iGEM 2023 — Team nwu-china-a

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

## Engineering

Inflammatory bowel disease (IBD) is a group of diseases that cause long-term inflammation in the colon
and small intestine, including ulcerative colitis and Crohn 's disease, which may further induce more
serious and fatal diseases, such as colorectal cancer. Reactive oxygen species (ROS) plays an important
role in the progression of IBD[1][2]. CAT (catalase) and SOD (super-oxide dismutase) can
eliminate reactive oxygen species and alleviate inflammation[3][4]. The inhibition
of elastase and protease-3 by Elafin can attenuate several key processes in the inflammatory
cascade[5]. Our ultimate goal is to construct a recombinant **Escherichia
coli** Nissle 1917 (EcN) that can simultaneously express CAT, SOD and Elafin. We
first studied on **E. coli** BL21 to confirm whether these genes can be normally
expressed in prokaryotes and whether they have activity. After that, we transformed three genes into EcN
to express and construct recombinant EcN that can alleviate inflammatory bowel disease.

****The first iteration:****

****Design****: pET-28a(+) is a commonly used vector, and we are
familiar with it, so we decided to use it as a vector to verify the feasibility of our design. We
inserted CAT and SOD genes into pET-28a(+) plasmid respectively , and verified their expression in
**E. coli**.

****Build****: Using pET-28a(+) as a vector, pET-28a(+)-CAT and pET-28a
(+)-SOD expression vectors were constructed respectively.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image1.jpg)

****Figure 1-**** The plasmid map of pET-28a(+)-CAT

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image2.jpg)

****Figure 2-**** The plasmid map of pET-28a(+)-SOD

****Test********:****

1.Two plasmids were transformed into **E. coli** DH5α, respectively.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image3.jpg)

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image4.jpg)

****Figure 3-****Left image: The DH5α colonies containing pET-28a(+)-CAT
on LB solid culture medium ; Right image : The DH5α colonies containing pET-28a(+)-SOD on LB solid
culture medium

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image5.jpg)

****Figure 4-**** Theagarose gel
electrophoresis results of plasmid pET-28a(+)-CAT and pET-28a(+)-SOD

2.These two plasmids were transformed into BL21 respectively.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image6.jpg)

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image7.jpg)

****Figure 5-**** Left image: The BL21 colonies containing pET-28a(+)-CAT
on LB solid culture medium ; Right image : The BL21 colonies containing pET-28a(+)-SOD on LB solid
culture medium

3.(1) The two plasmids were transformed into BL21 respectively, and the expression of different time
periods was verified after IPTG induction.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image8.jpg)

****Figure 6-****The two control groups were BL21 induced by IPTG for 12 h and
the supernatant of BL21 containing pET-28a(+)-CAT plasmid without induction for 12 h, respectively. The
experimental group was the supernatant of the engineering strain BL21 containing pET-28a(+)-CAT plasmid
 broken at different time periods.

The results showed that we successfully expressed CAT in BL21 strain. After gray scale analysis, we
found that the expression of CAT was the highest at 6h.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image9.jpg)

****Figure 7-****The control group was the supernatant of BL21 with
pET-28a(+)-SOD without induction for 12 h. The experimental group was the supernatant of the engineering
strain BL21 containing pET-28a(+)-SOD plasmid  broken at different time periods.

The results showed that we successfully expressed SOD in BL21 strain. After gray analysis, we found
that the expression of SOD protein was the highest at 6h.

(2) The activity of CAT and SOD was detected:

1. The activity of CAT was detected by bubble method.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image10.jpg)

****Figure 8****-The diagram of the activity of CAT protein expressed in
**E.****coli** BL21 transformed with
recombinant plasmid pET-28a(+)-CAT.  From left to right, the control groups were LB medium,
BL21 bacterial solution without plasmid introduction and BL21(CAT) bacterial solution without induction
of CAT expression. And the experimental groups were the BL21(CAT) bacterial liquid that was induced and
cultured for 2, 6, 10, 14 and 18h. (Video: [NWU-CHINA-A: Detection of CAT activity in
E.coli BL21(DE3) transformed by pET-28a(+)-CAT (2023) [English] - iGEM Video Universe](https://video.igem.org/w/49Np6hUhcb9g9LWyMa3tR6))

We expressed active CAT, and the activity was higher at 14 hours.

1. The activity of SOD in different time periods was tested by Pyrogallol solution method.

 Pyrogallol solution can be rapidly self-oxidized under alkaline conditions, releasing superoxide
anion radicals (O2-), generating intermediate products with special absorption at 325 nm. The addition
of SOD enzyme solution can scavenge superoxide anion free radicals, prevent the accumulation of
intermediate products, and reduce the absorbance at 325 nm.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image11.jpg)

****Figure 9-****The calculation formula of SOD activity measured by Pyrogallol
solution method

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image12.jpg)

****Figure 10****- The results of SOD activity were measured by Pyrogallol
solution method, and the inhibition rate of Pyrogallol solution reaction was induced by IPTG at
different time.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image13.jpg)

****Figure 11-****The activity of SOD protein induced by IPTG for 0-24 hours
was measured by Pyrogallol solution method. The abscissa represents the time we induce expression,
and the ordinate represents the inhibition rate.

Conclusion: The active SOD protein was expressed, and the activity of SOD protein was the highest at 8h.

****Learn****: Considering that plasmid incompatibility occurs when two
homologous plasmids are introduced into a bacteria at the same time, we want to co-express the two
genes by inserting them on the same vector.

****The second iteration****

****Design:**** The CAT and SOD genes were simultaneously inserted on the
pET-28a(+) vector, so that they can be expressed simultaneously and  our engineered probiotics
have a stronger role in alleviating IBD.

****Build:**** We added the SOD gene sequence downstream of the CAT gene
sequence. (Both CAT and SOD are preceded by RBS sequences.). Considering that this vector needs to play
a role in EcN, and the absence of T7 RNA polymerase in EcN makes T7 promoter unable to work, so we
changed the promoter of the vector from T7 promoter to T5 promoter. The
vector pET-28a(+)-T5-CAT-SOD was obtained.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image14.jpg)

****Figure 12-****Theplasmid map of
pET-28a(+)-T5-CAT-SOD

****Test：****The plasmid was transformed into **E.
coli** DH5α.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image15.jpg)

****Figure 13-**** The DH5α colonies containing pET-28a(+)-CAT-SOD
on LB solid culture medium

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image16.jpg)

****Figure 14-**** Theagarose gel
electrophoresis results of plasmid pET-28a(+)-CAT-SOD

****Learn：****Because we finally plan to connect the three genes together, this
iteration does not perform protein expression detection.

****The third iteration****

****Design:**** In order to enhance the role of our engineered bacteria,
we introduced another peptide, Elafin, which can alleviate inflammatory bowel disease. At the same time,
in order to avoid the problem of incompatibility of homologous plasmids, we selected pCDFDuet-1 from
different sources of pET-28a(+) as the expression vector of Elafin.

****Build:**** We selected the plasmid vector pCDFDuet-1 with different sources
from pET-28a(+) as the expression vector of Elafin. The amino acid sequence of Elafin protein was
obtained on UniProt, and its nucleic acid sequence was obtained by reverse transcription on NCBI, and
codon optimization was performed to finally obtain pCDFDuet-1-Elafin.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image17.jpg)

****Figure 15-****The plasmid map of pCDFDuet-1-Elafin

****Test：****The plasmid pCDFDuet-1-Elafin was transformed into **E.
coli** DH5α.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image18.jpg)

****Figure 16-**** The DH5α colonies containing pCDFDuet-1-Elafin on
LB solid culture medium

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image19.jpg)

****Figure 17-**** Theagarose gel
electrophoresis results of plasmid pCDFDuet-1-Elafin

We successfully transferred pCDFDduet-1-Elafin into DH5α.

****Learn:**** Because we finally plan to connect the three genes
together, this iteration does not perform protein expression detection. Next, we need to verify whether
our three genes can play a role in **E. coli** at the same time.

****The fourth iteration****

****Design********:****In
order to make the three proteins play a role at the same time and enhance the ability of engineering
bacteria to treat diseases, we integrated CAT, SOD and Elafin genes into a vector.

****Build:**** We ligated the Elafin gene sequence to the downstream of the
vector pET-28a(+)-T5-CAT-SOD to construct pET-28a(+)-T5-CAT-SOD-Elafin to achieve their co-expression.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image20.jpg)

****Figure 18-****The plasmid map of pET-28a(+)-T5-CAT-SOD-Elafin

****Test：****

1.The pET-28a(+)-T5-CAT-SOD-Elafin was transformed into DH5α ****and****BL21, respectively.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image21.jpg)

****Figure 19-****The DH5α colonies containing pET-28a(+)-CAT-SOD-Elafin
on LB solid culture medium

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image22.jpg)

****Figure 20-****Theagarose gel
electrophoresis results of plasmid pET-28a(+)-CAT-SOD-Elafin

The pET-28a(+)-T5-CAT-RBS-SOD-Elafin was transformed into BL21 coated LB solid plate by heat shock
method.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image23.jpg)

****Figure 21-**** The BL21 colonies containing pET-28a(+)-CAT-SOD-Elafin
on LB solid culture medium

2. Test whether the three proteins can be normally expressed, and whether the expression level and
   activity of the expressed proteins are changed.

(1) SDS-PAGE was used to detect protein expression.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image24.jpg)

****Figure 22-**** The control group was the supernatant of BL21 with
pET-28a(+)-CAT-SOD-Elafin without induction for 10h and BL21 bacterial solution without plasmid
introduction for 10h. The experimental group was the supernatant of the engineering strain BL21
containing pET-28a(+)-CAT-SOD-Elafin plasmid  broken at different time periods.

We successfully expressed CAT, SOD and Elafin in BL21, and the expression of 6-8h was more.

(2) The activity of CAT was detected by bubble method.

We determined the activity of CAT by adding hydrogen peroxide at the same time to observe bubble
production. It was verified that we successfully expressed active CAT.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image25.jpg)

****Figure 23****-The diagram of the activity of CAT protein expressed in
**E. coli** BL21 transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From
left to right, the control groups were LB medium, BL21 bacterial solution without plasmid introduction
and BL21(CAT) bacterial solution without induction of CAT expression. The experimental group was induced
and cultured for 4, 8, 10, 12, 14 and 18h. (video: [NWU-CHINA-A: Detection of CAT activity in
E. coli BL21(DE3) transformed by pET-28a(+)-CAT-SOD-Elafin. (2023) [English] - iGEM Video
Universe](https://video.igem.org/w/kYBJMyesEmfyYP5kQCnpPm))

The results showed that we successfully expressed CAT in BL21 strain, and It was verified that we
successfully expressed active CAT.

(3) The activity of SOD was detected by Pyrogallol solution method.

We reflected the SOD content in the sample by the inhibition rate of Pyrogallol solution autoxidation
rate. It was verified that we successfully expressed active SOD. The calculation formula and the change
of inhibition rate with the induced expression time are as follows:

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image11.jpg)

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image26.jpg)

****Figure 24****-The calculation method of SOD activity detected by Pyrogallol
solution method

****Figure 25-****The plasmid pET-28a(+)-T7-SOD measured by Pyrogallol solution
method was transformed into BL21 strain and IPTG was added to induce the activity of SOD protein for
0-20 hours. The abscissa represents the time we induce expression, and the ordinate represents the
inhibition rate.

We expressed active SOD, and the activity was the highest at 2h.

(4)Determination of Elafin Activity by Egg White Assay

Protease K is a highly efficient serine proteinase. Elafin can act on serine proteinase and inhibit its
activity[5], so when we mix a certain amount of active Elafin with protease K, the effect of
protease K will be inhibited, thus reducing the efficiency when acting on egg white. After a certain
period of reaction, the viscosity of egg white will be lower than that of the control group. The
viscosity of the egg white after the reaction can be used to determine whether Elafin is active.

We took 1ml egg white and added it to a 1.5ml centrifuge tube. In the experimental group, 10μL of 20mg
/ ml protease K and 100μL of pET-28a(+)-CAT-SOD-Elafin BL21 bacterial solution induced for 8, 10 and
14h were fully mixed and reacted at 37 °C for 10 minutes. Then the mixture was added to 1ml egg
white and reacted at 55 °C for 25 minutes. In the control group, 10μL 20mg / ml protease K and
100μL double distilled water were fully mixed at 37 °C for 10 minutes, and then the mixture was
added to 1ml egg white and reacted at 55 °C for 25 minutes. Blank control 110μL double distilled
water was reacted at 37 °C for ten minutes, and then the mixture was added to 1ml egg white and
reacted at 55 °C for 25 minutes. Viscosity was observed after the reaction.

## [Vidoe](https://video.igem.org/w/3qspwMZ2Xb8hsPijepGu7n)

NWU-CHINA-A: Elafin activity from
pET-28a(+)-CAT-SOD-Elafin transformation into E.coli BL21(DE3). (2023) [English] - iGEM Video
Universe

It can be clearly seen from the video that the egg white of the 14 h experimental group was still sticky
after the reaction, indicating that our bacterial supernatant inhibited the activity of protease K. It
is proved that we have expressed the active Elafin protein in BL21.

****Learn:**** Our expression is successful, and the use of our engineered
strains should be considered next.

****The fifth iteration****

****Design:**** Considering the safety of food, we transformed
pET-28a(+)-T5-CAT-SOD-Elafin into probiotic EcN.

****Build:**** The pET-28a(+)-T5-CAT-SOD-Elafin was transformed into the
probiotic EcN.

****Test:****

1. pET-28a(+)-T5-CAT-SOD-Elafin was transformed into EcN, respectively.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image27.jpg)

****Figure 26-**** The EcN colonies containing pET-28a(+)-CAT-SOD-Elafin
on LB solid culture medium

2. ****Expression and activity detection****

****(1) SDS-PAGE****

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image28.jpg)

****Figure 27-**** The control group was the supernatant of EcN with
pET-28a(+)-CAT-SOD-Elafin without induction for 8h and EcN bacterial solution without plasmid
introduction for 8h. The experimental group was the supernatant of the engineering strain EcN containing
pET-28a(+)-CAT-SOD-Elafin plasmid  broken at different time periods.

It was proved that we successfully expressed CAT, SOD and Elafin in EcN, and the expression level of 6-8h
was higher.

****(2) The activity of CAT was detected by bubble method.****

We determined the activity of CAT by adding hydrogen peroxide at the same time to observe bubble
production. It was verified that we successfully expressed active CAT.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image29.jpg)

****Figure 28****-The diagram of the activity of CAT protein expressed in
**E. coli** EcN transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From
left to right, the control groups were LB medium, EcN bacterial solution without plasmid introduction
and EcN (CAT) bacterial solution without induction of CAT expression. The experimental group was induced
and cultured for 4, 8, 12, 16and 20h.  (video: [NWU-CHINA-A: Detection of CAT activity in
EcN transformed by pET-28a(+)-CAT-SOD-Elafin. (2023) [English] - iGEM Video
Universe](https://video.igem.org/w/sMk4CKtbseHib9XicELUS4))

The bubbles generated in the experimental groups were much higher than those in the control groups, which
verified that we successfully expressed active CAT.

****(3) The activity of SOD was detected by Pyrogallol solution method.****

We reflected the SOD content in the sample by the inhibition rate of Pyrogallol solution autoxidation
rate. It was verified that we successfully expressed active SOD. The calculation formula and the change
of inhibition rate with the induced expression time are as follows:

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image30.jpg)

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image11.jpg.jpg)

****Figure 29-****The calculation method of SOD activity detected by Pyrogallol
solution method

****Figure 30-****The recombinant plasmid pET-28a(+)-CAT-SOD-Elafin was
transformed into EcN strain to express SOD activity.

It was proved that SOD was active, and the activity was the highest at 4h.

****(4) Determination of Elafin Activity by Egg White Assay****

Protease K is a highly efficient serine proteinase. Elafin can act on serine proteinase and inhibit its
activity[5], so when we mix a certain amount of active Elafin with protease K, the
effect of protease K will be inhibited, thus reducing the efficiency of acting on egg white. After a
certain period of reaction, the viscosity of egg white will be lower than that of the control group. The
viscosity of the egg white after the reaction can be used to determine whether Elafin is active.

We took 1ml egg white and added it to a 1.5ml centrifuge tube. In the experimental group, 10μL of
20mg/ml protease K and 100μL of pET-28a(+)-CAT-SOD-Elafin EcN bacterial solution induced for 14,18
and 20h were fully mixed and reacted at 37 °C for 10 minutes. Then the mixture was added to 1ml egg
white and reacted at 55 °C for 25 minutes. In the control group, 10μL 20mg/ml protease K and
100μL double distilled water were fully mixed at 37 °C for 10 minutes, and then the mixture was
added to 1ml egg white and reacted at 55 °C for 25 minutes. Blank control 110μL double distilled
water was reacted at 37 °C for ten minutes, and then the mixture was added to 1ml egg white and
reacted at 55 °C for 25 minutes. Viscosity was observed after the reaction.

Video- [NWU-CHINA-A: Elafin activity
from pET-28a(+)-CAT-SOD-Elafin transformation into EcN (2023) [English] - iGEM Video
Universe](https://video.igem.org/w/aEYiF2mF6qLZypKPDM7tRY)

It can be clearly seen from the video that the egg white of the 20 h experimental group was still sticky
after the reaction, indicating that our bacterial supernatant inhibited the activity of protease K. It
was proved that we expressed the active Elafin protein in EcN.

****The sixth iteration****

****Design:**** In order to enable our engineered bacteria to specifically
play a role in the location of inflammatory bowel disease, we plan to change the promoter of the plasmid
to HemH promoter.

****Build********:****We
obtained a hydrogen peroxide-inducible promoter ([Part:BBa K1104202 - parts.igem.org](https://parts.igem.org/Part%3ABBa_K1104202)) from
the iGEM part. Seamless cloning primers were designed to reinsert the original T5 promoter with a
hydrogen peroxide-inducible promoter. The pET-28a(+)-HemH promoter-CAT-RBS-SOD-Elafin was obtained.

![](https://static.igem.wiki/teams/4762/wiki/project-engineering/image31.jpg)

****Figure 31-****Plasmid map of pET-28a(+)-HemH promoter-CAT-SOD-Elafin

****Learn********:****We
will finish this work in the near future.

****The seventh iteration****

****Design********:**** In order to prevent the
engineering bacteria from leaking to the outside world and polluting the environment, we decided to set
a suicide switch for the engineering bacteria. In the design of suicide switch, we decided to choose
different suicide switches from previous years in order to innovate.

****Build********:**** Suicide switch has two
main parts: protein synthesis inhibitor (RelE) and its antitoxin (RelB). The translation of the gene
RelE was promoted by conventional RBS, while the translation of the gene RelB was promoted by
temperature-sensitive RBS. The expression of RelB is completed only when the temperature reaches the
body temperature. When the engineered bacteria leave the human body, the temperature drops, inhibiting
the expression of the antitoxin RelB, and the bacteria die due to excessive toxin RelE to prevent
pollution.

****Figure 32-****Action principle diagram of suicide switch

****Learn********:**** Due to limited time, we
failed to complete this iteration. We will apply this suicide switch to our project in the future.

****Quote****

[1]Ramos, G. P., & Papadakis, K. A. (2019). Mechanisms of Disease: Inflammatory Bowel Diseases. Mayo
Clinic proceedings, 94(1), 155–165.

[2]陈丽霏,张世倡,肖林,吕凯结 & 黎志晴.(2020).炎症性肠病中活性氧及抗氧化的研究进展. 中国当代医药(09),24-27.

[3]McCord JM. Superoxid edismu tase：Anenzymatic function for erythrocuprein（hem ocup rein）[J]. J Biol
Chem，1969，244： 6049-6055.

[4]ZHANG Xiaoyan. Research status and application of superoxide dismutase in food[J].Journal of Yangzhou
Vocational University, 2002(01):34-37.

[5]Lee S,Oliver W. Therapeutic potential of human elafin.[J]. Biochemical Society
transactions,2011,39(5).

{% endblock %}

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

## LB Liquid Medium (100 mL)

1. Prepare the following mixture in an Erlenmeyer flask.

   | Element | Volume or Weight |
   | --- | --- |
   | NaCl | 1g |
   | Peptone | 1g |
   | Yeast | 0.5g |
   | Water | 100mL |
2. After they fully dissolve, transfer the liquid to a 250mL Erlenmeyer flask and seal it.
3. Sterilize in a high-pressure steam sterilizer.

## LB Solid Medium (100 mL)

1. Prepare the following mixture in an Erlenmeyer flask.

   | Element | Volume or Weight |
   | --- | --- |
   | NaCl | 1g |
   | Peptone | 1g |
   | Yeast | 0.5g |
   | Agar Powder | 1.5g |
   | Water | 100mL |
2. After they fully dissolve, transfer the liquid to a 250mL Erlenmeyer flask and seal it.
3. Sterilize in a high-pressure steam sterilizer.

## Agarose Gel Electrophoresis (30 mL, 1% Pore Gel)

1. Prepare a small gel plate and a small comb.
2. Weigh 0.3g of agarose.
3. Add 30mL of 1× TAE.
4. Heat in a microwave oven until the agarose is completely dissolved.
5. When the solution cools to below 60°C, add 3μL of GelGreen nucleic acid staining dye and mix thoroughly.
6. Pour the solution onto the flat plate with the comb inserted and use a pipette to remove any bubbles.
7. After solidification (about 30 minutes), carefully remove the comb and soak the gel in 1× TAE buffer.
8. Mix the sample with 6× DNA loading buffer at a volume ratio of 5:1. Add the appropriate amount of DNA marker
   and sample to the sample wells.
9. Open the power supply, set the voltage to 100V, and start running until the bromophenol blue dye reaches the
   bottom of the gel.
10. Remove the gel from the electrophoresis instrument and observe it under a fluorescence projector.

## Polyacrylamide Gel Electrophoresis

1. Assemble the gel-making device and perform a leakage test (add water to observe any leaks).
2. Prepare the separation gel using the following concentrations:

   | Gel Concentration | Water (mL) | 30% Acr-Bis (mL) | Separation Gel Buffer (mL) | Modified Ammonium Persulfate (μL) |
   | --- | --- | --- | --- | --- |
   | 10% | 2.08 | 1.67 | 1.25 | 20 |
   | 12% | 1.75 | 2.0 | 1.25 | 20 |
3. Note: Add modified ammonium persulfate at the end and shake it immediately before pouring it into the glass
   plate.
4. Seal the liquid surface with water or isopropanol.
5. Prepare the concentrated gel using the following volumes:

   | Gel Volume | Water (mL) | 30% Acr-Bis (mL) | Separation Gel Buffer (mL) | Modified Ammonium Persulfate (μL) |
   | --- | --- | --- | --- | --- |
   | 2mL | 1.14 | 0.34 | 0.5 | 10 |
6. Note: Add modified ammonium persulfate at the end and shake it immediately before pouring it into the glass
   plate.
7. Insert the comb and wait for the gel to solidify completely.
8. Prepare samples:
   1. Preparation of bacterial samples:
      * Centrifuge 200μL of bacterial culture for a certain time at 12,000 rpm for 3 minutes, discard the
        supernatant, resuspend the pellet with 20μL of sterile water, and then mix with 5μL of 5× SDS loading
        buffer.
      * Boil the sample in boiling water for 8 minutes, immediately ice-bath for 3 minutes, and then
        centrifuge at 12,000 rpm for three minutes. Take the supernatant when loading.
   2. Preparation of supernatant samples after cell disruption:
      * Fully mix 20μL of supernatant with 5μL of 5× SDS loading buffer.
      * Heat the sample at 98°C for 12 minutes in a PCR instrument, and load the samples after cooling.
9. After gel solidification, carefully remove the comb, place the plate into the electrophoresis tank, add 1×
   SDS buffer to the electrophoresis tank to fill it, and then carefully add the sample to the sample wells.
10. Open the power supply, set the voltage to 100V, and change to 120V when the dye enters the separation gel.
    Stop electrophoresis when the dye moves to 1cm from the bottom.
11. Remove the gel, cut off the corners, dye with Coomassie brilliant blue for 30 minutes, and rinse with
    double-distilled water until the eluent becomes transparent. Gels are ready for subsequent analysis.

## Bacterial Transformation (e.g., transformation of plasmid containing kanamycin resistance gene into E. coli DH5α)

1. Thaw a tube of 100μL DH5α competent cells on ice and add 10μL of the plasmid solution. Gently mix cells and
   DNA by pipetting.
2. Place the mixture on ice for 30 minutes.
3. Perform a 42°C heat shock for 90 seconds.
4. Place the mixture on ice for 2 minutes.
5. Add 800μL LB culture medium to the mixture and incubate at 37°C in a shaking incubator for 1 hour.
6. Centrifuge the culture at 12,000 rpm for 5 minutes to concentrate the cells. Remove a certain amount of
   supernatant and leave 100μL of solution. Gently mix the cells.
7. Take 100mL of LB solid medium, autoclave, and heat in a microwave oven until the medium is transparent and
   free of colloidal precipitation.
8. Place it in a sterile petri dish and add 100μL of kanamycin at a concentration of 50 ng/mL. Shake well and
   immediately pour into the petri dish. Allow it to solidify.
9. Divide the 100μL of cells into several portions and add them to the solidified solid medium, spreading them
   with an applicator. Incubate at 37°C overnight.

## Extraction of DNA from Bacterial Cultures

Omega Plasmid Mini Kit I is used to extract DNA from bacterial cultures.

Application object: Used to extract 1-5 mL of E. coli cultured overnight.

### Preparing

1. Heat the Elution Buffer to 70°C if the plasmid DNA is >10kb.
2. Add a bottle of RNase A to Solution I and store it at 4°C.
3. Dilute DNA Wash Buffer with 120mL absolute ethanol and store it at room temperature.
4. Dilute HBC Buffer with 16mL isopropanol.

### Steps

1. Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 1-5 mL LB medium
   containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (220
   rpm).
2. Take the bacterial solution into a new 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 minute at
   room temperature.
3. Decant or aspirate and discard the culture media. Repeat the operation until all the bacteria are
   centrifuged.
4. Add 250μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of the
   cell pellet is vital for obtaining good yields.
5. Add 250μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3
   minute incubation may be necessary.
6. Add 350μL Solution III. Immediately invert several times until a flocculent white precipitate forms. Make
   sure that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid
   localized precipitation.
7. Perform high-speed centrifugation at 14,500 rpm for 10 minutes.
8. Transfer the supernatant to a HiBind Miniprep DNA binding column with a 2mL collection tube, centrifuge at
   room temperature for 1 minute, and then pour the filtrate into the collection tube.
9. Reinstall the adsorption column into the collection tube, add 500μL HBC buffer, centrifuge at room
   temperature for 1 minute at maximum speed, and then discard the filtrate.
10. Repeat the above step.
11. Discard the filtrate and repeat the above step once more.
12. Discard the filtrate. Transfer the HiBind DNA Mini Column to a 2 mL collection tube. Centrifuge the empty
    HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. After centrifugation, transfer
    the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube, open the lid, and dry at room temperature
    for three minutes.
13. Place the adsorption column in a clean 1.5 mL centrifuge tube, add 40μL of elution buffer to the column
    matrix, let it stand for 1 minute, and centrifuge at 12,000 rpm for 1 minute to elute DNA.
14. The eluted DNA can be stored at -20°C.

## Polymerase Chain Reaction (PCR)

Mix various components before use.

### Components (Volume for 50μL Reaction)

* Q5 High-fidelity 2X Master Mix: 25μL
* 10μM Forward Primer: 2.5μL
* 10μM Reverse Primer: 2.5μL
* Template DNA: 5μL
* Nuclease-Free Water: To 15μL

Mix the reactants gently and rotate quickly to collect the liquid at the bottom of the PCR tube.

The reaction is quickly transferred to a preheated PCR instrument and operated under the provided thermal cycle
conditions.

## Touchdown PCR

Mix various components before use.

### Components (Volume for 50μL Reaction)

* Q5 High-fidelity 2X Master Mix: 25μL
* 10μM Forward Primer: 2.5μL
* 10μM Reverse Primer: 2.5μL
* Template DNA: 5μL
* Nuclease-Free Water: 15μL

Mix the reactants gently and rotate quickly to collect the liquid at the bottom of the PCR tube.

The reaction is quickly transferred to a preheated PCR instrument and operated under the provided thermal cycle
conditions.

## Restriction Endonuclease Digestion (Double Digestion)

### Components (Volume for 50μL Reaction)

* 10× rCutSmart Buffer: 5μL
* Not I-HF Restriction Enzyme: 1μL
* Hind III-HF Restriction Enzyme: 1μL
* DNA Solution: 1μg (Converted into volume)
* Deionized Distilled Water: To 50μL

1. Mix the ingredients on ice.
2. Incubate the mixture in a thermostatic water bath at 37°C for 15-60 minutes.
3. Run gel electrophoresis to check the length of the cut DNA fragment.

## DNA Ligation

1. Set the following reaction system in a micro-centrifugal tube on ice.

### Components (Volume for 20μL Reaction)

* T4 DNA Ligase Buffer (10X): 1μL
* Vector DNA: 50ng (0.020 pmol)
* Insert DNA: 37.5ng (0.060 pmol)
* Deionized Distilled Water: To 10μL
* T4 DNA Ligase: 1μL

2. The solution is gently mixed and incubated at 16°C for 16 hours or overnight at 4°C.

## Induction by IPTG

1. Select a single colony from the plate and grow it in LB liquid medium containing kanamycin (or streptomycin)
   at 37°C overnight.
2. Dilute the overnight culture into 100mL of LB liquid medium and incubate the cells at 37°C until the OD600
   reaches 0.6-0.8.
3. Add 100μL of IPTG (1 mol/L) to the culture and incubate in a shaker at 37°C for 12-16 hours.
4. Store at 4°C.

## Preparation of Competent Cells (Taking E. coli Nissle 1917 as an Example)

1. Select a single colony from the EcN plate and inoculate it into a shaker tube containing 5mL LB liquid
   medium. Culture overnight at 37°C.
2. Transfer 1mL of bacterial culture to an Erlenmeyer flask containing 100mL LB liquid medium and incubate at
   37°C for 2-3 hours until the OD600 reaches about 0.4.
3. Transfer the bacterial culture to a 50mL centrifuge tube that has been sterilized and ice bath for 10
   minutes.
4. Centrifuge the cells at 4°C, 4000 rpm for 10 minutes.
5. Pour out the medium as much as possible with a pipette.
6. Suspend the pellet with 10mL of cold 0.1mol/mL CaCl2. Centrifuge at 4°C, 4000 rpm for 1 minute, and then
   recover the cells.
7. Suspend the bacterial cells with 2mL of cold 0.1mol/mL CaCl2, gently agitate on ice.
8. Use the suspension immediately for plasmid transformation or add an equal volume of pre-cooled 60% glycerol
   solution and store at -80°C for long-term use.

## Ultrasonic Crushing of Bacteria

1. After induction, centrifuge the bacterial culture at 4°C, 4000 rpm for 5 minutes and transfer it to a
   centrifuge tube.
2. Discard the supernatant and retain the bacterial pellet. Add 5mL of PBS buffer to resuspend the bacteria.
3. Centrifuge at 4°C, 4000 rpm for 5 minutes, then discard the supernatant and retain the bacteria.
4. Repeat the washing step once more.
5. Add PBS buffer at a ratio of 10mL of buffer to 1g of bacteria to resuspend the bacteria.
6. Insert the ultrasonic probe and place the surrounding area in ice water. Use the ultrasonic pulverizer for
   15 minutes with cycles of 1 second at 100-150W and 2-second rests at 25.2°C.
7. Centrifuge at 4°C, 12,000 rpm for 20 minutes.
8. Discard the supernatant. The supernatant can be used for SDS-PAGE or activity detection.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Result

****Plasmid Construction****

Firstly, we constructed two plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image1.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image2.jpg)

****Figure 1-****The plasmid profiles of pET-28a(+)-T7-CAT and
pET-28a(+)-T7-SOD

After they were transferred to DH5α, the colony growth on LB medium was observed. The bacteria
containing recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully grown in the
plate.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image3.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image4.jpg)

****Figure 2****-Left image: The DH5α colonies containing pET-28a(+)-CAT
on LB solid culture medium ; Right image: The DH5α colonies containing pET-28a(+)-SOD on LB solid
culture medium

We performed agarose gel electrophoresis to verify the successful introduction of the plasmid.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image5.jpg)

****Figure 3-****The agarose gel electrophoresis result of the recombinant
plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD transformed into DH5α.

Electrophoresis results showed that pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully transferred
into **E.coli** DH5α.

Then, we constructed two plasmids, pET-28a(+)-T5-CAT-SOD and pCDFDuet TM-1-Elafin.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image6.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image7.jpg)

****Figure 4****-The plasmid profiles of pET-28a(+)-T5-CAT-SOD and pCDFDuet
TM-1-Elafin.

After we transferred them to DH5α respectively, their growth on LB medium was observed. The
bacteria containing recombinant plasmids pET-28a(+)-CAT-SOD and pCDFDuet-1-Elafin were successfully
grown in the plate.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image8.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image9.jpg)

****Figure 5****-Left image: The DH5α colonies containing
pET-28a(+)-CAT-SOD on LB solid culture medium ; Right image: The DH5α colonies containing
pCDFDuet-1-Elafin on LB solid culture medium.

We performed agarose gel electrophoresis to verify the successful introduction of the plasmid.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image10.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image11.jpg)

****Figure 6-****The agarose gel electrophoresis results of the recombinant
plasmids pET-28a(+)-CAT-SOD and pCDFDuetTM-1-Elafin transformed into DH5α.

Electrophoresis results showed that pET-28a(+)-T5-CAT-SOD and pCDFDuet™-1-Elafin were successfully
transferred into **E.coli** DH5α.

Finally, in order to make the three proteins play a role at the same time and enhance the ability of the
engineering bacteria to alleviate intestinal inflammation, we cut the Elafin gene on the plasmid
pCDFDuet™-1-Elafin, and connected it to the downstream of the gene SOD in the plasmid
pET-28a(+)-CAT-SOD. The three genes were integrated into a vector to form pET-28a(+)-T5-CAT-SOD-Elafin.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image12.jpg)

****Figure 7****-The plasmid profile of pET-28a(+)-T5-CAT-SOD-Elafin.

We transferred it into DH5α, and then its growth on LB medium was observed. The bacteria
containing the recombinant plasmid pET-28a(+)-CAT-SOD-Elafin were successfully grown in the plate.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image13.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image14.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image15.jpg)

****Figure 8****-The DH5α colonies containing
pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium.

We performed agarose gel electrophoresis to verify the successful introduction of the plasmid.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image16.jpg)

****Figure 9-****The agarose gel electrophoresis result of the recombinant
plasmid pET-28a(+)-CAT-SOD-Elafin transformed into DH5α.

We see the correct size of the stripe. The electrophoresis result showed that we successfully transformed
pET-28a(+)-CAT-SOD-Elafin into DH5α.

****Expression of plasmid****

Firstly, we separately transformed the recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD into
**E.coli** BL21(DE3), and the transformed colonies on LB medium were observed.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image17.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image18.jpg)

****Figure 10****-The left image: the BL21 coated LB solid plate transformed by
pET-28a(+)-CAT was coated with LB solid plate by heat shock method ; right picture: The BL21 transformed
by pET-28a(+)-SOD was coated on LB solid plate by heat shock method.

We performed agarose gel electrophoresis to verify that the plasmids were successfully introduced.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image19.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image20.jpg)

****Figure 11****-The agarose gel electrophoresis results of the recombinant
plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD transformed into BL21 respectively.

The results of electrophoresis showed that pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully
transferred into BL21 .

We measured the growth curves of three strains. As we have seen, the growth curves of all these bacteria
experienced lag phase, logarithmic phase, stationary phase and decline phase.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image21.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image22.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image23.jpg)

****Figure 12****-The growth curves of BL21 transformed with recombinant
plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD and BL21 without plasmid in OD600(the

dotted line in the figure is the trend line).([Raw
data](https://kdocs.cn/l/cmJiL1lLZAfq))

We exogenously added IPTG to induce the expression of related proteins, and broken the cells to take the
supernatant. We observed the correct size of the band by SDS-PAGE.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image24.jpg)

****Figure13****-The SDS-PAGE map of BL21 where the recombinant plasmid
pET-28a(+)-CAT transferred.The two control groups were BL21 induced by IPTG for 12h and the supernatant
of BL21 introduced with pET-28a(+)-CAT without induction for 12h, respectively. The experimental
groups were the supernatant of the recombinant strain CAT broken at different time periods.

We detected the successful expression of CAT in BL21. After gray scale analysis, we found that the
expression of CAT was the highest at 6h.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image25.jpg)

****Figure 14****-The SDS-PAGE map of BL21 where the recombinant plasmid
pET-28a(+)-SOD transferred. The control group was the supernatant of BL21 with pET-28a(+)-SOD
without induction for 12h, and the experimental groups were the

supernatant of recombinant strain SOD in BL21 induced at different time periods.

We detected the successful expression of SOD in BL21. After gray analysis, we found that the expression
of SOD protein was the highest at 6h.

Then, we transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD into EcN, and observed the
recombinant plasmid transformed colonies on LB medium.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image26.jpg)

****Figure 15****-The EcN colonies containing pET-28a(+)-CAT-SOD on
LB solid culture medium

We performed agarose gel electrophoresis to verify its successful transfer.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image27.jpg)

****Figure 16****-The agarose gel electrophoresis result of the recombinant
plasmids pET-28a(+)-T5-CAT-SOD transformed into EcN .

Secondly, we ligated the Elafin gene sequence to the downstream of the vector
pET-28a(+)-T5-CAT-SOD to construct pET-28a(+)-T5-CAT-SOD-Elafin to achieve their co-expression. We
transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin into BL21, and observed the recombinant
plasmid transformed colonies on LB medium.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image28.jpg)

****Figure 17****-The BL21 colonies containing
pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium

We measured the growth curve of the strain. As we can see, the growth curves of all these bacteria
experienced lag phase, logarithmic phase, stationary phase and decline phase.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image29.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image30.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image31.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image32.jpg)

****Figure 18****-The growth curves of BL21 transformed with recombinant
plasmids pET-28a(+)-T5-CAT-SOD-Elafin(induced expression and not induced expression)and BL21 without
plasmid in OD600(the dotted line in the figure is the trend line).

([Raw data](https://kdocs.cn/l/canRsNTXOvq6))

We exogenously added IPTG to induce the expression of related proteins, then broke the cells and took the
supernatant for SDS-PAGE.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image33.jpg)

****Figure 19-****The SDS-PAGE map of BL21 where the recombinant plasmid
pET-28a(+)-T5-CAT-SOD-Elafin transferred. The two control groups were BL21 which was induced for
10h and BL21 which was introduced into pET-28a(+)-T5-CAT-SOD-Elafin without induction for 10h.The
experimental groups were to induce the expression of recombinant strains in BL21 at different
time periods.

We observed the correct bands of CAT, SOD and Elafin, which proved the successful expression of the
three. And the expression of CAT in the bacterial solution of 6-8h was more.

Finally, we transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin into EcN, and the
transformed colonies on LB medium were observed.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image34.jpg)

****Figure 20****-The EcN colonies containing
pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium

We measured the growth curve of the strain. As we can see, the growth curves of all these bacteria
experienced lag phase, logarithmic phase, stationary phase and decline phase.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image35.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image36.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image37.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image38.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image39.jpg)

****Figure 21-****The growth curves of EcN transformed with recombinant plasmid
pET-28a(+)-T5-CAT-SOD-Elafin(Induced expression and not induced expression)and

EcN without plasmid in OD600(the dotted line in the figure is the trend line)

[(Raw data)](https://kdocs.cn/l/ceK7I17RXk2e)

We exogenously added IPTG to induce the expression of related proteins, then broke the cells and took the
supernatant. SDS-PAGE showed a band of the correct size.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image40.jpg)

****Figure 22****-The SDS-PAGE map of EcN where the recombinant plasmid
pET-28a(+)-T5-CAT-SOD-Elafin transferred. The two control groups were EcN introduced into
pET-28a(+)-T5-CAT-SOD-Elafin with EcN induced for 8h and without induction for 8h.The experimental
groups were to induce the expression of recombinant strains in EcN at different time periods.

We successfully expressed CAT, SOD and Elafin in EcN, and the expression of 6-8h was more.

****Activity determination****

1.The recombinant plasmid pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were transformed into BL21 strain to
express CAT and SOD activity.

****Detecting the activity of CAT in BL21 by bubble method.****

Firstly, we conducted a preliminary experiment to explore the relationship between the amount of
bacteria and hydrogen peroxide and the amount of bubbles produced, so as to determine the amount of
bacteria and hydrogen peroxide added in our formal experiment.

* Pre-experiment :We judged the activity of CAT by adding hydrogen peroxide at the same time to
  observe bubble production. It was verified that we successfully expressed active CAT.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image41.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image42.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image43.jpg)

(a)

(b)                               (c)

****Figure 23****-The diagram of the activity of CAT protein expressed in
**E.coli** BL21(CAT) transformed with recombinant plasmid pET-28a(+)-CAT.(a)From
left to right, 5ml bacterial solution was cultured with IPTG for 4h, 8h, 12h, 16h and 18h, and 1ml
volume fraction of 7.5 % hydrogen peroxide was added to it at the same time.(b)From left to right, the
control groups : the supernatant of LB medium and BL21(CAT) bacterial solution without IPTG
after centrifugation ; the experimental groups : The supernatant of BL21(CAT) bacterial
solution centrifuged at 4h, 8h, 12h, 16h and 18h was cultured with IPTG. Each group of 5ml was added
with 1ml volume fraction of 7.5 % hydrogen peroxide at the same time.(c)From left to right, the control
groups : LB liquid medium, supernatant after centrifugation of BL21(CAT) cultured without
IPTG, sediment resuspension after centrifugation of BL21(CAT) cultured without IPTG, BL21(CAT)
cultured without IPTG, supernatant after centrifugation of BL21(CAT) cultured with IPTG, sediment
resuspension after centrifugation of BL21(CAT) cultured with IPTG, and BL21(CAT) cultured with IPTG
;

each group of 6ml, while adding 1ml 7.5 % hydrogen peroxide.

Analysis :(a) A large number of bubbles were generated after the bacterial solution was directly added
with hydrogen peroxide. It shows that the bacterial solution containing CAT or hydrogen peroxide
destroys the cells to release CAT. We expressed active CAT. The supernatant after centrifugation of the
bacterial solution produced bubbles after hydrogen peroxide, but the amount was very small.(b)The
bubbles generated by directly adding hydrogen peroxide to the bacterial solution were much more than
those generated by adding hydrogen peroxide to the supernatant after centrifugation, indicating that 7.5
% hydrogen peroxide could destroy BL21 cells.(c)The amount of oxygen produced by adding hydrogen
peroxide after the centrifugal precipitation of the bacterial solution was re-suspended was less than
the amount of oxygen produced by adding hydrogen peroxide directly to the bacterial solution, but it was
greater than the amount of oxygen produced by adding hydrogen peroxide to the supernatant of the
bacterial solution by centrifugation. It can be seen from the results that BL21 without induction
culture can produce CAT itself, but the amount is relatively small. It shows that CAT protein is mainly
present in the cell and rarely secreted outside the cell.

The results of preliminary experiments showed that we successfully expressed CAT protein in BL21. The
preliminary experiment provides a reference for our formal experiment. We chose to add 1ml 7.5 %
hydrogen peroxide to 4ml bacterial solution.

* Formal experiment: 4ml of LB medium, BL21 bacterial solution without plasmid, BL21 bacterial
  solution without inducing expression of CAT and BL21 bacterial solution induced for 2,6,10,14 and
  18h were added respectively, and 1ml 7.5 % hydrogen peroxide was added at the same time. The results
  are as follows:

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image44.jpg)

****Figure 24****-The diagram of the activity of CAT protein expressed in
**E. coli** BL21 transformed with recombinant plasmid pET-28a(+)-CAT.  From left
to right, the control groups were LB medium, BL21(CAT) bacterial solution without plasmid
introduction and BL21(CAT) bacterial solution without induction of CAT expression. And the
experimental groups were the BL21(CAT) that was induced and cultured for 2, 6, 10, 14 and 18h.
(****Video****: [NWU-CHINA-A: Detection of CAT activity in
E.coli BL21(DE3)transformed by pET-28a(+)-CAT(2023)[English] - iGEM Video
Universe](https://video.igem.org/w/49Np6hUhcb9g9LWyMa3tR6))

The results showed that we successfully expressed CAT protein in BL21, and the activity of CAT protein
was higher.

****Detecting the activity of SOD by**** ****pyrogallol solution
solution********method.****

We reflected the SOD content in the sample by the inhibition rate of pyrogallol solution
solution autoxidation rate. It was verified that we successfully expressed active SOD. The
calculation formula and the change of inhibition rate with the induced expression time are as follows:

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image45.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image46.jpg)

****Figure**** ****25****-The diagram about the
determination of SOD activity after the recombinant plasmid pET-28a(+)-T7-SOD was transformed into
BL21(SOD). The activity of SOD protein induced by IPTG for 0-24 hours was measured by pyrogallol
solution solution method. The abscissa represents the time we induce expression,

and the ordinate represents the inhibition rate. ([Raw
data](https://kdocs.cn/l/caHNp7paeZ2u))

The results showed that our recombinant strain could express active SOD protein, and the activity was the
best at 8 hours.

2.The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into BL21 to express CAT, SOD
and Elafin activity determination.

****Detecting the activity of CAT in BL21 by bubble method.****

We judged the activity of CAT by adding hydrogen peroxide at the same time to observe bubble production.
It was verified that we successfully expressed active CAT.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image47.jpg)

****Figure 26****-The diagram of the activity of CAT protein expressed in
**E. coli** BL21 transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From
left to right, the control groups were LB medium, BL21(CAT) bacterial solution without
plasmid introduction and BL21(CAT) bacterial solution without induced expression of CAT. The
experimental groups were induced to culture BL21(CAT) bacterial solution for 4,8,10,12,14
and 18h.([NWU-CHINA-A: Detection of CAT
activity in E. coli BL21(DE3)transformed by pET-28a(+)-CAT-SOD-Elafin.(2023)[English] - iGEM
Video Universe](https://video.igem.org/w/kYBJMyesEmfyYP5kQCnpPm))

The bubbles generated in the experimental groups were much higher than those in the control groups,
and the CAT activity was high. It was verified that we successfully expressed active CAT.

****Detecting the activity of SOD by**** ****pyrogallol solution
solution********method.****

We reflected the SOD content in the sample by the inhibition rate of pyrogallol solution
solution autoxidation rate. It was verified that we successfully expressed active SOD. The
calculation formula and the change of inhibition rate with the induced expression time are as follows :

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image48.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image49.jpg)

****Figure 27****-The diagram about the determination of SOD activity after the
recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into BL21. The plasmid
pET-28a(+)-T7-SOD measured by pyrogallol solution solution method was transformed into
BL21(SOD) and IPTG was added to induce the activity of SOD protein for 0-20 hours. The abscissa
represents the time we induce expression, and

the ordinate represents the inhibition rate.([Raw
data](https://kdocs.cn/l/cv13KLhxUHuE))

The results showed that our recombinant strain could express active SOD protein, and the activity was
highest at 2 hours.

****Determination of Elafin Activity by Egg White Assay****

According to the principle that protease can hydrolyze protein, and Elafin can inhibit the activity of
protease, we designed the reaction experiment of egg white and protease K. After the reaction, the
viscosity of the solution was added to the BL21 bacterial solution that we induced and cultured for 8,
10 and 14h, respectively, to react, and observed the viscosity after the reaction.

****Video-****[NWU-CHINA-A: Elafin activity from
pET-28a(+)-CAT-SOD-Elafin transformation into E.coli BL21(DE3).(2023)[English] - iGEM Video
Universe](https://video.igem.org/w/3qspwMZ2Xb8hsPijepGu7n)

The experimental results showed that the viscosity of BL21 bacterial solution at 14h was higher and the
Elafin activity was higher. It is proved that we have expressed the active Elafin protein in BL21.

****3****.The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed
into EcN to express CAT, SOD and Elafin activity determination.

****Detecting the activity of CAT in BL21 by bubble method.****

We judged the activity of CAT by adding hydrogen peroxide at the same time to observe bubble production.
It was verified that we successfully expressed active CAT.

****Figure 2********8****-The diagram of the activity
of CAT protein expressed in EcN transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin.
From left to right, the control groups were LB medium, EcN(CAT) bacterial solution
without plasmid introduction and EcN(CAT) bacterial solution without induced expression of CAT. The
experimental groups were induced to culture EcN(CAT) bacteria solution for
4,8,12,16,20h.([NWU-CHINA-A: Detection of
CAT activity in EcN transformed by pET-28a(+)-CAT-SOD-Elafin.(2023)[English] - iGEM Video
Universe](https://video.igem.org/w/sMk4CKtbseHib9XicELUS4))

The bubbles generated in the experimental groups were much higher than those in the control groups,
which verified that we successfully expressed active CAT.

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image49.jpg)

![](https://static.igem.wiki/teams/4762/wiki/preject-result/image50.jpg)

****Detecting the activity of SOD by**** ****pyrogallol solution
solution********method.****

We reflected the SOD content in the sample by the inhibition rate of pyrogallol solution
solution autoxidation rate. It was verified that we successfully expressed active SOD. The
calculation formula and the change of inhibition rate with the induced expression time are as follows :

****Figure 2********9****-The diagram about the
determination of SOD activity after the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed
into EcN. The recombinant plasmid pET-28a(+)-CAT-SOD-Elafin was transformed into EcN to express SOD
activity. The abscissa represents the time we induce expression, and the

ordinate represents the inhibition rate.([Raw data](https://kdocs.cn/l/cbQFwZMaMRRd))

The results showed that we successfully expressed the active SOD in the EcN, and the activity was highest
at 4 hours.

****Determination of Elafin Activity by Egg White Assay****

According to the principle that protease can hydrolyze protein, and Elafin can inhibit the activity of
protease, we designed the reaction experiment of egg white and protease K. After the reaction, the
viscosity of the solution was added to the BL21 bacterial solution that we induced and cultured for
14,18 and 20 h, respectively, to react, and observed the viscosity after the reaction.

****Video-****[NWU-CHINA-A: Elafin activity from
pET-28a(+)-CAT-SOD-Elafin transformation into EcN(2023)[English] - iGEM Video Universe](https://video.igem.org/w/aEYiF2mF6qLZypKPDM7tRY)

The experimental results showed that the viscosity of EcN bacterial solution at 20 h was higher and the
activity of Elafin was higher. It was proved that we expressed the active Elafin protein in EcN.

{% endblock %}