, the adhesion protein, we saw that cp19k, as a new type of adhesion protein, not only has
a strong adhesion ability, but also can self-assemble to complete adhesion, which is very simple (compared to other
Adhesion proteins - may require other enzyme catalysis to have an adhesion effect).

The asr promoter (or Pasr promoter)
In view of the immune overstimulation in the intestine caused by 33-mer becoming an antigen, which leads to celiac
disease, we try to place engineered bacteria that secretes gluten enzyme into the intestine to alleviate this immune
response; the first step should be to ensure that the bacteria can stay in the intestine- colonization.

The first question to ensure colonization is: where to colonize? Because the immune response caused by 33-mer is mainly
in the small intestine, we want to break down the 33-mer by gluten enzyme before it reaches the small intestine - at
least before it causes a comprehensive immune response. Therefore, we chose to let the engineered bacteria colonize the duodenum in the front part of the small intestine.

However, the pH fluctuates greatly at this location and is acidic, so in the end we chose the asr promoter induced by an
acidic environment to express the adhesion protein cp19k.

GS linker
While we were performing the literature research of cp19k, we thought that a GS linker should be added between bacteria
and cp19k to provide more space for cp19k to stretch and show its adhesion ability; otherwise, cp19k is likely to stick
directly to the surface of the bacteria because there is not enough space to stretch, thereby weakening the ability of
bacteria to adhere to the intestine - imagine when you tear off a long piece of tape ready to use, the long tape will be
very easy to stick to our hands first instead of where we want to stick - GS linker is like a stick to pick the adhesion
protein cp19k away, reducing the probability of cp19k all sticking to the bacteria.

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-2.png)

Figure 2.2.2 The work of GS linker.

mSandy (mSandy2)
This is a new type of protein, and we want to use this reporter protein, which may be brighter than mCherry, to show our
recombinant protein expression, hoping to aid our experiments. Because cp19k with a size of 519 bp may be expressed at a
low level, a reporter protein with too little brightness may not be detected, so ultimately we intend to use mSandy.

Release module

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-3.png)

Figure 2.2.3 Biobircks of release primary design, ① pET-Pasr-Fvp-sfGFP; ② pET-Pasr-PEP-sfGFP; ③ pET-J23119-Fvp-sfGFP;
④pET-J23119-Fvp-sfGFP; ⑤ pET-Pasr-sfGFP-Control; ⑥ pET-J23119-sfGFP-Control. Our key proteins are gluten enzymes (Fvp
and PEP); The asr promoter(Pasr) expression protein is then still selected.

Gluten enzyme There are a lot of oral gluten enzymes for celiac disease, and we start with oral enzymes to find the protein we want.
At present, the more widely used on the market are AN-PEP and PEP enzymes, and we can find these two enzymes from the
market or from a large number of studies. However, these two enzymes also have some defects, such as (1) they cannot
adapt to the environment of pH instability in the gastrointestinal tract (close to the stomach) - we know that enzymes
are extremely sensitive to changes in pH, and even a slight change in acidity may greatly reduce the activity of
enzymes; (2) The decomposition efficiency of gluten is low, if most of the 33-mer cannot be removed in time due to low
decomposition efficiency, it will still cause the patient's immune response. In the end, we chose Fvp and PEP as
the secreted proteins of engineered bacteria as the core proteins of our treatment. PEP is resistant to petroprotein
hydrolysis and adapts to acidic environment (pH = 2-3 is still stable and active); Fvp has more pairs of 33-mer pairs of
shear sites, giving it efficient decomposition efficiency; Selected by us.

# Secondary design

Adhesion module

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-4.png)

Figure 2.2.4 Biobircks of adhesion secondary design. When we were detecting cp19k expression with
pSB-J23119-cp19k-mSandy, we found a problem with J23119.

|  |  |
| --- | --- |
| pSB-J23119-mSandy-Control: | g.2055\_2056del |
| pSB-J23119-cp19k-mSandy-Control: | g.2055\_2071del |

Then we modified the plasmid with primer and got a new, correct plasmid; Finally, perform our experiment with the
correct plasmid.

Release module

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-5.png)

Figure 2.2.5 Biobircks of release primary design. ① pET-NorR-ProA-PnorV-Fvp-his; ② pET-NorR-ProA-PnorV-Fvp-his; ③
pET-PnorR-ProA-PnorV-his-Control. The key protein still is our gluten enzymes; The highlight is the NO sensor, which was
added by paper study. Another small improvement is the use of sfGFP reporter proteins into his tags for subsequent protein
expression detection - because the sequence of sfGFP proteins is too long, resulting in a large size of the entire
plasmid, which can seriously affect the expression of key proteins.

NO sensor
While maintaining the original plan, we are also exploring more targeted and sensitive activation switches for celiac
disease conditions; After a period of literature review and understanding of similar projects by previous iGEM teams, we
decided to conduct the first iteration of our release module through the relationship between celiac disease and
intestinal inflammation, and finally chose the most suitable module for NO sensor. Here's a brief description of how it
starts.

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-6.gif)

Figure 2.2.6 NO sensor mechanism. Molecular mechanism of NorR-mediated σ54-dependent transcriptional activation. (A)
PnorV promoter in its off state. Integration host factors (yellow) bend DNA with a near 160° sharp angle to allow the
NorR (Red) homohexamer to come in contact with the σ factor at its binding sites (−24 and −12). NorR binding with the
upstream enhancer sequence is shown in the pink block. In the absence of the inducer, the RNAp complex is closed from
transcription. Upon nitric oxide binding, NorR exposes its AAA+ ATPase domain and activates σ into its active form for
DNA opening and transcriptional initiation. (B) The way NorR interacts with nitric oxides is through its nonhaem iron
center (blue), which signals a reversible conformation change that opens up the ATPase domain upon binding.

# Further study:

Adhesion module

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/2-2-7.png)

Figure 2.2.7 Biobrick of the new part. We tried to compare the adhesion effect of cp19k with mefp5, so we replaced the
key protein with mefp5 to become a new plasmid.

When we conducted cp19k-related experiments, we found that the UM\_Macau iGEM team in 2019 also used an adhesion protein
with strong adhesion ability-mefp5, and the relevant literature resources are rich and the application scenarios seem to
be more extensive. So we want to try to compare cp19k and mefp5 in future studies to observe the adhesion effect of the
two adhesion proteins in the intestinal environment. However, due to the short schedule and lack of time, we only
completed the design of the plasmid and did not fully carry out the experiment.

Safety control

![](https://static.igem.wiki/teams/4716/wiki/wiki/hp-ihp/figure-2-2-8.png)

Figure 2.2.8 Plasmid map of the safety control.

The safety of the engineered bacteria in the environment and in patients themselves are also important considerations in
the realization of the most important functional modules of our project. Our safety module is divided into two main
parts: in vitro and in vivo.

![](https://static.igem.wiki/teams/4716/wiki/wiki/hp-ihp/figure-2-2-9.png)

Figure 2.2.9 Temperature control switch diagram.

The toxin/antitoxin system, widely used both inside and outside of the iGEM competition, is a proven and robust system
for building flora suicide switches. When our implanted intestinal flora is expelled from the body for various reasons,
we ensure that it will not be potentially contaminated to other natural environment microorganisms due to the
uncontrolled horizontal gene transfer of our additional adhesion and releasing modules we have added to the intestinal
flora. Based on the human body temperature of around 37 degrees Celsius, we chose a temperature controlled suicide
switch based on Part:BBa\_K3247005 RNA thermometer NoChill-06. When our engineered bacteria are expelled from patient’s
body, the module that manipulates the expression of the antitoxin stops due to the temperature deviation from 37 degrees
Celsius, terminating the inhibition of the toxin, and realising its suicide in the natural environment after it is
expelled.

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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* Experiments
* [Experiments & Study](#P1)
* [Material](#P3)
* [References](#P4)

# Experiments & Study:

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/figure-2-3-1-experiment-final-aim.png)

Figure 2.3.1 After experimental verification based on E. coli, we replaced the engineered bacteria chassis with
probiotics; Modified probiotics are added to foods to help improve intestinal stress in celiac disease.

Perspective of the process of the whole projects:

(1) Biobricks creation —> in E.coli —> in probiotic —> yogurt (to go
further)

* After the carefully designed, repeatedly verified and continuously improved multiple
  cycle processes of chassis based on
  the expression of E. coli, we also learned the characteristics of other strains from the
  literature research, and
  planned to replace the chassis organisms with other probiotics if the project can be
  further advanced - after all, most
  of the E. coli bacteria are harmful to the human body and easily disrupt the balance of
  intestinal flora - such as
  bifidobacteria and Bacillus subtilis (studies have found that gluten-free drinking
  reduces lactobacilli and
  bifidobacteria in the gut).
* ![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/no-titile-probiotics.jpg)

  For the probiotic part, application of new engineered probiotics colonizing to the
  intestinal
  mucosal layer, regulating the state of intestinal microbiota. After the success of parts
  I and II, the best adhesion
  proteins and 33-mer-breaking enzymes will be applied to common factory probiotics, such
  as Bifidobacterial in yogurt.
  This part is possible to be tested by digital modeling. In addition, the range of
  applications can be expanded with
  computer calculations. That is what our project tries to do against the background of
  problems with celiac disease, it
  is possible for engineered bacteria to cut the root of the problem - gluten. Also, it
  can be used at the outside of the
  capsule to modify and balance ROS. In the context of cystic fibrosis problems, bacteria
  can colonize the respiratory
  mucus layer to help regulate Cl- concentration.

(2) All the [protocol](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/protocol-download.pdf) follow the steps (click to download)

* Plasmid members
* Recombinant plasmids
* Recombinant bacteria
* Gene expression testing
* DNA levels
* Protein levels

(3) [Notebook](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/notebook-download.pdf) (click to download)

# Material

* Bacterial strains: E.coli DH5α (Laboratory collection)
* Primers (pairs name, 5’ to 3’ primer sequence)

|  |  |  |
| --- | --- | --- |
| NO-his.FOR | gttagctgtgatataccatgtagcgtggtgatcagg | 36-mer |
| NO-his.REV | agcggatcctataaacgcagaaaggcccaccc | 32-mer |
| NO\_regulater.FOR | ccgcgaaatttaatccttcaatcccagacgtttcg | 35-mer |
| NO\_regulater.REV | acatggtatatcacagctaacaccacgtcgt | 31-mer |
| Vector.FOR | gcgtttataggatccgctgctaacaaagc | 29-mer |
| Vector.REV | gaaggattaaatttcgcgggatcgagatct | 30-mer |

* Promoters (name, characteristics(such as: arabinose inducible promoter pBAD), 5’ to 3’
  primer sequence)
* Imaging systems: Olympus IX83
* Microlate readers: INSA machine: Synergy H1 Biotek, Agilent

# References

* Letourneau J., Levesque C., Berthiaume F., Jacques M., Mourez M. (2011). In Vitro Assay of
  Bacterial Adhesion onto
  Mammalian Epithelial Cells. JoVE. 51.
  http://www.jove.com/details.php?id=2783, doi:
  10.3791/2783
* Francis, D. M., & Page, R. (2010). Strategies to optimize protein expression in E.
  coli. Current protocols in protein
  science, 61(1), 5-24.

See the Protocol file for details.

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# Results

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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* Results
* [The goals](#P1)
* [Plasmid design](#P2)
* [Results](#P3)

# The goals

* Make the Fvp/PEP can degraded protein and disgest the gluten in vitroly
* Use cp19k to help bacteria to adhere to mammalian cell surface;
* Place Fvp/PEP under the control of the NO sensor, which is consist of NO promoter and NO regulator, to induce their expression under inflammation (disease condition);
* Place cp19k under the control of the Pasr promoter, which is acid induced promoter, to adapt to the human intestinal
  environment;

# Plasmid design

Part I, the engineering bacteria (E. coli) colonization of the mucosal layer. For this part we choose to use the cp19k
and mefp-5 as the adhesive protein that present on the surface of E. coli. (by fused with CsgA). Ccp19k (moonlighting
protein) from Pollicipes pollicipes participates in colonization, like double-sided glue, sticking the bacteria and the
intestinal mucosal layer together to complete colonization. Mefp-5 (Mytilus edulis foot protein-5) from M-edulis is also
used in the colonization to compare with the effect of cp19k. The tyrosine residues on the mefp-5 protein could be
converted to amino acid residues containing the DOPA moiety by the action of tyrosinase, which is essential for the
colonization of the gut by bacteria using mefp-5 expressed on the surface of E. coli.

Part II, the engineering bacteria release 33-mer-breaking enzymes, belonging to serine peptidase (S28 family). As the
key pathogenicity of gluten protein is the 33-mer peptide chain, the 33-mer-breaking enzymes should be able to degrade
the 33-mer peptide chain with structural properties that are difficult to be thoroughly broken down, and therefore can
relieve the pathogenicity of gluten. Enzymes used in this project are endopeptidases from Flavobacterium
meningosepticum, Sphingomonas capsulate, and Aspergillus niger. PEP and FVP.. Since, gluten triggers an inflammatory
T-cell response in patients’ duodenum. In order to avoid T cell recognition, our groups let bacteria colonize the
surface of the mucus layer to secrete breaking enzymes, and let gluten degrade before being taken up by T cells through
M cells.

# Results

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-1.png)

(1) Detection of the protein expression by the Fluorescence

Figure 1.3.1 The upper box is the design of the plasmid insert part, and the lower box is a picture of bacteria standing
under a fluorescence microscope.

[Download : Download high-res image (click to download)](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-1.png)

We can clearly see that the microscope has barely detected the mSandy's red fluorescence, and we suspect that there may
be due to the following reasons:

(1) The fluorescence microscope is not optimized to detect the mSandy. We use the mCherry channel instead of detecting
the fluorescence of mSandy,. aAlthough the wavelengths of light differ by only a few nanometers, this may reduce the
sensitivity during the detection.:
mSandy 2 (𝜆em = 606 nm, quantum yield = 0.35)
mCherry (𝜆em = 609 nm, quantum yield = 0.23)

(2) We designed that the location of mSandy is between promotor and cp19k proteins, and may affect the conformation of
mSandy proteins and affect the signal. However, if mSandy is expressed after cp19k, there is a high probability of
affecting the adhesion effect of cp19k. Hence, o mSandy's location became a problem remained to be solved. As an
alternative, we can swap the reporter protein for a small tag (such as 6xHis) and the protein expression can be detected
by Western blotting.

(3) After seqeuencing, we found a mutation on the J23119 promoter, this may affect he transcription of the target gene.
However, even the protein expressed by the Pasr promoter has not detected fluorescence.

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-2.png)

Figure 1.3.2 The upper 2 boxes areis the design of the plasmid insert part, and the lower box is a picture of bacteria
standing under a fluorescence microscope.

[Download : Download high-res image (click to download)](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-2.png)

There are only 1 photo each of (1) pET-J23119-Fvp-his tag (2) pET-J23119-PEP-his tag bacteria, because of the
relationship between technology and time (the part of the NO sensor is the part added during the second design), the
plasmids related to the NO sensor have not been built for the time being, and there are no fluorescent photos of the
related bacteria.

We found that only bacteria containing Fvp plasmids had partial green fluorescence, while almost no fluorescence
compared to PEP-expressing bacteria, and we hypothesized that perhaps E. coli was more receptive to the expression of
the Fvp protein (the size of the Fvp protein is slightly smaller than PEP). However, we can see that the fluorescence
distribution is not uniform, and no fluorescence is found outside the bacterial cell, only there are obvious fluorescent
spots in the bacterial cell, which may indicate that the protein is not successfully secreted outside the cell when the
photo is taken.

(2) The adhesion assay to determine the function of the Cp19k aAdhesion protein

Brief introduction: We put the same bacterial concentration (OD600 = 0.5 ± 0.02) on the surface of different materials
to culture for 1h and 2h, and then wash off the floating and non-unadherent bacteria with 1XPBS, take pictures and count
the number of bacteria left on (sticking) the surface of the material.

Experiment I Adhesion assay with pH = 7 culture condition (1h)

Experiment II Adhesion assay with pH = 5 culture condition (1h)

Experiment III Adhesion assay with Caco-2 intestine epithelial cells (2h)

Summary of the adhesion protein result:

* Under acidic conditions, the Pasr promoter expressed the Cp19k and increase the adhesion ability of the bacteria.
* The adhesion effect of cp19k enhanced by collagen coating.

[Download the table Bacteria number. pdf (for all the number of bacteria left behind after being washed by
the slow water flow) (click to download)](bacteria-number.pdf)

Experiment I Adhesion assay with pH = 7 culture condition (Compare the adhesion properties on different plasmids )

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-3.png)

Figure 1.3.3 The figure shows the count of bacteria under a microscope (100X objective and 10X eyepiece, 1344pixel ×
1024pixel). After a period of culture, we washed the surface of bacteria where bacteria lived once with 1XPBS.

[Download : Download high-res image (click to download)](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-3.png)

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/graph-1-3-1.png)

We can observed that:

Summary of the adhesion protein result:

* Pasr v.s. J23119, bacterial adhesion with Pasr promoter much higher adhesion ability when compared to J23119 when the
  glasses with collagen coating.
* The bacteria with Cp19k has better adhesion when compare to the control (no Cp19k), regardless the promoter that used.
* Collagen coating can increase the bacteria adhesion, except the control plasmids (Pasr-Contol and J23119-control).

Experiment II Adhesion assay with pH = 5 culture condition

When pH = 5 in the environment, culture for 1h.

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-4.png)

Figure 1.3.4 The figure shows the count of bacteria under a microscope (100X objective and 10X eyepiece, 1344pixel ×
1024pixel). After a period of culture, we washed the surface of bacteria where bacteria lived once with 1XPBS.

[Download : Download high-res image (click to download)](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-4.png)

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/graph-1-3-2.png)

Graph 1.3.2 G = Glass, G + C = Glass + Collagen.

Experiment III Adhesion assay with Caco-2 intestine epithelial cells

When pH = 7/5 in the environment, culture for 2h. (Caco-2 cell line)

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-5.png)

Figure 1.3.5 The figure shows the count of bacteria under a microscope (100X objective and 10X eyepiece, 1344pixel ×
1024pixel). After a period of culture, we washed the surface of bacteria where bacteria lived once with 1XPBS.

[Download : Download high-res image (click to download)](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-5.png)

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/graph-1-3-3.png)

Graph 1.3.3 G = Glass, G + C = Glass + Collagen.

We observed that:

* There is no change of the adhesion after the expression of cp19k. This may be related the original affinity of the
  bacteria on the cells is strong, or due to the complex extracellular matrix environment of the mammalian cells.

(3) Effect of pH on bacteria growth curves with different plasmid

Brief introduction: We incubated the same number of bacteria overnight in LB medium (with chloromycetin as antibiotic)
with a certain pH gradient (pH=4.75-6.5, interval 0.25) for 20 h, and finally the OD600 value of the bacterial solution
was measured by microplate reader.

Summary of the result:

* Bacteria with J23119 promoter expression plasmids survive better
* Within each plasmids, there is no strong inhibition of cell growth at different pH condition, except Psar-cp19k and
  Psar-control, this indicate the acid promoter may affect the bacteria growth under strong acid condition (pH <5)

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/graph-1-3-4.png)

Graph 1.3.4 All the graphs here show the growth of our engineering bacteria. (A) Summary of growth. (B) J23119 promoter
(Part: BBa\_J23119) bacterial growth expressing cp19k. (C) Bacterial growth containing J23119 promoter but not expressing
special proteins. (D) Containing Pasr promoter (Part: BBa\_K1231000) bacterial growth without expressing specific
proteins. (E) Growth of bacteria expressing cp19k by Pasr promoter.

[Download : Download high-res image (click to download)](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/graph-1-3-4.png)

(4) Gibson assembly results

From the synthesis of plasmids at the beginning, to the addition of new fragments (such as the addition of NO sensor),
and the modification of some plasmids, it is necessary to go through PCR and Gibson assembly experiments. We have both
successful and failed plasmid builds.

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-6.png)

Figure 1.3.6 One result of template DNA running gel. After completing the PCR procedure for the template DNA, based on
the sequence size, we determined if the DNA was the correct size for the next step for Gibson assembly.

![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-7l.png)
![](https://static.igem.wiki/teams/4716/wiki/wiki/wetlab/1-3-7r.jpg)

Figure 1.3.7 Photographs of some completed Gibson's plasmids and transfected into medium for E. coli. We can see some
without long colonies, such as the left image; There are some that have successfully grown colonies, as shown in the
figure on the right.

For the analysis of the experiment:

* Perhaps it is a procedural problem (see Protocol for details), such as the possibility that the temperature and time
  may not be suitable for the ligase we are using;
* Maybe it's a problem with our personnel operation, most of the members are not skilled in experimental operation,
  maybe the experiment fails because of ignoring some small details during the experiment, such as mix is not mixed;
* Or maybe the template DNA is not pure enough, causing Gibson to fail; Wait a minute.
* But the experiment continues...

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