iGEM 2022 — Team bit-china

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# Notebook

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2022

◢
**August**

[August](#August)
[8/8 - 13/8](#8.8-8.13)
[15/8 - 21/8](#8.15-8.21)
[22/8 - 28/8](#8.22-8.28)
[29/8 - 4/9](#8.29-9.4)

◢
**September**

[September](#September)
[5/9 - 11/9](#9.5-9.11)
[12/9 - 18/9](#9.12-9.18)
[19/9 - 25/9](#9.19-9.25)
[26/9 - 2/10](#9.26-10.2)

◢
**October**

[October](#October)
[3/10 - 9/10](#10.3-10.9)

#### August

#### 8/8 - 13/8

* **Detection Module**
* Extracted E.coli genome
* Searched sequences and designed primers
* **Function Module**
* Prepared kanamycin culture medium
* Prepared kanamycin and chloramphenicol medium
* At the same time, introduced empty pET-28A(+) plasmid and nirB/icd plasmid as negative control

#### 15/8 - 21/8

* **Detection Module**
* Amplified nirB and icd sequences fragments via PCR
* Gibson assembly with corresponding fluorescent protein (nirB mRFP, icd eGFP) and loading into p5T7 plasmid
* sent for sequencing
* Transformation of recombinant plasmid harboring mRFP into E. coli
* **Function Module**
* Recombination of mRFP with Escherichia coli
* Resistance screening of kanamycin and chloramphenicol, liquid medium culture
* Detected the change of fluorescence intensity with time-variant and ploting A1
* Detected the change of OD (bacterial count) with time-variant and draw B1
* Preparation culture medium contains 5 μ M hemin
* Introduction of recombinant plasmid of leghemoglobin

#### 22/8 - 28/8

* **Detection Module**
* Designed primers for the amplifaction of fragments including nirB, eGFP, mRFP and icd, and interted them
  into plasmid P5T7
* Scribing of mRFP Escherichia coli transformation
* **Function Module**
* Kanamycin resistance screening and liquid medium culture
* Group 1: No hemin culture
* Detection of expression effect by SDS-PAGE: C21
* Detect the change of OD (bacterial count) with time-variant and draw B2
* Detect the change of fluorescence intensity with time-variant and draw A2
* Group 2: Add 5 μ M hemin
* Detection of expression effect by SDS-PAGE: C22
* Detect the change of OD (bacterial count) with time-variant and draw B2
* Detect fluorescence intensity and OD (bacterial load) over time and plot A22 and B22.

#### 29/8 - 4/9

* **Detection Module**
* Culture bacteria and Extract plasmid
* Two plasmids of nirB+mRFP and icd+eGFP were obtained by PCR using P5T7 plasmid as the vector
* DNA fragments recovery
* Design and verify primers
* Culture bacteria and Extract plasmid
* **Function Module**
* construction of plasmid with laccase carrying signal peptide
* Introduction of recombinant plasmid carrying signal peptide laccase
* Kanamycin resistance screening and liquid medium culture
* SDS-PAGE is used to detect the expression effect C3

#### September

#### 5/9 - 11/9

* **Detection Module**
* Carried out strain PCR, scribing and Gibson's large intestine transformation to obtain the required plasmid
* Scribed and added strain PCR to verify correctness
* Culture bacteria.
* Extracted the plasmid, and found the concentration is normal,which between 100 and 200 ng
* Culture bacteria and Genome extraction
* Design sequencing primer (about 1kb) and send it to the company for detection
* **Function Module**
* Introduction of signal peptide laccase plasmid and detection plasmid
* Screening with kanamycin and chloramphenicol
* Detect the change of fluorescence intensity with time-variant and draw A3
* Detect the change of OD (bacterial count) with time-variant and draw B3

#### 12/9 - 18/9

* **Detection Module**
* Through sequencing results, analyzed and judged whether the plasmid construction was successful to verify
  whether the interface is correct
* Culture bacteria and Extract plasmid
* Determined the culture conditions, as well as the subsequent test forms and data, and analyzed the data
* Design the evacuation unit and determine which two specific gases to use
* Determine the volume ratio of the gas to be charged and how long to incubate to start testing
* Import the plasmid including nirB+mRFP
* Culture nirB plasmid under two media
* **Function Module**
* Detect the change of fluorescence intensity with time-variant and draw A31
* Detect the change of OD (bacterial count) with time-variant and plot B31
* Introduction of laccase recombinant plasmid without signal peptide
* Kanamycin resistance screening and liquid medium culture
* SDS-PAGE to detect the expression effect C4

#### 19/9 - 25/9

* **Detection Module**
* Detects changes in fluorescence intensity over time and plots them
* Detection of OD (bacterial load) over time and plotting
* **Function Module**
* Continue to import the detection plasmid
* Screening with kanamycin and chloramphenicol
* Detect the change of fluorescence intensity with time-variant and draw A4
* Detect the change of OD (bacterial count) with time-variant and draw B4

#### 26/9 - 2/10

* **Detection Module**
* Introduce plasmids containing only nirB+mRFP
* Introduce a plasmid containing only icd+eGFP
* Screen with chloramphenicol
* Detect fluorescence intensity changes over time and plot A52
* Detect the change of OD (bacterial load) with time and plot B52
* **Function Module**
* Add 0.2 mmol/L copper ion to CueO+nirB and add IPTG after 3h
* Detect fluorescence intensity changes over time and plot A61
* Detect the change of OD (bacterial load) with time and plot B61
* Add 0.5 mmol/L copper ion to CueO+nirB and add IPTG after 3h
* Detect fluorescence intensity changes over time and plotting A62
* Detect OD (bacterial load) over time and plot B62

#### October

#### 3/10 - 9/10

* **Detection Module**
* Configure normal medium without cysteine
* Configure hypoxic medium with cysteine
* Culture the two plasmids under normal medium
* Sample the plasmids of both assay modules cultured on normal media at regular intervals
* Culture the two plasmids under hypoxic medium
* Sample the plasmids of both assay modules cultured on hypoxic medium at regular intervals
* Measure the absorbance and fluorescence intensity of each time gradient by enzyme marker
* **Function Module**
* cotransformation of leghemoglobin plasmid and CueO plasmid, noted as pcl-group
* Add 0.2 mmol/L copper ions to CueO+nirB+Lb and add IPTG after 3h
* Detect fluorescence intensity changes over time and plot A71
* Detect the change of OD (bacterial load) with time and plot B71
* Add 0.5 mmol/L copper ions to CueO+nirB+Lb and add IPTG after 3h
* Detect fluorescence intensity changes over time and plot A72
* Detect the change of OD (bacterial load) with time and plot B72

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{% block title %}Engineering Success{% endblock %}
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# Engineering

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Content

◢
**Construction of the function modules**

[Construction of the function modules](#title1-0)
[the choice of globins](#title1-1)
[the expression of leghemoglobin](#title1-2)
[heme/hemin addition of leghemoglobin](#title1-3)
[Laccase with copper ion expression](#title1-4)
[Cotransformation of laccase and leghemoglobin plasmids](#title1-5)

◢
**Construction of the detection module**

[Construction of the detection module](#title2-0)
[ArcA promoter expression iteration](#title2-2)
[Methods for judging detection modules](#title2-3)
[ArcA promoter expression iteration](#title2-4)

◢
**Future vision**

[Future vision](#title3-0)
[Construction of a graded expression-gene oscillation
device](#title3-1)
[signal peptide of laccase](#title3-2)
[new choices of promoters](#title3-3)
[Leghemoglobin targeting](#title3-4)
[Expansion of the project -- oxygen controller](#title3-5)

[**References**](#title4)

Our project mainly consists of two parts -- function module and Detection module.The role of the functional
energy module is to build a low oxygen environment in the cell. The detection module functions to detect the
oxygen concentration in the cell, in order to know whether the intracellular oxygen concentration has been
reduced to a low level.

#### Construction of the function modules

For the construction of a low-oxygen environment, we proposed two ideas: consuming the oxygen in the cell or
transporting it to go elsewhere.

#### the choice of globins

At the beginning of the experiment, we planned to transport the intracellular oxygen to build a low oxygen
environment.Given this, we initially planned to use vitreoscilla hemoglobin(VHb) and transfer it into to E.coli
to build the low oxygen environment in cell. However, after investigation and research, it was found that the
main function of VHb is to trap oxygen and strengthen the oxygen utilization rate in the low oxygen environment.
This does not fit our low oxygen construction purpose, and therefore, we abandoned the use of VHb at the
beginning. We also considered human hemoglobin or myoglobin. But In the end, after we read and analysed plenty
of articles, we finally choose leghemoglobin to carry and convey oxygen.

#### the expression of leghemoglobin

We found a special protein, leghemoglobin, from soybean *rhizobia*. It can directionally transport oxygen
close to
the cell periplasm to provide energy for cell growth. In that way, it can achieve the purpose of reducing the
intracellular oxygen concentration by transporting oxygen.

But leghemoglobin comes from soybean, which means it contains introns. Therefore, we removed the introns in the
process of reorganizing *E. coli*, allowing leghemoglobin to work normally. Later, we did codon
optimization according to the codon preference of *E. coli*. As shown in figure 1.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/01.png)

Figure 1: Intron removal and codon optimization of leghemoglobin

#### heme/hemin addition of leghemoglobin

The main role in leghemoglobin is played by heme, but heme may not be synthesized sufficiently in *E.
coli*, so
leghemoglobin may not bind to heme sufficiently after expression, and may have limited ability for oxygen
delivery. For this problem, we want to solve it by adding heme to improve the transport capacity of bean
hemoglobin for oxygen, and related studies have shown that this method is feasible, and the difference in
expression content is shown in Figure 2. Since heme is difficult to order commercially, we used hemin for
addition instead. Hemin is very similar to heme, and can be converted into heme.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/02.png)

Figure 2: Effect of hemin addition on leghemoglobin expression[1]

#### Laccase with copper ion expression

The active center of laccase CueO is copper-containing [Cu(MeCN)4]+. Initially, we did not add copper ions
within the culture medium when expressing CueO. Experiments showed that it could have some effect, but it was
not significant, as shown in Figure 3.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/03.jpg)

Figure 3: Fluorescence intensity comparison between control group and laccase group without
copper ion

After reviewing a lot of information and talking with our teacher Yu Yang, we determined that the successful
expression of laccase requires the addition of a certain amount of copper ions within the culture medium. We
tried adding 0.2 mmol/L and 0.5 mmol/L of copper ions to the medium. We found that the fluorescence intensity of
the group with the addition of 0.2 mmol/L copper ions was significantly enhanced when laccase was acting alone,
as shown in Figure 4, which confirms our
idea.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/04.png)

Figure 4: The fluorescence intensity of bacteria that are introduced nirB,
CueO+nirB+0,0.2,0.5mmol/L Cu2+

#### Cotransformation of laccase and leghemoglobin plasmids

We designed and built function modules for the experiments, i.e., laccase and soy hemoglobin were introduced
into *E. coli* strain BL-21, respectively, along with the nirB-mRFP recombinant plasmid capable of
detecting
intracellular oxygen concentration. After testing for fluorescence and OD, the results are shown in Figure 5.

![](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/u1.png)
![](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/u2.png)

Figure 5: Changes of OD and fluorescence intensity with time when laccase or leghemoglobinis
introduced separately

When the experiment came to
this point, we thought that the effect of reducing oxygen concentration brought by
the introduction of laccase and leghemoglobin failed to achieve our goal. On account of the difference between
the leghemoglobin and laccase in principle, we wondered whether the effect of
reducing oxygen concentration would be better when the two were introduced together. At this time, through data
modeling, we simulated the fluorescence intensity of the bacteria that are only introduced leghemoglobin or
laccase, which is basically consistent with the experimental data. Therefore, we simulated the fluorescence
intensity of the bacteria that are introduced both leghemoglobin and laccase in the same way, which is nearly
twice as high as that of the bacteria that are only introduced a single element. As is shown in the figure 6.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/07-1.png)

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/07-2.png)

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/07-3.png)

Figure 6: Oxygen consumption rate of bacteria with CueO (left), Lb (middle), CueO+Lb
(right).
It shows that bacteria that are introduced both Lb and CueO have the highest oxygen consumption
rate.

Therefore, we conducted cotransformation experiments under the guidance of modeling.

Both plasmids were
introduced at the same time, and the experimental results showed that better results were successfully obtained,
as shown in Figure 7.

It can be found that the fluorescence intensity can reach up to nearly 100,000, which is significantly higher
than that of the strains introducing leghemoglobin or laccase alone, indicating that the two proteins used
together play a very good role. On the other hand, the amount of bacteria shown by OD is not greatly influenced.
([click here to know more about
results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D))

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/u3.png)

Figure 7: Fluorescence and OD change with time when leghemoglobinand laccase work together

#### Construction of the detection module

Since we could not directly detect the oxygen concentration in the cell, we intended to construct a detection
module to visually show whether the low oxygen environment was constructed or not.

For the construction of a low-oxygen environment, we proposed two ideas: consuming the oxygen in the cell or
transporting it to go elsewhere.

The constructed detection module needs to meet two requirements: one is resistant to low oxygen environment, and
the other is able to respond to low oxygen environment. For requirement 2, we plan to attach fluorescent
proteins to the requirement one to react to the low oxygen environment. After investigation, we finally chose
nirB, which is sensitive to oxygen and resistant to low oxygen environment, binding with a fluorescent protein
to construct our low oxygen response module.

#### ArcA promoter expression iteration

In the course of the nirB investigation study, we found an ArcA promoter that was also resistant to low-oxygen
environments. Unlike nirB, ArcA is a promoter sensitive to the rate of oxygen consumption. Although nirB has
completed its corresponding function for low oxygen, the emergence of ArcA allows us to be able to react
cellular oxygen consumption rates in response to low oxygen, characterizing the activity of the cells. But since
the ArcA is a module coming from the icd promoter, For the icd promoter that contains not only cra modules but
also the ArcA. What's more, ArcA overlap with cra. This creates the dilemma of our inability to extract ArcA
alone during the PCR process. Therefore, in the actual experiment process, we chose to use the icd promoter
instead of ArcA for the detection of the oxygen consumption rate. We hope to find ways to extract ArcA
separately in later experiments to eliminate the interference of cra to the experiment.

#### Methods for judging detection modules

That is all about the building of the detection model. After building the detection module, how to judge whether
the detection module is really effective has become a difficult problem. At the beginning, we planned to use the
device shown in Figure 8 to build the external low oxygen and see whether the detection module is effective. The
specific working principle is as follows: Put the liquid culture medium in the bottom of the cone bottle, charge
the nitrogen and other inert gas into the bottle through the long gun head, and blow out the oxygen in the
culture medium to ensure the low oxygen in the cell growth environment; then, removing the longer gun head (as
shown in Figure 9), the inert gas such as nitrogen is passed through the shorter gun head to ensure the overall
low oxygen of the whole tapered bottle.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/08.png)

Figure 8: low oxygen device 1

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/09.png)

Figure 9: low oxygen device 2

However, because it is more difficult to access nitrogen with the above method in the school laboratory. So we
improved the experimental setup (Figure 10, Figure 11) -- After vacuuming the anaerobic bag, Add a protective
gas generation agent to it, and seal the protective gas to create a low oxygen environment.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/10.png)

Figure 10

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/11.png)

Figure 11

#### ArcA promoter expression iteration

Connect the nirB and icd ArcA modules in series to a plasmid vector (the circuit is shown in Figure 12). This
can reduce the number of plasmids, and also reflects the characteristics of synthetic biology that can integrate
different modules. According to the principle of chemical reaction, the higher the oxygen concentration, the
higher the oxygen consumption rate. As stated in the experimental design, the lower the oxygen concentration,
the higher the nirB activity; The lower the oxygen consumption rate, the higher the ArcA activity. Therefore,
the ideal state is that when the oxygen concentration and oxygen consumption rate are high, the red and green
fluorescence intensities are weak; when the oxygen concentration and oxygen consumption rate are low, the two
fluorescence intensities are strong. The fluorescence intensity of the two bands measured by the microplate
reader can reflect the oxygen concentration and oxygen consumption rate, which can improve the detection
efficiency.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/12.png)

Figure 12: Plasmid vector constructed in tandem by nirB and ArcA

#### Future vision

#### Construction of a graded expression-gene oscillation device

According to some teacher's advice, We plan to use laccase to reduce the oxygen concentration first, and then
then further reduce the concentration of oxygen with leghemoglobin. The reason is that laccase is an enzyme,
which can play a role in high oxygen
concentration and consume a lot of oxygen; However, leghemoglobin is combined with oxygen. If the oxygen
concentration is too high, it will be saturated with leghemoglobin, so it can not have a good oxygen reduction
effect. Therefore, laccase is suitable for decreasing oxygen at high oxygen concentration, and leghemoglobin is
suitable for decreasing oxygen at low oxygen concentration.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/13.png)

Figure 13: Schematic Diagram of rocking equipment

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/14.png)

Figure 14: The final effect of the rocking equipment

Therefore, inspired by the use of gene oscillator by BUCT China team, we designed a metabolic pathway based on
the principles of synthetic biology (Fig. 13). Among them, O2 sensitive promoter I/II (hereinafter referred to
as OS promoter) is a hypoxia inducible promoter. The oxygen concentration of OS promoter I is higher than that
of OS promoter II. It is assumed that the oxygen partial pressure required for induction is p1 and p2
respectively. T7 promoter guides the expression of laccase, OS promoter I promoter guides the expression of
leghemoglobin, and OS promoter II is used as an anti-sense promoter[2], which is reversely connected
to laccase and
leghemoglobin.

At first, laccase and leghemoglobin were not expressed. Then we used IPTG induction to make laccase begin to
express and reduce intracellular oxygen concentration. When the partial pressure of oxygen decreases below p1,
OS promoter I is activated and leghemoglobin is expressed. When the partial pressure of oxygen further decreases
below p2, the oxygen concentration no longer needs to be further reduced. Therefore, OS promoter II is also
activated and starts to reverse express, producing anti-sense mRNA chains that complement and pair with the
original mRNA chains, so that the translation process of leghemoglobin and laccase is inhibited, thus realizing
regulation. After a period of time, the oxygen concentration gradually recovers, and the activity of OS promoter
II decreases. Transcription and translation begin to work normally, and the oxygen concentration starts to
decrease again, so as to achieve the effect that a gene expression oscillates with time.

In the period of high oxygen concentration, cells mainly grow and reproduce. In the period of hypoxia, the cells
mainly produce hydrogen and nitrogen fixation. Thus, the cycle of growth and production is realized, as is shown
in figure 14.

#### signal peptide of laccase

Since the use of VHb to transport oxygen to reduce the intracellular oxygen concentration did not work, we
turned our eyes to the direction of consuming the intracellular oxygen. After the investigation and study, we
finally intend to choose laccase to build a low oxygen environment. Laccase is a reductive enzyme that can
catalyze the reaction of oxygen with reducing substances to achieve the effect of consuming intracellular
oxygen. Since we finally intended to conduct experiments in *E. coli*, the laccase we selected was the
polycopper
oxidase -- CueO in *E. coli*. Based on the above ideas, we started the experiment.

Later, we found that the laccase would be localized to the cellular periplasm. The main reason is that the
laccase contains a 28-residue N-terminal signal peptides.For this problem, we reconstructed the laccase plasmid
to remove the signal peptide sequences contained in the laccase. The reconstructed laccase plasmids are shown in
Figure 15.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/15.png)

Figure 15: Laccase without a signal peptide

#### new choices of promoters

Figures 16 and 17 show the final laccase and leghemoglobin plasmid constructs. But during the experiment, we
found that the promoters used were not very ideal. Initially, we used the default hypoxia promoter, but the
coupling is complex.For control convenience, we redesigned both of the promoter araBAD and tacThe araBAD
promoter is a promoter from Escheric. Coli, transduced growth to 0.6-0.8OD, the inducer was arabinose at an
induction concentration of 7.9%. The tac promoter is a promoter heterozygous for the trp and lac promoters,
transduced growth to 0.6-0.8OD, the induction was IPTG at an induction concentration of 0.1%. These two
promoters are universal, and the mechanistic studies are relatively clear. However, due to insufficient
experimental time, we finally did not test the induced expression effect of these two primes in the actual
experiment. We hope to be able to refine this part in the subsequent experiments.

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/16.jpg)

Figure 16: laccase plasmid

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/17.jpg)

Figure 17: leghemoglobin plasmid

In subsequent experiments, we hope to allow for a detailed partition of the promoter function. When the
intracellular oxygen concentration is high, hyperoxic promoter can be used to induce the expression of bean
hemoglobin alone, making the laccase not working. However, when the intracellular oxygen is low, low oxygen
promoters such as nirB are able to induce laccase expression alone, making leghemoglobin not work。Finally,
making the leghemoglobin and laccase play a division of labor and cooperation effect.

#### Leghemoglobin targeting

This project has confirmed that the introduction of leghemoglobin can reduce the intracellular oxygen
concentration, but there is still doubt whether leghemoglobin can deliver oxygen to the respiratory site.
Therefore, in the subsequent experiment, it is necessary to investigate whether the leghemoglobin has the
function of transporting oxygen to the respiratory site. If not, it is necessary to introduce a signal peptide
to locate the leghemoglobin on the cell membrane in the later stage, so as to deliver oxygen to the respiratory
site.

#### Expansion of the project——oxygen controller

We "oxygen hunter" have added leghemoglobin (that transports
oxygen) and laccase (that consumes oxygen) into cells, but if we add some parts that enrich oxygen (eg. VHb)
(figure 18) into
cells,we can also fulfill the intracellular high oxygen pressure environment. By using different promoters,
the devices that regulate oxygen can work together, so that the
oxygen concentration in cells can be controlled in different ranges. Finally we can construct an oxygen
controller system.

We hope our project can achieve our second idea and achieve the effect of "oxygen controller".

![img](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/18.jpg)

Figure 18: the molecular structure of VHb

![](https://static.igem.wiki/teams/4232/wiki/engineering-image/engineering-image/up1.png)

#### Reference

[1]Yue Su. Study on efficient expression of heterologous leghemoglobin in microorganisms(2020)

[2]Grant Logan , Christine M. Smyth, et al. 298. Characterization of a Novel Anti-Sense Promoter Element in
Adeno-Associated Virus: Implications for Hepatocellular Carcinoma(2016)Molecular Therapy

{% endblock %}

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

# Experiments

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

Content

◢
**Detection module**

[Detection module](#title1-0)
[Oxygen concentration detection](#title1-1)
[Detection of oxygen consumption rate](#title1-2)

◢
**function module**

[function module](#title2-0)
[legemoglobin](#title2-1)
[laccase](#title2-2)
[Detection of reduced oxygen concentration](#title2-3)

[**References**](#title3)

#### Detection module

#### Oxygen concentration detection

In the detection module, we first used the oxygen sensitive promoter nirB. Its characteristic is that the lower
the oxygen concentration, the higher the expression activity. It is a gene sequence with more than 2000 bp, and
its activity under hypoxia is much higher than that under normal oxygen concentration. nirB's principle
operation is there are two critical regions on nirB for induction by anaerobic conditions. The first is a
hexamer region, TAAGGT, which is necessary for anaerobic activation. The second region is the fumarate and
nitrate reductase regulatory (FNR) protein-binding site. FNR protein is a general regulatory protein that
activates transcription initiation in some anaerobic promoters such as nirB under anaerobic conditions.

We then inserted the red fluorescent protein (mRFP) gene into the plasmid vector p5T7 and transferred it to E.
coli. In this way, by detecting the fluorescence intensity in the red light wavelength range, the
intracellular oxygen concentration can be reflected, thus realizing a convenient and high flux detection. That
is, the stronger the fluorescence, the higher the nirB activity, the lower the oxygen concentration.

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/01.png)

nirB-red fluorescent protein expression vectors

We use anaerobic bags with different oxygen concentrations, or use hypoxic culture media. First, we detect the
corresponding fluorescence intensity under different oxygen concentrations by conducting gradient hypoxia tests,
and then build a detection system. We can reasonably extrapolate the existing data. Later, we can predict the
oxygen concentration in the system at that time by directly measuring the fluorescent protein activity, After
that, we will judge and detect the hypoxia effect of the system we built, and visualize the data that is
difficult to be visualized, so as to vividly display our experimental results.

#### Detection of oxygen consumption rate

The second part of the detection module uses icd promoter and ArcA transcription factor to detect oxygen
consumption rate. The experiment showed that the activity of ArcA had no significant relationship with oxygen
concentration, but had a significant negative correlation with oxygen consumption rate (R2=0.93)[1].
This is because ArcA is closely related to cell respiration and oxygen metabolism.

ArcA activity is regulated by fermentation products via the inhibition of ArcB phosphorylation. Since the
NADH/NAD+ ratio increases under oxygen limited conditions, fermentation reactions occur to facilitate NADH
regeneration. The accumulated fermentation product(s) would inhibit ArcB phosphorylation. The measurement of the
phosphorylated state of ArcA and ArcB would make this mechanism more reliable.

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/02.png)

Relationships between ArcA activity and qO2.[5]

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/03.png)

The structure of icd promoter

We extracted the icd promoter and the ArcA fragment after it, and connected it with green fluorescent protein
(eGFP) to measure its expression activity, then transferred it to plasmid p5T7, and then transferred it to E.
coli. Find the method to calibrate the oxygen consumption rate. In this way, the lower the oxygen
consumption rate, the higher the fluorescence intensity.

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/04.png)

icd-green fluorescent protein expression vectors

Similarly, we artificially constructed a culture environment with different oxygen concentrations to detect the
functions of icd and ArcA modules. However, due to time constraints, we have not been able to further explore
the relationship between ArcA activity and oxygen consumption rate. As expected, we will rely on different
oxygen concentrations and their corresponding oxygen consumption rates to obtain a chart that can accurately
represent the degree of oxygen concentration decline and oxygen consumption efficiency by curve fitting through
software, so as to judge the effectiveness of the system we have built.

In order to detect the effects of nirB and icd, we need to create a hypoxic environment artificially. After our
engineering improvement, we purchased anaerobic bags and configured anaerobic culture media with a specific
formula. Among
them, the anaerobic bag uses specific substances to absorb oxygen, thus creating a hypoxic environment in a
closed environment inside the bag, while the anaerobic culture medium contains a large amount of reducing
substances.

![img](https://static.igem.wiki/teams/4232/wiki/addnewpic/draw-bag.png)

anaerobic bag we used

After these specific promoter sequences are inserted into specific fluorescent proteins, they are introduced
into the plasmid p5T7, and then inoculated onto the solid medium with a lattice. They are cultured under normal
conditions and in anaerobic bags that create an oxygen poor environment, respectively. After a period of time,
take out the medium in the anaerobic bag, and pick it together with the normally cultured Escherichia coli in
the super clean bench to a new culture dish for the convenience of subsequent fluorescence detection. Pick up
some strains on the cover glass, place the cover glass in the dark room under a fluorescent microscope with a
magnification of 400X and observe through the computer screen.First, find the places where the strains are
enriched in large quantities in the normal light band. Then turn on the excitation light source in the blue
light band, and observe it in the red light band and green light band. It can be found that the nirB promoter
emits red fluorescence, and the icd promoter emits green fluorescence. At the same time, The fluorescence
intensity of bacteria cultured in anaerobic bags was significantly higher than that of bacteria cultured in
normal atmosphere with 21% oxygen concentration.This can prove that the nirB and icd promoters can indeed
express, and can express better under hypoxia.

After that, we prepared normal and hypoxic liquid culture media, respectively, and connected the nirB and icd
promoters into them, placed them in a shaker for culture, and took samples at regular intervals. Finally, put it
into the microplate reader for detection, and measure its absorbance and fluorescence intensity under different
wave bands. The specific effects of hypoxia on the two promoters can be obtained more accurately.

Intracellular oxygen concentration and oxygen consumption rate can be used to characterize the status of
intracellular oxygen metabolism, provide data support for modeling work, and can later cooperate with functional
module.

#### function module

In the function module, we used legemoglobin and laccase to reduce the oxygen concentration in cells.

#### leghemoglobin

leghemoglobin, which exists in soybean root nodules in nature, is a protein composed of 144 amino acids.
*rhizobia* need oxygen to supply energy, but nitrogenase activity is easily inhibited by oxygen. The
leghemoglobin
in the root nodule can transport oxygen to the cell respiration site in a directional manner, provide sufficient
energy for nitrogen fixation, and reduce the oxygen concentration around the nitrogenase to ensure the
nitrogenase activity[2].

We introduced the leghemoglobin gene into *E. coli* cells for recombinant expression. We did codon
optimization
for leghemoglobin gene, and
integrated it into the plasmid pET-28A (+) with lactose operon. We introduced the recombinant plasmid into E.
coli BL-21 strain for culture.

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/05.jpg)

Leghemoglobin expression vectors

#### laccase

Laccase is a kind of oxidoreductase which widely exists in microorganisms, animals and plants. Its basic
principle is to catalyze the reaction of reductive substrate and oxygen to reduce the concentration of oxygen,
which can directly consume oxygen, thus helping to build a hypoxic environment in cells[3]. It is a
516 amino acid protein, of which the first 28 amino acid residues are signal peptides, which can locate CueO in
the periplasm[4].

We are going to choose the multiple oxidase CueO contained in Escherichia coli as laccase. We constructed the
recombinant plasmid pET-28A(+)-CueO and transferred it into **E. coli* BL 21(DE3)* strain for culture.

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/06.png)

Ribbon representation of the CueO molecule (PDB code 1N68). (a) Overall structure
highlighting three structural domains (D1, red; D2, green;D3, gold), four defined copper centers (T1, blue; T2,
teal; T3 (binuclear), purple; T4, red), and the location of methionine residues (in gray-yellow sticks).(b)
Molecular structure around T4 highlighting the ligands for CuII, possible ligands for CuI (including M440), and
a hydrogen bond connecting the T4 copper ligand D439 and the T1 copper ligand H443[4].

#### Detection of reduced oxygen concentration

We have observed the experimental effect by combining the function module and the detection module during the
experiment. The basic principle is that when induced with IPTG, CueO or leghemoglobin will be expressed in large
amounts, thus reducing the intracellular oxygen concentration. The detection module will detect the decrease in
oxygen concentration and "report" it in the form of fluorescence production.

We constructed the plasmid of leghemoglobin and laccase, and then introduced it into *E. coli* and the
detection
module. The experimental results are detailed in the result section.

In order to promote the better expression of function modules, we also considered some problems: first, part
of the leghemoglobin is heme, and we considered the influence of heme on the experiment.

We have conducted the following experimental design: we will only import one group of nirB,
leghemoglobin+nirB+hemin, and leghemoglobin+nirB, and carry out a control experiment. The results are listed in
the results.

Secondly, laccase contains copper ions. Adding copper ions in the culture will help laccase expression and
function, and the concentration of copper ions may have a certain impact.

We have conducted the following experimental design: we will only introduce nirB, laccase+nirB+copper ions, with
the concentration of 0mmol/L, 0.2mmol/L and 0.5mmol/L respectively, and carry out the control experiment. The
results are listed in the results.

In the design experiment, we take into account that laccase and leghemoglobin use different mechanisms to create
a hypoxic environment, which can complement each other: leghemoglobin is on the membrane, laccase is in the
cell, and external input is prevented to ensure respiration; Internal leakage prevention and oxygen free. It can
better solve the oxygen paradox problem.

Therefore, we introduced laccase and leghemoglobin into the chassis strain for experimental measurement, and
compared with the previous experimental results. We introduced laccase+leghemoglobin+nirB, added copper ions
(0mmol/L, 0.2mmol/L, 0.5mmol/L) and hemin, and carried out three groups of control experiments. See results for
details.

![img](https://static.igem.wiki/teams/4232/wiki/experiment-image/07.jpg)

We cultured these strains and detected the expression of fluorescent protein with fluorescence intensity to
judge the actual effect of adding function modules (leghemoglobin, laccase or both) on the intracellular
oxygen concentration. We judge the concentration of bacteria in the liquid culture medium by the data of
bacterial liquid absorbance (i.e. OD value) to reflect the growth of bacteria. In order to reduce the chance of
the experiment, we set up three parallel experiments for each group of data, and finally took the average value
of the three groups as the experimental data of the sampling time of this group.

We conducted data analysis on the experimental results, and the experimental results were basically the same and
basically consistent with the data after we modeled and predicted through Matlab. The growth curve of
Escherichia coli grew in accordance with the S-shape. At first, the growth was slow due to the lack of strain
concentration. Later, with the increase of strain concentration, it gradually reached the maximum propagation
rate. Later, due to the lack of nutrients in the culture medium, intensified intraspecific competition and other
reasons, the growth of the strain gradually slowed down. Through data analysis, we basically determined that
laccase and leghemoglobin did indeed play a role in reducing the oxygen concentration, and successfully achieved
the goal of constructing the hypoxic environment in cells, while making the growth and reproduction rate of
bacteria almost unaffected. Then the effects of copper ion concentration and heme addition on the construction
of hypoxic environment were analyzed.([Click here to know about result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D))

#### References

[1] Prost JF, Nègre D, Oudot C, Murakami K, Ishihama A, Cozzone AJ, et al. Cra-dependent transcriptional
activation of the icd gene of Escherichia coli. Journal of Bacteriology. 1999.

[2] Ryu M-H, Zhang J, Toth T, Khokhani D, Geddes BA, Mus F, et al. Control of nitrogen fixation in bacteria that
associate with cereals. Nature Microbiology. 2019.

[3] Djoko KY, Chong LX, Wedd AG, Xiao Z. Reaction Mechanisms of the Multicopper Oxidase CueO from Escherichia
coli Support Its Functional Role as a Cuprous Oxidase. Journal of the American Chemical Society. 2010.

[4] Karrera Y. Djoko,# Lee Xin Chong, Anthony G. Wedd, and Zhiguang Xiao. Reaction Mechanisms of the Multicopper
Oxidase CueO from Escherichia coli Support Its Functional Role as a Cuprous Oxidase. JACS. 2009.

[5] Reza, N., & Akbari Eidgahi, M. R. (2014). Construction of a Synthetically Engineered nirB Promoter for
Expression of Recombinant Protein in Escherichia coli. Jundishapur Journal of Microbiology, 7(6).
doi:10.5812/jjm.15942

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

# Results

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

Content

[**Overview**](#title1)
◢
**Detection module result analysis**

[Detection module result analysis](#title2-0)
[Functional verification of nirB](#title2-1)
[Function verification of icd](#title2-2)

[**Function module data analysis**](#title3)

#### Overview

First of all, we tested that the selected hypoxic starting parts had successfully played the function of
responding to oxygen in *E. coli*.

Secondly, through SDS-PAGE, we can see that the leghemoglobin of the source plant is successfully expressed in
*E. coli*, indicating that our codon optimization has achieved the effect; Laccase was also successfully
expressed, although its molecular weight was high and its expression was low.

![img](https://static.igem.wiki/teams/4232/wiki/result-image/sds-page.png)

Figure 1: SDS-PAGE

1,2: CueO+nirB, sediment(1) and supernatant(2).
3,4: CueO+nirB+0.2mM Cu2+, sediment(3) and supernatant(4).
5,6: CueO+Lb+nirB+hemin, sediment(5) and supernatant(6).
7,8: CueO+Lb+nirB+0.2mMCu2+ +hemin, sediment(7) and supernatant(8).
9,10: CueO+Lb+nirB+0.5mMCu2+ +hemin, sediment(9) and supernatant(10).
11,12: only nirB, sediment(11) and supernatant(12).
13,14: Lb+nirB+hemin, sediment(13) and supernatant(14).

Next, we verified the function of the gene line designed by the project. From the growth curve, the strain
density of the experimental group with leghemoglobin is 82.3% of the control group containing only nirB, and the
number of cells with laccase is 88.6%. No matter which experimental group, compared with the control group
containing only nirB,the growth rate descent of the experimental group is only about 10% to 20%, which
is acceptable,
because the expression of foreign protein will have a certain impact on the growth of *E. coli*, but
there is no significant reduction.

From the perspective of fluorescence protein intensity, the fluorescence intensity of Escherichia coli
introduced with leghemoglobin and laccase part was significantly higher than that of the control group, and the
effect
of co-introduction was more significant than that of single introduction.

Based on the functional verification of the hypoxia response part, it is proved that the two parts of
leghemoglobin and laccase play a role in reducing the intracellular oxygen without affecting the cell growth,
and the combination of the two parts is more effective in reducing oxygen.

#### Detection module result analysis

First, we verified the function of the selected oxygen response parts nirB and icd.

#### Functional verification of nirB

In the functional verification of nirB part, we created a hypoxic environment through the cysteine culture
medium. As an experimental strain, the control strain was cultured under normal oxygen concentration. The OD600
of the two groups of strains were measured at different culture times, and the red fluorescence intensity of the
experimental group and the control group was detected after 22 hours of culture.

From the experimental results, after introducing nirB part into *E. coli*, compared with the control strain
without part, the OD value has no significant change in its growth process, which proves that nirB part has no
effect on the growth of *E. coli*. When cultured for 22 h, the test results of fluorescence microscope
showed that
the strains in the experimental group after the introduction of nirB part did produce red fluorescence.
Moreover, through the detection of fluorescence intensity, the red fluorescence intensity of the experimental
group was significantly higher than that of the control group, which proved that the hypoxia response part nirB
we selected successfully played its role, and the ability to express genes under hypoxic conditions was higher
than the normal oxygen concentration.

![img](https://static.igem.wiki/teams/4232/wiki/result-image/01-1.jpg)

Figure 2: OD and fluorescence intensity of nirB

![img](https://static.igem.wiki/teams/4232/wiki/result-image/01-2.jpg)

Figure 3: nirB Observation image by fluorescence microscope

#### Function verification of icd

In the function verification of icd part, we mainly verified the reaction of oxygen consumption rate of the
part. In the hypoxic culture environment created by using cysteine medium, the change of fluorescence intensity
with cell growth after the introduction of icd part was detected. From the experimental results, it can be seen
that the cells enter a stable phase after 12 hours, and the cell growth before the stable phase is a logarithmic
phase. At this stage, the cells grow rapidly and the oxygen consumption rate is high. It can be seen from the
intensity of green fluorescent protein that the intensity of green fluorescent protein decreases with the
logarithmic growth process, which proves the negative correlation between icd and oxygen consumption rate, and
verifies that the icd part has successfully played its role. At the same time, it can be seen from the results
of fluorescence microscopy that the green fluorescence in cells was indeed expressed in the late logarithmic
growth period of cells under the condition of hypoxia culture medium.

![img](https://static.igem.wiki/teams/4232/wiki/result-image/02-1.jpg)

![img](https://static.igem.wiki/teams/4232/wiki/result-image/02-2.jpg)

Figure 4: OD and fluorescence intensity of icd-Cys

![img](https://static.igem.wiki/teams/4232/wiki/result-image/03.jpg)

Figure 5: icd Observation image by fluorescence microscope

#### Function module data analysis

The OD value of the experimental data was measured from the third hour to the end of the 12th hour, that is, six
measurements were made, while the fluorescence intensity was measured from the fourth hour to the end of the
12th hour, a total of five measurements were made.

In the experimental group of leghemoglobin (as shown in Fig. 6), the addition of hemin can significantly reduce
the intracellular oxygen, which is 29.6% lower than that of the control group only introducing nirB. After
adding hemin in the culture, we found that the growth curve of the experimental group with hemin was higher than
that of the experimental group without hemin, indicating that the direction of adding hemin in our engineering
iteration was correct.

![img](https://static.igem.wiki/teams/4232/wiki/result-image/04.png)

Figure 6: Fluorescence intensity and OD of bacteria with Lb,with or without hemin

In the laccase experimental group (as shown in Figures 7), we first conducted the experiment of only
introducing laccase. The oxygen reduction efficiency was only 9.9%, and the growth was also affected. Later,
through literature review, it was found that adding copper ions would help laccase catalysis, so we carried out
the experiment of adding 0.2 mmol/L and 0.5 mmol/L copper ions. It was found that the oxygen reduction
efficiency of the experimental group added 0.2 mmol/L copper ion reached 37.3%, significantly improving the
laccase effect; Although the experimental group added 0.5mmol/L copper ion is better than 0.2mmol/L in growth,
it is obviously insufficient in oxygen reduction efficiency.

![img](https://static.igem.wiki/teams/4232/wiki/result-image/05.png)

Figure 7: Fluorescence intensity and OD of bacteria with CueO, adding 0/0.2/0.5 mmol/L
Cu2+ in the culture

Because leghemoglobin and laccase are different in the principle of consuming oxygen, if co culture complements
each other in effect, the effect of reducing oxygen will be better than that of introducing them alone, so we
carried out combined expression. In the experimental group containing both leghemoglobin, heme and laccase (in
Figures 8), the concentration of 0.5mmol/L copper ion has the largest role in promoting the production of
hypoxic environment, while the effects of different concentrations of copper ion on hypoxic environment are
different. Considering the effects of cell growth and oxygen reduction, we chose the experimental group of
0.5mmol/L copper ion. Its oxygen reducing effect reached 45.8%, which was 19.2% and 8.5% higher than that of
single leghemoglobin and laccase, respectively. It proved that the combined expression effect was better than
that of single functional part.

![img](https://static.igem.wiki/teams/4232/wiki/result-image/06.png)

Figure 8: Fluorescence intensity and OD of bacteria with Lb and CueO, adding hemin and
0/0.2/0.5 mmol/L Cu2+ in the culture

At the same time, we found an interesting phenomenon in the experiment. When 0.5mmol/L copper ion alone acts
with laccase, the oxygen reduction effect is not good, but after the introduction of leghemoglobin, the best
effect is achieved. Our guess is that copper ions can promote the expression of leghemoglobin to a certain
extent. Although 0.5 mmol/L copper ions have poor oxygen reducing effect, they may promote the expression of
leghemoglobin to a greater extent, making it a group that plays the largest role in promoting the expression of
leghemoglobin when both leghemoglobin and laccase are contained. Later, we also found a similar phenomenon in a
literature, which supports our guess.

In our communication with teachers, the above four analyses were praised and affirmed by Dr. Yang Yu.

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