iGEM 2024 — Team nku-china

![loading](https://static.igem.wiki/teams/5332/homeimage/loading.gif)

[May](#May)

[June](#June)

[July](#July)

[Augest](#Augest)

## May

#### In Vitro Experiments

[5.2 Macrophage
Thawing](https://static.igem.wiki/teams/5332/project/notebook/5-2-macrophage-thawing.pdf)

[5.3-5.4 Macrophage Passage](https://static.igem.wiki/teams/5332/project/notebook/5-3-5-4-macrophage-passage.pdf)

[5.5 Macrophage Plating](https://static.igem.wiki/teams/5332/project/notebook/5-5-macrophage-plating.pdf)

[5.5 S.Tm Liquid Culture](https://static.igem.wiki/teams/5332/project/notebook/5-5-s-tm-liquid-culture.pdf)

[5.6 S.Tm Subculture](https://static.igem.wiki/teams/5332/project/notebook/5-6-s-tm-subculture.pdf)

[5.6 Macrophages Starvation
Treatment](https://static.igem.wiki/teams/5332/project/notebook/new/5-6-macrophages-starvation-treatment-1.pdf)

[5.6 S. Tm-macrophage
Infection](https://static.igem.wiki/teams/5332/project/notebook/new/5-6-s-tm-macrophage-infection-1.pdf)

[5.6 Cell Condition
Determination](https://static.igem.wiki/teams/5332/project/notebook/5-6-cell-condition-determination.pdf)

[5.7 Macrophages Passage](https://static.igem.wiki/teams/5332/project/notebook/5-7-macrophages-passage.pdf)

[5.8 Macrophages Plating](https://static.igem.wiki/teams/5332/project/notebook/5-8-macrophages-plating.pdf)

[5.8 S.Tm Liquid Culture](https://static.igem.wiki/teams/5332/project/notebook/5-8-s-tm-liquid-culture.pdf)

[5.9 S.Tm Subculture](https://static.igem.wiki/teams/5332/project/notebook/5-9-s-tm-subculture.pdf)

[5.9 Macrophages Starvation
Treatment](https://static.igem.wiki/teams/5332/project/notebook/new/5-9-macrophages-starvation-treatment-1.pdf)

[5.9 Preparation of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-9-preparation-of-culture-medium.pdf)

[5.9 Sterilization of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-9-sterilization-of-culture-medium.pdf)

[5.9 Pouring of Culture Medium](https://static.igem.wiki/teams/5332/project/notebook/5-9-pouring-of-culture-medium.pdf)

[5.9 S. Tm-macrophage
Infection](https://static.igem.wiki/teams/5332/project/notebook/5-9-s-tm-macrophage-infection.pdf)

[5.9 Macrophage Collecting](https://static.igem.wiki/teams/5332/project/notebook/5-9-macrophage-collecting.pdf)

[5.9 Cell Number Counting](https://static.igem.wiki/teams/5332/project/notebook/5-9-cell-number-counting.pdf)

[5.9 Determination of Reactive
Oxygen Species (ROS)
Content](https://static.igem.wiki/teams/5332/project/notebook/5-9-determination-of-reactive-oxygen-species-ros-content.pdf)

[5.9 Cell Lysis, Bacterial
Suspension Dilution](https://static.igem.wiki/teams/5332/project/notebook/5-9-cell-lysis-bacterial-suspension-dilution.pdf)

[5.9 Bacterial Plating](https://static.igem.wiki/teams/5332/project/notebook/5-9-bacterial-plating.pdf)

[5.10-5.11 Bacterial Colony Number
Counting](https://static.igem.wiki/teams/5332/project/notebook/5-10-5-11-bacterial-colony-number-counting.pdf)

[5.11 RNA Extraction](https://static.igem.wiki/teams/5332/project/notebook/5-11-rna-extraction.pdf)

[5.11 Reverse Transcription](https://static.igem.wiki/teams/5332/project/notebook/5-11-reverse-transcription.pdf)

[5.12 RT-qPCR](https://static.igem.wiki/teams/5332/project/notebook/5-12-rt-qpcr.pdf)

[5.13-5.14 Data and Results
Analysis](https://static.igem.wiki/teams/5332/project/notebook/5-13-5-14-data-and-results-analysis.pdf)

[5.16-5.17 Macrophage Passage](https://static.igem.wiki/teams/5332/project/notebook/5-16-5-17-macrophage-passage.pdf)

[5.18 Macrophages Plating](https://static.igem.wiki/teams/5332/project/notebook/5-18-macrophages-plating.pdf)

[5.18 S.Tm Liquid Culture](https://static.igem.wiki/teams/5332/project/notebook/5-18-s-tm-liquid-culture.pdf)

[5.19 S.Tm Subculture](https://static.igem.wiki/teams/5332/project/notebook/5-19-s-tm-subculture.pdf)

[5.19 Macrophages Starvation
Treatment](https://static.igem.wiki/teams/5332/project/notebook/new/5-19-macrophages-starvation-treatment-1.pdf)

[5.19 Preparation of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-19-preparation-of-culture-medium.pdf)

[5.19 Sterilization of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-19-sterilization-of-culture-medium.pdf)

[5.19 Pouring of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-19-pouring-of-culture-medium.pdf)

[5.19 S. Tm-macrophage
Infection](https://static.igem.wiki/teams/5332/project/notebook/new/5-19-s-tm-macrophage-infection-1.pdf)

[5.19 Peptide Dilution](https://static.igem.wiki/teams/5332/project/notebook/5-19-peptide-dilution.pdf)

[5.19 Cell Status Observation](https://static.igem.wiki/teams/5332/project/notebook/5-19-cell-status-observation.pdf)

[5.23-5.24 Macrophage Passage](https://static.igem.wiki/teams/5332/project/notebook/5-23-5-24-macrophage-passage.pdf)

[5.25 Macrophages Plating](https://static.igem.wiki/teams/5332/project/notebook/5-25-macrophages-plating.pdf)

[5.25 S.Tm Liquid Culture](https://static.igem.wiki/teams/5332/project/notebook/5-25-s-tm-liquid-culture.pdf)

[5.26 S.Tm Subculture](https://static.igem.wiki/teams/5332/project/notebook/5-26-s-tm-subculture.pdf)

[5.26 Macrophages Starvation
Treatment](https://static.igem.wiki/teams/5332/project/notebook/new/5-26-macrophages-starvation-treatment-1.pdf)

[5.26 Preparation of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-26-preparation-of-culture-medium.pdf)

[5.26 Sterilization of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-26-pouring-of-culture-medium.pdf)

[5.26 Pouring of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/5-26-pouring-of-culture-medium.pdf)

[5.26 S. Tm-macrophage
Infection](https://static.igem.wiki/teams/5332/project/notebook/new/5-26-s-tm-macrophage-infection-1.pdf)

[5.26 Peptide Dilution](https://static.igem.wiki/teams/5332/project/notebook/5-26-peptide-dilution.pdf)

[5.26 Cell Status Observation](https://static.igem.wiki/teams/5332/project/notebook/5-26-cell-status-observation.pdf)

[5.26 Macrophage Collecting](https://static.igem.wiki/teams/5332/project/notebook/new/5-26-macrophage-collecting-1.pdf)

[5.26 Cell Number Counting](https://static.igem.wiki/teams/5332/project/notebook/5-26-cell-number-counting.pdf)

[5.26 Determination of Reactive
Oxygen Species (ROS)
Content](https://static.igem.wiki/teams/5332/project/notebook/5-26-determination-of-reactive-oxygen-species-ros-content.pdf)

[5.29-5.30 Macrophages Passage](https://static.igem.wiki/teams/5332/project/notebook/5-29-5-30-macrophages-passage.pdf)

## June

#### In Vitro Experiments

[6.1 Macrophages Plating](https://static.igem.wiki/teams/5332/project/notebook/6-1-macrophages-plating.pdf)

[6.1 S.Tm Liquid Culture](https://static.igem.wiki/teams/5332/project/notebook/6-1-s-tm-liquid-culture.pdf)

[6.2 S.Tm Subculture](https://static.igem.wiki/teams/5332/project/notebook/6-2-s-tm-subculture.pdf)

[6.2 Macrophages Starvation
Treatment](https://static.igem.wiki/teams/5332/project/notebook/new/6-2-macrophages-starvation-treatment-1.pdf)

[6.2 Preparation of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/6-2-preparation-of-culture-medium.pdf)

[6.2 Sterilization of Culture
Medium](https://static.igem.wiki/teams/5332/project/notebook/6-2-sterilization-of-culture-medium.pdf)

[6.2 Pouring of Culture Medium](https://static.igem.wiki/teams/5332/project/notebook/6-2-pouring-of-culture-medium.pdf)

[6.2 S. Tm-macrophage
Infection](https://static.igem.wiki/teams/5332/project/notebook/6-2-s-tm-macrophage-infection.pdf)

[6.2 Macrophage Collecting](https://static.igem.wiki/teams/5332/project/notebook/new/6-2-macrophage-collecting-1.pdf)

[6.2 Cell Number Counting](https://static.igem.wiki/teams/5332/project/notebook/6-2-cell-number-counting.pdf)

[6.2 Determination of Reactive
Oxygen Species (ROS)
Content](https://static.igem.wiki/teams/5332/project/notebook/6-2-determination-of-reactive-oxygen-species-ros-content.pdf)

[6.2 Cell Lysis, Bacterial
Suspension Dilution](https://static.igem.wiki/teams/5332/project/notebook/6-2-cell-lysis-bacterial-suspension-dilution.pdf)

[6.2 Bacterial Plating](https://static.igem.wiki/teams/5332/project/notebook/6-2-bacterial-plating.pdf)

[6.3-6.4 Bacterial Colony Number
Counting](https://static.igem.wiki/teams/5332/project/notebook/6-3-6-4-bacterial-colony-number-counting.pdf)

[6.5 RNA Extraction](https://static.igem.wiki/teams/5332/project/notebook/6-5-rna-extraction.pdf)

[6.5 Reverse Transcription](https://static.igem.wiki/teams/5332/project/notebook/6-5-reverse-transcription.pdf)

[6.7 RT-qPCR](https://static.igem.wiki/teams/5332/project/notebook/6-7-rt-qpcr.pdf)

#### In Vivo Experiment

[6.21-6.23 Adhesion factor
screening assay](https://static.igem.wiki/teams/5332/project/notebook/6-21-6-23-determination-of-adhesion-efficiency-of-engineered.pdf)

## July

#### Molecular Cloning(Both anti-inflammatory factors were done)

[7.4 Target gene amplification](https://static.igem.wiki/teams/5332/project/notebook/7-4-target-gene-amplification.pdf)

[7.4 Plasmid enzyme digestion](https://static.igem.wiki/teams/5332/project/notebook/7-4-plasmid-enzyme-digestion.pdf)

[7.4 Gel recycling](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/7-4-gel-recycling-1.pdf)

[7.4 Connection between target
gene and plasmid](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/7-4-connection-between-target-gene-and-plasmid-1.pdf)

[7.4-7.5 Plasmid
transformation](https://static.igem.wiki/teams/5332/project/notebook/new/7-4-7-5-plasmid-transformation-1.pdf)

[7.6 Plasmid extraction](https://static.igem.wiki/teams/5332/project/notebook/7-6-plasmid-extraction.pdf)

7.11-7.13 REPEAT

#### In Vivo Experiment

[7.22-7.24 Establishment of
colitis mouse model](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/7-22-7-24-establishment-of-colitis-mouse-model-1.pdf)

[7.25-7.27 Peptide gavage](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/7-25-7-27-peptide-gavage-1.pdf)

7.29 Measurement of colon length in mice

[7.29 lsolation of cells from
intestinal lamina
propria of
mice](https://static.igem.wiki/teams/5332/project/notebook/7-29-lsolation-of-cells-from-intestinal-lamina-propria-of-mice.pdf)

[7.29-8.1 Tissue embedding and
sectioning](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/7-29-8-1-tissue-embedding-and-sectioning-1.pdf)

[7.29 Flow cytometry detection in
in vivo
experiments](https://static.igem.wiki/teams/5332/project/notebook/7-29-flow-cytometry-detection-in-in-vivo-experiments.pdf)

## Augest

#### In Vivo Experiment

[8.2 HE staining](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/8-2-he-staining-1.pdf)

[8.2 Immunofluorescence
staining](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/8-2-immunofluorescence-staining-1.pdf)

[8.12-8.14 Establishment of
colitis mouse model](https://static.igem.wiki/teams/5332/project/notebook/8-12-8-14-establishment-of-colitis-mouse-model.pdf)

[8.15-8.17 Engineering bacteria
gavage](https://static.igem.wiki/teams/5332/project/notebook/new/8-15-8-17-engineering-bacteria-gavage-1.pdff)

8.18 Measurement of colon length in mice

[8.18 lsolation of cells from
intestinal lamina
propria of
mice](https://static.igem.wiki/teams/5332/project/notebook/8-18-lsolation-of-cells-from-intestinal-lamina-propria-of-mice.pdf)

[8.18-8.21 Tissue embedding and
sectioning](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/8-18-8-21-tissue-embedding-and-sectioning-1.pdf)

[8.18 Flow cytometry detection in
in vivo
experiments](https://static.igem.wiki/teams/5332/project/notebook/8-18-flow-cytometry-detection-in-in-vivo-experiments.pdf)

[8.22 HE staining](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/8-22-he-staining-1.pdf)

[8.22 Immunofluorescence
staining](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/8-22-immunofluorescence-staining-1.pdf)

[8.18-9.21 Microbial diversity](https://static.igem.wiki/teams/5332/project/notebook/new/newnew/8-18-9-21-microbial-diversity-1.pdf)

![loading](https://static.igem.wiki/teams/5332/homeimage/loading.gif)

[Introduction](#intro)

### **Three Components**

[ROSpromotor](#ROSpromotor)

[Ideas Of Anti-inflammatory Factors](#Ideas)

[How to determine effective drugs?](#how)

[Basic information on melittin](#basic)

[Action principle of melittin](#action)

[Therapeutic effect of melittin on ulcerative colitis](#the)

[Artificial modification of melittin](#arti)

[Conclusion](#c)

[Indolicidin](#i)

[Anti-inflammatory Factors](#anti-inflammatory-factors)

[Peptide cell experiment](#a1)

[In vivo peptide experiments](#a2)

[Adhesion Factor CMC](#AdhesionFactorCMC)

### **Idea Library**

[DL-endopeptidase](#DL-endopeptidase)

[miRNA](#miRNA)

[Idea library](#idealibrary)

# Introduction

Inflammatory bowel disease (IBD) is a chronic and recurrent intestinal disease, mainly including Crohn's
Disease (CD) and Ulcerative Colitis (UC). Although these two diseases have some similarities in clinical
manifestations, such as chronic diarrhea, abdominal pain, and possible extraintestinal complications, they have
significant differences in pathological features. CD mainly manifests as segmental inflammation of the
intestine, accompanied by granuloma formation and fissure like ulcers. UC, on the other hand, presents as
continuous mucosal inflammation without the formation of granulomas

The etiology of IBD is complex, involving multiple aspects such as genetic susceptibility, immune system
dysfunction, imbalance of gut microbiota ecology, and environmental factors. These factors, either individually
or in combination, contribute to the occurrence of IBD. Especially the disruption of intestinal barrier function
and abnormal immune response to gut microbiota are considered key links in the development of diseases.

![](https://static.igem.wiki/teams/5332/project/engineering/introduction1532.png)

**Figure:Metagenomic, metatranscriptomic, and stool metabolomic profiles are disrupted during IBD activity.**

*a,Relative abundance distributions for ten of the most cross-sectionally significantly differentially abundant
metabolites in samples from individuals with IBD, as a ratio to the median relative abundance in individuals
without IBD. Left, fraction of samples below detection limit. b, Relationships between two measures of disease
activity: patient-reported (Harvey–Bradshaw index (HBI) in CD; simple clinical colitis activity index (SCCAI) in
UC and host molecular (faecal calprotectin (cal)43. Linear regression shown with 95% confidence bound. c, d,
Distribution of microbial dysbiosis scores as a measure of disease activity. e, Kaplan-Meier curves for the
distributions of the durations of (left) and intervals between (right) dysbiotic episodes in UC and CD. Both are
approximately exponential (fits in dashed lines), with means of 4.1 and 17.2 weeks, respectively, for UC, and
7.8 and 12.8 weeks for CD. f, Relative abundance distributions of significantly different metagenomic species,
metabolites, and microbial transcribers in dysbiotic samples compared to non-dysbiotic samples from the same
disease group . Also shown are antibody titres for ANCA, ASCA (IgG or IgA), anti-OmpC, and anti-CBir1 antibodies.
(from Jason Lloyd-Price, Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases)*

Recent studies have revealed multiple gene loci associated with IBD, such as NOD2/CARD15, IL23R, and IL10.
These genes are involved in immune regulation, intestinal barrier function, and microbial community balance.
Among them, the variation of NOD2/CARD15 is significantly associated with the risk of CD, while the polymorphism
of IL23R and IL10 affects the susceptibility of UC. In addition, the reduction of gut microbiota diversity is
also one of the important factors contributing to the onset of IBD.

The incidence rate and prevalence of IBD are on the rise worldwide, especially in western developed countries.
It is reported that there are more than 3 million IBD patients in the United States, with 85387 new cases in
2019, accounting for 21% of the global incidence rate. Although the incidence of IBD is similar between males
and females, genetic factors are significantly associated with familial clustering, especially in CD.
Environmental factors such as diet, smoking, and lifestyle are also key external drivers of IBD.

![](https://static.igem.wiki/teams/5332/image/introduction2730.jpg)

In terms of clinical treatment, although Crohn's Disease (CD) and Ulcerative Colitis (UC) both belong to
inflammatory bowel disease (IBD), there are significant differences in treatment plans and prognosis between the
two. Traditional anti-inflammatory drugs have poor therapeutic effects on CD patients and may require the use of
immunosuppressants or biologics to assist in treatment. In contrast, UC patients typically have a better
response to aminosalicylic acid drugs and glucocorticoids. In terms of prognosis, due to the complex
pathological process and varied clinical manifestations of CD, its prognosis is relatively poor and it is more
likely to require surgical intervention, while UC has a relatively better prognosis, especially in early
diagnosis and treatment.

In general, current treatment options for IBD are often non-specific and diffuse, and can only target a single
factor in the treatment of the disease. In addition, the treatment is expensive and the process is often very
painful. Therefore, we hope to use the technology of synthetic biology to design an engineered bacterium that
can treat with spatiotemporal specificity, solve the problem from the source and is not limited to a single
factor, reduce the side effects of treatment, simplify the complex course of treatment, and provide a
cost-effective option!

After reviewing the literature, we took the gastrointestinal tract, the pathogenic location of IBD, as the
entry point. Combined with the studies of intestinal microflora in recent years, we expected to design a plasmid
that could express IBD therapeutic drugs on the basis of Escherichia coli as engineering bacteria.

We started by selecting the products of the genes. The most common symptom of IBD patients is inflammation of
the digestive tract. From this point of view, anti-inflammatory therapy is a direct and rapid treatment choice.
Therefore, we chose the base sequence encoding the anti-inflammatory factor as the main genetic element.

Once the bacteria were able to express the anti-inflammatory factors needed for treatment, the next question
was how to make the modified bacteria survive in the gut. The intestinal environment is complex, in which there
are a large number of intestinal flora. It is required that engineered bacteria should be at advantage in this
community. Based on the original design, we added the adhesion factor which can adhere bacteria to the gut, so
that the engineered bacteria can stay in the gut for a long time for recruitment and multiplication.

Gene expression requires not only its own coding sequence, but also the regulation of cis-acting elements and
trans-acting factors on both sides of genes. Promoters play an important role in fast responding to the
inflammatory environment and initiating gene expression. After meeting these conditions, the engineered bacteria
is able to play a therapeutic effect in the right place and time, and reduce the adverse effects of the
expressed anti-inflammatory factors in other parts of the human body. Therefore, we divided the selection range
of promoters into genes that can respond to ROS, that is, select an ROS promoter.

The following schematic diagram of this engineered bacterial plasmid shows our three main components. Our whole
component design process runs perfectly in the Design-Build-Test-Learn cycle, where we do a lot of literature
research, design a lot of ideas, discuss the most appropriate ones to carry out the construction of the components,
and test the effectiveness of the components, and then choose the most appropriate ones for optimization.Below we
will introduce the design process of our components, and the ideas that were not selected will also be introduced
in the idea library. Please follow our text through our Design-Build-Test-Learn cycle!

![](https://static.igem.wiki/teams/5332/image/introduction3.png)

![](https://static.igem.wiki/teams/5332/image/introduction4.png)

Three Components
Idea Library

After selecting the target gene, we need a suitable "switch" to make it express at the right
place and time. According to the characteristic that the concentration of ROS in the intestine of patients
with enteritis is often higher than that in other parts of the body, this "switch" should be able to
increase the expression of target genes in response to ROS, and hardly enhance the expression in the absence
of ROS environment.

## ROS Promotor

ROS, or reactive oxygen species, are molecules commonly found in organisms undergoing aerobic respiration.
Under normal circumstances, it does not threaten the health of the organism. Typically, a single electron is
initially transferred to oxygen to form a superoxide (O2-), which is then catalyzed by superoxide dismutase
to become a hydroperoxide (H2O2). Superoxide is less likely to cross the membrane, which limits its range of
influence. However, H2O2can easily spread to other sites. When the human body is in a state of disease, ROS
production is greatly increased, and H2O2 which is of high diffusion will spread from the lesion to other
healthy parts, causing oxidative damage to the Escherichia coli in this place [1].

![](https://static.igem.wiki/teams/5332/image/ros-fig1.png)

**Fig1. ROS are highly reactive and quickly transition between species.**

*(Picture resource: Brieger K, Schiavone S, Miller FJ Jr, Krause KH. Reactive oxygen species: from health
to disease. Swiss Med Wkly. 2012 Aug 17;142:w13659. doi: 10.4414/smw.2012.13659. PMID: 22903797.)*

To counter this adverse effect, organisms have evolved relative clearance systems. The well-known ways in
which Escherichia coli eliminate H2O2 are glutathione reduction, the thioredoxin system, and the gludoxin
system. The genes that respond to ROS and encode related enzymes differs within organisms. In E. coli, the
key regulators of adaptive responses to hydrogen peroxide and superoxide anions are OxyR and SoxR and SoxS.
Among them, OxyR direct oxidation is the mechanism by which Escherichia coli sense hydrogen peroxide and
induce the production of OxyR regulon. OxyR proteins are homologous to the LysR-NodD family of bacterial
regulatory proteins and bind to the promoter of the oxyR regulatory gene. OxyR protein can activate the
transcription of oxyR regulatory genes in vitro [2]. As shown in Fig2, there are multiple cysteine residues
on the reduced OxyR protein, containing many sulfhydryl groups. These groups can be oxidized by H2O2 to form
disulfide bonds. Under the action of glutathione and gludoxin, OxyR can be regenerated in reduced form [3].
This suggests that oxidation of the OxyR protein brings about conformational changes [2]. The changes are
mainly due to the intramolecular disulfide bond Cys199-Cys208[3]. The activated OxyR binds upstream of the
promoters −35 sequence of the target genes such as dps，katG and gorA in the form of tetramers [3][4].
Transcription is stimulated by direct interaction with the carboxyl terminal domain of the alpha subunit of
RNA polymerase [4]. In addition, both states of OxyR is capable to bind to the oxyR promoter as repressors
that limit the amount of regulatory protein synthesis [3]. OxyR regulates at least eight genes, including
gorA encoding glutathione, dps encoding non-specific DNA-binding protein, katG encoding hydroperoxidase I
and ahpCF encoding alkyl hydroperoxide reductase [5]. When the expression of OxyR regulators increased, the
expression of these genes also augmented. Therefore, we conducted comparative analysis in the genes of katG,
dps, gorA and ahpCF to select the final promoter base sequence.

![](https://static.igem.wiki/teams/5332/project/engineering/ros-promoter4634.png)

**Fig2. The oxyR regulon**

*(Picture resource: Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR
transcription factors. Trends Biotechnol. 2001 Mar;19(3):109-14. doi: 10.1016/s0167-7799(00)01542-0. PMID:
11179804.)*

In a series of related studies, we founded that CARMEN MICHAÂN et al. used wild-type Escherichia coli in a
culture medium containing 10μM H2O2 and measured gene expression of katG，dps，gorA and ahpCF over a period of
time [5]. As shown in Fig3, we can find that katG has a rapid response to reactive oxygen stress and the
longest duration among the genes to be selected. Therefore, it is the first choice as the promoter of
engineered bacteria.

![](https://static.igem.wiki/teams/5332/image/ros-fig2.png)

**Fig3.In vivo transcription of the Escherichia coli oxyR regulon**

*Wild-type Escherichia coli from an overnight culture in M9 minimal medium were diluted in fresh
medium and incubated at 37°C with shaking at 150 rpm. At an OD600 of 0.2, H2O2 was added to half of the
culture, to make a final 10 M solution, and the rest was used as a control. Samples were collected
immediately (1min) after the addition of H2O2 and at 5, 10, 15, and 20 min of exposure. Samples were frozen
with liquid nitrogen, and total RNA was purified. The fluorescence signal of each PCR product was compared
to that of gapA. Data are from an average of eight MPCR amplifications. Values from treated samples were
divided by those from the corresponding control. All genes were analyzed, but only those genes for which
statistically significant (P＜0.05) increases were observed at a given time are represented. Error bars were
estimated from the corresponding SEM. (Picture resource: Michán, C., Manchado, M., Dorado, G., & Pueyo, C.
(1999). In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in
response to oxidative stress. Journal of bacteriology, 181(9), 2759–2764.
https://doi.org/10.1128/JB.181.9.2759-2764.1999)*

There is an experimental analysis of reactive oxygen stress response of katG gene conducted separately. we
could see that with the multiplication of bacteria (the curve indicated the change of OD600 value), the
expression of katG gradually increased at first, and then showed a trend of decline after the number of
bacteria was saturated. The activity of peroxidase, a gene expression product, had the same trend as the
gene, but remained stable instead of decreasing later. This phenomenon further indicates that the expression
of katG is related to H2O2 concentration. When H2O2 increased, the expression of katG went up. While the
reaction between peroxidase and H2O2 reaches equilibrium, the expression of katG will gradually decrease.
This mechanism can reduce the extra energy expenditure of cells. Therefore, the promoter of katG is suitable
for the design of engineered bacteria.

In this paper, the changes of katG gene expression due to the increase of H2O2 caused by aerobic
respiration during bacterial proliferation were analyzed by the RNA content expressed by katG. Escherichia
coli cultured overnight in LB broth were diluted in fresh medium and incubated at 37°C and shaken at 150
rpm. Samples are collected at regular intervals and quickly cooled to 0°C. The culture growth is monitored
by measuring OD600 for a specified time and is measured in the usual RNA purification and measurement
manner.There is an experimental analysis of reactive oxygen stress response of katG gene conducted
separately. With the multiplication of bacteria (the curve indicated the change of OD600 value), the
expression of katG gradually increased at first, and then showed a trend of decline after the number of
bacteria was saturated. The initial upregulation was the result of increased H2O2 output by aerobic
metabolism under favorable conditions. The activity of peroxidase, a gene expression product, had the same
trend as the gene, but remained stable instead of decreasing later. This phenomenon further indicates that
the expression of katG is related to H2O2 concentration. When H2O2 increased, the expression of katG went
up. While the reaction between peroxidase and H2O2 reaches equilibrium, the expression of katG will
gradually decrease. This mechanism can reduce the extra energy expenditure of cells. Therefore, the promoter
of katG is suitable for the design of engineered bacteria [5].

![](https://static.igem.wiki/teams/5332/image/ros-fig3.png)

**Fig4.Peroxidase activity during course of growth of wild-type bacteria in nutrient LB medium.**

*Activity data (darkly shaded bars) are from an average of four independent determinations. Error bars
represent SEM. The katG expression data are shown in light bars. Statistical significance (P＜0.05), for
comparisons with minimal values at 1.5 h of outgrowth, is marked with asterisks. Bacterial growth, monitored
as OD600, is indicated by the line. (Picture resource: Michán, C., Manchado, M., Dorado, G., & Pueyo, C. (1999).
In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to
oxidative stress. Journal of bacteriology, 181(9), 2759–2764.https://doi.org/10.1128/JB.181.9.2759-2764.1999)*

[1] Brieger K, Schiavone S, Miller FJ Jr, Krause KH. Reactive oxygen species: from health to disease. Swiss
Med Wkly. 2012 Aug 17;142:w13659. doi: 10.4414/smw.2012.13659. PMID: 22903797.

[2] Storz G, Tartaglia LA, Ames BN. The OxyR regulon. Antonie Van Leeuwenhoek. 1990 Oct;58(3):157-61. doi:
10.1007/BF00548927. PMID: 2256675.

[3] Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors.
Trends Biotechnol. 2001 Mar;19(3):109-14. doi: 10.1016/s0167-7799(00)01542-0. PMID: 11179804.

[4] Tao K. In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative
stress-inducible regulon in Escherichia coli. FEBS Lett. 1999 Aug 20;457(1):90-2. doi:
10.1016/s0014-5793(99)01013-3. PMID: 10486570.

[5] Michán C, Manchado M, Dorado G, Pueyo C. In vivo transcription of the Escherichia coli oxyR regulon as
a function of growth phase and in response to oxidative stress. J Bacteriol. 1999 May;181(9):2759-64. doi:
10.1128/JB.181.9.2759-2764.1999. PMID: 10217765; PMCID: PMC93716.

---

## Ideas Of Anti-inflammatory Factors

---

### How to determine effective drugs?

---

As mentioned before, the engineered bacteria we designed have three important elements: reactive oxygen
species initiation factors, adhesion factors, and anti-inflammatory factors. Among them, anti-inflammatory
factors are the key to the therapeutic effect of engineered bacteria. How to determine effective and
easy-to-express anti-inflammatory drug molecules is an important issue we have to face.

In the early stages of the project, we considered a variety of drugs that may have anti-inflammatory
effects, such as Short chain fatty acids (SCFAs), DL-endopeptidase, IL-10, etc（We will display these ideas
in the idea library）. However, taking into account factors such as the absorption capacity of the
pharmaceutical molecules, their anti-inflammatory capabilities and the size of the introduced gene, we
believe that peptide-based drugs should be chosen. Peptides are small in molecular weight, easily absorbed,
have a small gene length to save vector space, they are ideal pharmaceutical molecules.

Peptides with anti-inflammatory effects are currently less applied, and we have screened two peptides with
anti-inflammatory effects through extensive literature review, namely melittin (MEL) and Indolicidin (Indo).
Among them, melittin is the main active substance of bee venom, which has long been used in traditional
Chinese medicine to treat inflammatory diseases, and modern researchers have also confirmed the
anti-inflammatory effects of melittin. This has inspired us to apply melittin in the treatment of IBD. Below
are the introductions to these two drugs.

---

### Basic information on melittin:

---

![](https://static.igem.wiki/teams/5332/image/fig1.png)

Figure 1

Melittin is an amphipathic, water-soluble peptide consisting of 26 amino acid residues and is the
principal active component of natural bee venom. The N-terminal region contains four positively charged
residues, while the C-terminal region contains two positively charged residues. Due to the uneven
distribution of polar and non-polar amino acid residues, melittin exhibits both hydrophilic and lipophilic
amphipathic properties. The first 20 amino acids (from the N-terminus) are hydrophobic, whereas the
subsequent 6 amino acids are hydrophilic. The complete sequence is as follows:

NH2-Gly-ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Gly-Leu-Pro-Ala-Leu-ile-Ser-Trp-ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH2

![](https://static.igem.wiki/teams/5332/image/fig2.png)

Figure 2

*Reference: Ceremuga M, Stela M, Janik E, Gorniak L, Synowiec E, Sliwinski T, Sitarek P, Saluk-Bijak J,
Bijak M. Melittin-A Natural Peptide from Bee Venom Which Induces Apoptosis in Human Leukaemia Cells.
Biomolecules. 2020 Feb 6;10(2):247.*

Each melittin chain exhibits a helix-hinge-helix motif or bent rod structure. Amino acid residues
3-10 form an α-helix, while residues 12-14 create a hinge region that connects to another α-helix containing
residues 15-24. This unique structure, along with its amphipathic characteristics, enables melittin to
penetrate cell membranes and interact with cellular substructures at the molecular level.

---

### Action principle of melittin:

---

#### Effect of melittin on cell membrane:

---

![](https://static.igem.wiki/teams/5332/image/fig3.png)

Figure 3

*Reference: Zhang HQ, Sun C, Xu N, Liu W. The current landscape of the antimicrobial peptide melittin and
its therapeutic potential. Front Immunol. 2024 Jan 22;15:*

Melittin monomers can attach to the membrane surface and spontaneously integrate into natural or
artificial phospholipid bilayers, thereby reducing the rigidity between the polar and non-polar regions and
decreasing the permeability barrier. It has been proposed that melittin-induced membrane permeability may
result from the formation of toroidal pores or fissures within the membrane.

---

#### Anti-inflammatory effect of melittin:

---

![](https://static.igem.wiki/teams/5332/image/fig4.png)

Figure 4

*Reference: Liu Minchen & Du Ruofei, Chinese Journal of Modern Applied Pharmacy, 2023*

In terms of anti-inflammatory effects, melittin plays a crucial role in regulating inflammatory
signaling pathways. It inhibits the activity of the NF-κB pathway, reducing the expression of inflammatory
cytokines such as TNF-α, IL-1β, and IL-6. Melittin can suppress the phosphorylation of IKK and IκB, thereby
blocking the nuclear translocation of NF-κB and interfering with inflammatory signal transduction.
Additionally, melittin affects the MAPK signaling pathway by inhibiting the phosphorylation of JNK and p38,
thus regulating the production of pro-inflammatory cytokines and reducing the activity of inflammatory cells
and the secretion of inflammatory mediators. Furthermore, melittin interferes with the JAK/STAT pathway,
inhibiting the activity of STAT transcription factors decreasing the secretion of inflammatory cytokines,
which helps alleviate inflammatory diseases. Melittin also reduces inflammation by inhibiting Akt
phosphorylation, which interferes with the expression of inflammatory proteins and suppresses the production
of inflammatory mediators such as COX-2, iNOS, and cPLA2. Additionally, melittin promotes the recruitment of
immune cells to the site of inflammation and induces T-cell apoptosis, thereby modulating the immune
response and further alleviating inflammation.

Current experiments have demonstrated that melittin can be used to treat various inflammatory diseases,
such as arthritis, skin inflammation, ulcerative colitis, and other inflammatory conditions.

![](https://static.igem.wiki/teams/5332/image/fig5.png)

**table 1: The inflammatory diseases can be treated by melittin.**

*Reference:Liu Minchen &Du Ruofei, Chinese Journal of Modern Applied Pharmacy, 2023*

[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/engineering/table1.pdf)

This project focuses on the treatment of IBD, with particular attention to ulcerative colitis (UC), a form
of IBD characterized by persistent inflammation primarily affecting the superficial layers of the rectum and
colon.

Interviews with clinicians reveal that conventional therapies for UC often fail to achieve satisfactory
efficacy and have notable side effects. Therefore, more effective treatments for UC are needed. We think
melittin may be a good choice for treatment, our and other experiments have proved this.

---

### Therapeutic effect of melittin on ulcerative colitis:

---

Ahmedy et al. (2020) evaluated the anti-inflammatory effects of melittin through oral administration in an
acetic acid-induced colitis mouse model. Melittin treatment ameliorated acetic acid-triggered decreases of
body weight and an increase of colon mass index in colitis model mice （Figure 5）. Compared to untreated
colitis model mice, the melittin-treated group showed significantly reduced levels of tumor necrosis factor
(TNF-α) and IL-6 (Figure 6). Melittin amended changes induced by acetic acid in the upstream inflammatory
signaling pathways in colon such as NF-κB, Toll-like receptor 4 (TLR4), p38 MAPK, TNF receptor-associated
factor 6 (TRAF6) (Figure 7). Melittin also reversed acetic acid-induced alterations in PGE2 and enzymes
involved in its synthesis pathway in colon inculding secretory phospholipase A2 (sPLA2), and
cyclooxygenase-2 (COX-2) (Figure 8). Furthermore, Melittin attenuated acetic acid-induced oxidative stress
(Figure 9) and protected colon mucosa and submucosa against acetic acid-induced damage (Figure 10).

*5-10Refernce:Ahmedy OA, Ibrahim SM, Salem HH, Kandil EA. Antiulcerogenic effect of melittin via mitigating
TLR4/TRAF6 mediated NF-κB and p38MAPK pathways in acetic acid-induced ulcerative colitis in mice. Chem Biol
Interact. 2020 Nov 1;331:109276.*

![](https://static.igem.wiki/teams/5332/image/fig6.png)

Figure 5

Melittin inverted changes induced by acetic acid in body weight and colon mass index. a Change in
body weight from day 0 to day 5 induced by acetic acid and melittin administration. b Effect of acetic acid
and melittin on colon mass index in mice. Each bar with vertical line represents the mean ± S.E.M. of 10
mice per group; \* significantly different from control group at p < 0.05, \*\* significantly different from
control group at p < 0.001, \*\*\* significantly different from control group at p < 0.0001, significantly
different from acetic acid group at p < 0.05, significantly different from acetic acid group at p < 0.0001
using One-Way ANOVA followed by Tukey multiple comparisons test.####

![](https://static.igem.wiki/teams/5332/image/fig7.png)

Figure 6

Melittin mitigated acetic acid-induced alterations in TNF-α and IL-6 levels in colon. Each bar
with vertical line represents the mean ± S.E.M. of 6 mice per group; \*\*\* significantly different from
control group at p < 0.0001, significantly different from acetic acid group at p < 0.0001 using One-Way
ANOVA followed by Tukey multiple comparisons test.###

![](https://static.igem.wiki/teams/5332/image/fig8.png)

Figure 7

Melittin amended changes induced by acetic acid in the upstream inflammatory signaling
pathways in colon. a Western blot analysis for the protein expression levels of TLR4, TRAF6 and p38
MAPK using β-actin as loading control (n = 3) b NF-κB level as measured by ELISA (n = 6). Each bar
with vertical line represents the mean ± S.E.M.; \* significantly different from control group at p <
0.05, \*\* significantly different from control group at p < 0.001, \*\*\* significantly different from
control group at p < 0.0001, significantly different from acetic acid group at p < 0.0001 using
One-Way ANOVA followed by Tukey multiple comparisons test.###

![](https://static.igem.wiki/teams/5332/image/fig9.png)

Figure 8

Melittin reversed acetic acid-induced alterations in PGE2 and enzymes involved in its
synthesis pathway in colon. a PGE2 level as estimated by ELISA (n = 6). b Western blot analysis
for the protein expression level of sPLA2 using β-actin as loading control (n = 3). c COX-2
level as estimated by ELISA (n = 6). Each bar with vertical line represents the mean ± S.E.M.; \*
significantly different from control group at p < 0.05, \*\* significantly different from control
group at p < 0.001, \*\*\* significantly different from control group at p < 0.0001,
significantly different from acetic group at p < 0.001 using One-Way ANOVA followed by Tukey
multiple comparisons test.###

![](https://static.igem.wiki/teams/5332/image/fig10.png)

Figure 9

Melittin attenuated acetic acid-induced oxidative stress. Each bar with vertical
line represents the mean ± S.E.M. of 6 mice per group; \*\*\* significantly different from
control group at p < 0.0001, significantly different from acetic acid group at p < 0.05,
significantly different from acetic acid group at p < 0.001, significantly different from
acetic acid group at p < 0.0001 using One-Way ANOVA followed by Tukey multiple comparisons
test.######

![](https://static.igem.wiki/teams/5332/image/fig11.png)

Figure 10

Melittin protected colon mucosa and submucosa against acetic acid-induced
damage. (A) H & E staining of histological sections in the colon of different
experimental groups. Control mice as well as those receiving melittin alone showed
normal histological structures of colon wall with apparent intact mucosal lining
including glandular elements and lining enterocytes, normal submucosa as well as outer
muscular coat with minimal inflammatory cells infiltrates and normal vasculatures.
However, mouse received acetic acid showed focal areas of erosion and damaged lining
mucosa including glandular elements (arrows) with abundant intraluminal necrotic
depresses and marked congestion of mucosal as well as submucosal blood vessels (dotted
arrows), moderate submucosal edema and inflammatory cells infiltrates (star). On the
other hand, mouse treated with melittin showed apparent intact intestinal mucosa, intact
submucosal and outer muscular layers with occasional focal mononuclear inflammatory
cells aggregates (H & E X 200 and X 50). (B) Histological scoring of colon mucosal
damage. Results are expressed as median and range of 3 mice per group; \*\* significantly
different from control group at p < 0.001, # significantly different from acetic acid
group at p < 0.05, using Kruskal–Wallis one-way ANOVA followed by Dunn's multiple
comparisons test.

Additionally, melittin reduced oxidative stress in colitis model mice. Atieh
Yaghoubi et al. (2022) evaluated the anti-inflammatory properties of melittin in a
dextran sulfate sodium (DSS)-induced colitis model.(Figure 11) They also analyzed
the synergistic effects of melittin and sulfasalazine (SSZ) to assess whether
combination therapy could improve clinical symptoms in UC model mice. The results
indicated that both melittin alone and in combination with sulfasalazine
significantly alleviated pathological damage in the colon tissues of UC model
mice.(Figure 12-15) Melittin treatment exhibited antioxidant effects, suggesting
that melittin could be a potential alternative therapy for UC intervention.

*11-15Reference:Yaghoubi A, Amel Jamehdar S, Reza Akbari Eidgahi M, Ghazvini K.
Evaluation of the therapeutic effect of melittin peptide on the ulcerative colitis
mouse model. Int Immunopharmacol. 2022 Jul;108:108810.*

![](https://static.igem.wiki/teams/5332/image/fig12.png)

Figure 11

Melittin peptide improve the clinical symptoms of DSS-induced colitis in
mice. (A, B, and D) Therapy with melittin peptide effect on colon weight and colon
shortening that induced by DSS; (C) Evaluation of the colon weight to length ratio
in a different group in comparison to the colitis group. [(\*p < 0.05; \*\*\*p < 0.001
compare to the control group); (#p < 0.05; ##p < 0.01; ### p < 0.001 compare to
the colitis group)]. (DSS=dextran sulfate sodium; SSZ=Sulfasalazine).

![](https://static.igem.wiki/teams/5332/image/fig13.png)

Figure 12

Melittin peptide ameliorated the disease activity index. (A and B)
Therapy with melittin peptide (2.4 mg/kg/day) alone or along with SSZ (100
mg/kg/day) was measured in a different group in comparison to colitis group.
[(\*\*p < 0.01; \*\*\*p < 0.001 compare to the control group); (##p < 0.01; ### p <
0.001 compare to the colitis group); (@@p < 0.01; @@@p < 0.001 compare to the
Melittin group); (++p < 0.01; +++p < 0.001 compare to the Sulfasalazine
group); ($p < 0.05; $$p < 0.01; $$$p < 0.001 compare to the Melittin in
combination with sulfasalazine group)].

![](https://static.igem.wiki/teams/5332/image/fig14.png)

Figure 13

Melittin peptide attenuated edema and inflammation of colon
tissue induced by DSS in colitis mice. Evaluation of the effect of the
therapy with melittin peptide alone or along with SSZ on (A) inflammation
score; (B) Mucosal damage score; (C) and crypt loss in different groups.
[(\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001 compare to the control group); (##p <
0.01; ### p < 0.001 compare to the colitis group); (+p < 0.05; ++p < 0.01
compare to the Sulfasalazine group)].

![](https://static.igem.wiki/teams/5332/image/fig15.png)

Figure 14

Melittin peptide effect on histological damage to colon
tissue in DSS-induced colitis mice. (A) Pathological damage ranges were
measured in the different groups; (B) Evaluation of histological score
of the colon in different treatment groups; (C) Submucosal edema and
infiltration of the inflammatory cell in the histological result of
different groups. [(\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001 compare to the
control group); (### p < 0.001 compare to the colitis group); (+p <
0.05; ++p < 0.01 compare to the Sulfasalazine group)].

![](https://static.igem.wiki/teams/5332/image/fig16.png)

Figure 15

Melittin effect on oxidative stress markers in
DSS-induced colitis mice. The outcomes of melittin and melittin
along with SSZ (A) MDA; (B) SOD activity; (C) catalase enzyme
activity; and (D) total thiol of the serum in comparison to the
colitis group. [(\*\*\*p < 0.001 compare to the control group); (#p <
0.05; ### p < 0.001 compare to the colitis group); (@@p < 0.01;
@@@p < 0.001 compare to the Melittin group); (+++p < 0.001 compare
to the Sulfasalazine group)] (DSS=dextran sulfate sodium;
SSZ=Sulfasalazine; MDA=malonyl dialdehyde; SOD=Superoxide
dismutase).

---

### Artificial modification of melittin

---

To further enhance the performance of melittin, we conducted
in-depth research and precise modifications. In the modification
process, we employed an innovative approach by connecting two
melittin monomers with a linker to form a unique hairpin
structure. This modification resulted in multiple performance
improvements. The specific amino acid and polypeptide sequences
are shown in Figure 16.

![](https://static.igem.wiki/teams/5332/image/mel16.png)

Figure 16

The artificial modification significantly reduced the
cytotoxicity of melittin. By introducing this structure, we
effectively decreased the peptide's toxicity to normal cells,
greatly enhancing its application safety. This improvement
allows melittin to be used in a wider range of scenarios,
particularly in treatments requiring prolonged contact with
biological tissues. Additionally, the modified melittin shows a
marked increase in immunostimulatory capacity (building on
previous laboratory work). This feature enhances its potential
in regulating immune and inflammatory responses. The enhanced
immunostimulatory ability not only helps activate the immune
system more effectively but may also facilitate appropriate
inflammation modulation, offering new possibilities for treating
IBD.

---

### Conclusion

---

Therefore, due to its anti-inflammatory and antioxidant
properties, we have selected melittin as one of the drug
molecules for this project.

---

### Indolicidin

---

Indolicidin, as a naturally occurring compound with significant
bioactivity, is gradually revealing its potential applications
in the treatment of colitis. Studies indicate that due to its
unique anti-inflammatory and immunomodulatory properties,
indolicidin can act through various mechanisms, particularly
excelling in modulating the gut microbiota and enhancing
intestinal barrier function. The synergistic effects of these
functions enable indolicidin to effectively alleviate symptoms
of colitis, thereby improving patients' quality of life.

---

#### Basic information:

---

![](https://static.igem.wiki/teams/5332/image/fig17.png)

Figure 17

Indolicidin, discovered by Selsted et al. (1992), is a natural
peptide belonging to the cathelicidin family isolated from
bovine neutrophils. This peptide consists of 13 amino acid
residues, nearly half of which are hydrophobic, including 5
tryptophan residues. These tryptophan residues are responsible
for insertion and distribution within biological membranes,
leading to the peptide's hemolytic activity (Subbalakshmi et
al., 1996). The physicochemical properties are shown in Table 2.

**Table 2: Sequence and physicochemical properties of indolicidin**

![](https://static.igem.wiki/teams/5332/image/fig18.png)

Reference:Batista Araujo J, Sastre de Souza G, Lorenzon EN.
Indolicidin revisited: biological activity, potential
applications and perspectives of an antimicrobial peptide not
yet fully explored. World J Microbiol Biotechnol. 2022 Jan
12;38(3):39.

---

#### Action principle:

---

Representation of indolicidin's mechanism of action after
transient membrane perturbation,intracellular potential targets
are proteins and nucleic acids (a), indolicidin-LPS
interactionrelated to anti-sepsis and anti-biofilm activity (b)
and the indolicidin anion carrier mechanismis thought to explain
hemolytic activity(c).

![](https://static.igem.wiki/teams/5332/project/engineering/fig16.png)

Figure 18

(a)The bacteriostatic effect of indolicotin: Indolicidin has a
broad range of biological targets, including both Gram-positive
and Gram-negative bacteria (Vergis et al., 2020). Additionally,
Indolicidin can disrupt established biofilms and inhibit biofilm
formation. At the molecular level, although Indolicidin can
insert into the plasma membrane, it does not directly cause
membrane rupture. Instead, it enters the cytoplasm, binds to
DNA, and inhibits the synthesis of proteins and DNA.

(b)Indolicidin neutralizes the pro-inflammatory effects of LPS:
Lipopolysaccharide (LPS) is considered an endotoxin and a major
outer membrane component of almost all Gram-negative bacteria,
acting as a potent immune system stimulant (Cohen 2002). LPS
activates inflammatory cells, such as monocytes and macrophages,
leading to the release of cytokines (e.g., TNF-α, IL-1β, and
IL-6) and nitric oxide, thereby triggering an inflammatory
cascade (Nan et al., 2009a; Park et al., 2009). Indolicidin can
bind and neutralize LPS, making it a potential anti-inflammatory
agent.

(c)The indolicidin anion carrier mechanismis thought to explain
hemolytic activity.

Reference:Batista Araujo J, Sastre de Souza G, Lorenzon EN.
Indolicidin revisited: biological activity, potential
applications and perspectives of an antimicrobial peptide not
yet fully explored. World J Microbiol Biotechnol. 2022 Jan
12;38(3):39.

---

### Conclusion:

---

Melittin and indolicidin, as novel peptide-based drugs, exhibit
significant anti-inflammatory potential. Both inhibit
inflammatory responses through different mechanisms, improving
gut health. Our project focuses on these two anti-inflammatory
agents, aiming to fully utilize their properties and enhance
clinical efficacy and safety, providing new therapeutic options
for IBD patients.

---

## Anti-inflammatory Factors

---

### Peptide cell experiment

---

Next, we will use the cell model constructed above for
preliminary drug screening experiments. Thaw macrophages and
culture them in wells on a 24 well plate after two passages.
Wait for the cells to grow full again and perform the same
starvation treatment on the cells. After infection, different
concentrations of Mel and Indo were applied to the experimental
group, and the cells were collected after four hours of
cultivation. Compared with the control group treated only with
PBS solution, the experimental group cells showed varying
degrees of decreased levels of reactive oxygen species（ROS）,
decreased levels of some inflammatory factors, and increased
expression of anti-inflammatory factor IL-10.

Use flow cytometry to detect cellular ROS levels. As shown in
Figure 1A, the experimental groups treated with 5, 10, and 15
ppm Mel and 5 and 15 ppm Indo showed a significant decrease in
the level of ROS in cells. However, the experimental results of
applying different concentrations of peptides to uninfected
macrophages showed that Mel and Indo did not reduce the level of
ROS in healthy cells (Figure 1B). The above results indicate
that appropriate concentrations of Mel and Indo can
significantly reduce the levels of ROS in inflammatory cells.

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1638.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1637.png)
![图片3](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1636.png)
![图片4](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1634.png)

Figure 1A

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1635.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1651.png)
![图片3](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1649.png)
![图片4](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor1650.png)

Figure 1B

The expression levels and changes of four inflammatory factors
in cells were detected by RT-qPCR technology,
and the results are shown in Figure 2A. Only concentrations of 5
and 10 ppm in the Indo group showed
significant decrease in IL-1 β. In the Mel group, a
concentration of 15ppm of Mel significantly upregulated
all four inflammatory factors, while a concentration of 10ppm
significantly downregulated the expression
levels of IL-1 β and IL-6. We speculate that both peptides at
appropriate concentrations can downregulate the
expression of some inflammatory factors. Excessive
concentrations may lead to peptides acting as antigenic
substances to stimulate macrophages to generate inflammatory
response, resulting in a significant increase in
the expression level of inflammatory factors.

In addition, we also conducted cell counts and intracellular
cell counts after infection. As shown in Figure
2B, there was no significant change in the number of cells after
experimental treatment in all groups. Except
for a significant decrease in the number of intracellular
bacteria in cells treated with Mel at concentrations
of 5 and 10 ppm, there was no significant change in the number
of intracellular bacteria in the other groups.

![](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor3225.png)

Figure 2A

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor3238.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor3239.png)

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor3236.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor3237.png)

Figure 2B

---

### In vivo peptide experiments

---

The above experiments have demonstrated that Mel and Indo at
appropriate concentrations have certain
anti-inflammatory effects in an in vitro cellular inflammation
model. Next, we will further validate the
anti-inflammatory effects of these two drugs in mice.

Firstly, a mouse intestinal inflammation model was constructed. A batch of female Balb/cJNju-Foxn1nu/Nju Hfh11nu mice were ordered and experimental procedures were carried out according to the table and all fluids were administered into the mice by gavage. The concentration of Salmonella is , Mel and Indo is 10ppm. The intestinal specimens taken were from the cecum to the colon.

![](https://static.igem.wiki/teams/5332/project/engineering/392f12ffd2bd17b76b3300517da42ed.png)

We measured the colon length of each group of mice to
characterize the level of intestinal inflammation. The
results showed that both Mel and Indo could significantly
alleviate the shortened colon length in mice caused
by intestinal inflammation, and Mel had better alleviating
effect (Figure 3A).

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor5095.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor5099.png)

Figure 3A

Figure 3B

Take some colon tissue for microscopic observation. After dehydration, embedding, and sectioning of colon tissue, HE staining was performed. The results are shown in Figure 3B. It can be seen that compared with the PBS group, the S.Tm group had damaged intestinal epithelial cells and disrupted intestinal mucosal integrity. However, the symptoms of the treatment group were significantly relieved, and the Mel group had better effects. Some tissue sections were processed for immunofluorescence staining. The results are shown in Figure 3C. It can be seen that the IL-6 fluorescence signal intensity of the tissues treated with Salmonella by gavage was significantly higher than that of the PBS group, indicating more severe intestinal inflammation. However, the IL-6 fluorescence intensity of the Mel and Indo groups was significantly lower than that of the S.Tm group, and both Mel and Indo had certain anti-inflammatory effects.

![](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor5513.png)

Figure 3C

Cut off a portion of colon tissue, completely dissociate it,
stain immune factors and immune cell marker
proteins, and then measure their expression levels using flow
cytometry. As shown in Figure 3D, Mel can
significantly reduce the levels of IFN-γ, IL-6, TNF-α, F4/80,
and Ly6G in the colon tissue of mice with
enteritis, while Indo can only reduce the levels of IFN-γ,
TNF-α, and F4/80

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6834.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6830.png)

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6835.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6829.png)

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6836.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6828.png)

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6833.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6832.png)

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6831.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor6838.png)

Figure 3D

Take a portion of colon tissue, extract cellular RNA, and
perform RT qPCR to detect the expression levels of
various inflammatory factors inside the cells. The results are
shown in Figure 3E. Compared with the control
group, the expression levels of IL-6, IL-1 β, and IL-8 in the
intestinal tissue after Mel treatment were
significantly reduced, while after Indo treatment, only IL-6
expression levels were significantly reduced, and
other inflammatory factors showed no significant changes or
significant increases. In addition, compared to
the positive control group, there was no significant change in
IL-10 levels in the Mel group, while the Indo
group showed a significant increase.

Through in vitro and in vivo experiments with the two peptides
mentioned above, we found that compared to
Indo, Mel exhibited better anti-inflammatory performance in all
the indicators we tested. Therefore, we
selected Mel for further experimental research.

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor8044.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor8051.png)
![图片3](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor8043.png)

![图片1](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor8042.png)
![图片2](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor8041.png)
![图片3](https://static.igem.wiki/teams/5332/image/anti-inflammatory-factor8053.png)

Figure 3E

---

## Adhesion Factor CMC

---

The key to the efficient functioning of FMK is to make the
engineered bacteria FMK stay in the intestine
longer and express anti-inflammatory factors in response.
Therefore, the team simulated the binding between
the glucan binding protein and the glucan substrate, and
designed the artificial glucan binding protein CMC.

Glucan is an extremely important polysaccharide produced by a
large number of bacteria and fungi. At the same
time, the surface of intestinal epithelial cells secretes mucus,
which plays a protective role as a barrier.
The main component of mucus is the highly glycosylated
glycoprotein MUC2, which has various polysaccharide
structures such as Core1, Core2 and Core3 [1]. At the same time,
the beneficial properties of probiotics are
related to the extracellular polysaccharide (EPS) they produce
[2], and the surface of most probiotics can be
exposed to glucan. Lactobacillus in Firmicutes has various
functions, such as regulating the balance of
intestinal microbial community, promoting food digestion and
nutrient absorption, alleviating intestinal
inflammation and infection, etc. Its cell wall is mainly
composed of peptidoglycan and polysaccharide. For
example, Lactobacillus can produce glucan sucrase (GS), and
synthesize EPSs through glucose-transfer glycogen
[3], such as α-glucan [4]. Salmonella is a common food-borne
pathogen that uses a variety of virulence factors
to overcome colonization resistance and induce intestinal
inflammation [5], all of which are Gram-negative
bacteria. Lipopolysaccharide, an important component of cell
wall, is an endotoxin and contains O antigen [6].
S.typhimurium is a zoonotic pathogen that causes a large
proportion of non-typhoid Salmonella (NTS)
infections, accounting for a quarter of infections, second only
to Salmonella enteritidis serotype in
incidence [7]. Therefore, we thought of combining this
characteristic of glucan as the core idea for designing
synthetic proteins, so that the adhesion factors we designed can
serve as a "bridge" between
intestinal microorganisms and the intestines, recruit intestinal
probiotics, and achieve stable attachment of
engineered bacteria to the intestines. This idea inspired us to
design CBMcipc domain, which is derived from
the scaffold protein CipC of Clostridium cellulolyticum and
contains a group III cellulose binding domain
(CBD), a hydrophilic domain and two hydrophobic domains. The CBD
domain confers CBMcipc with the ability to
bind to glucan[8].

In order to select the best glucan-binding protein, fluorescent
protein mCherry sequence was introduced in
the experiment, and proteins M, CM, CMC and CMCC with different
copy numbers of Clostridium cellulolyticum
CipC (CBMcipc) were designed. By introducing pET-28a plasmid
carrying corresponding genes (namely M, CM, CMC,
CMC, CMCC) into Escherichia coli BL21, the synthetic Escherichia
coli strains EcM, EcCM, EcCMC, EcCMCC were
constructed. The proteins M, CM, CMC and CMCC are composed of
mCherry, mCherry and one copy of CBMcipc,
mCherry and two copies of CBMcipc, mCherry and three copies of
CBMcipc (Fig1a). The N-terminal connected
outer membrane protein A (OmpA) signal peptide helps the protein
to pass through the intima and locate on the
outer membrane surface [8], thus giving the protein surface
display ability.

To verify the above properties, cells were stained with
FITC-labeled glucan for flow cytometry and confocal
microscopy. After incubation with FITC-glucan for 10 minutes,
EcM cells showed almost no FITC fluorescence,
while EcCM, EcCMC and EcCMCC cells showed obvious FITC
fluorescence. Among the three CBMcipc containing
strains, EcCMC cells showed the highest FITC fluorescence (Fig
1b, c), indicating that EcCMC had the highest
glucan-binding capacity.

Confocal microscopy images and fluorescence quantification
further revealed that EcM, EcCM, and EcCMCC cells
had a whole-cell distribution of mCherry fluorescence, while
EcCMC cells had a bilateral distribution of
mCherry, indicating that CMC exhibited a stronger surface
display ability(Fig1d). Since the cell-surface
display of the binding protein is necessary for it to bind to
dextran outside the cell, this property of EcCMC
may help enhance its dextran binding ability.

![](https://static.igem.wiki/teams/5332/image/adhesion-factor-cmc-4570.png)

Fig1. Design and characterization of the synthetic bacteria
used for glucan binding[7]. (Based on the
preliminary research findings from the laboratory)

Gavage experiments with EcCM, EcCMC, EcCMCC mixed with
Lactobacillus plantarum (probiotics) were done on mice
to assess the ratio of the number of beneficial intestinal
bacteria to the amount of beneficial bacteria in
the gavage after 12 hours of gavage (adherence rate) and the
results showed that EcCMC had the highest
adherence rate (Fig2). Whereas, after replacing the probiotics
with Salmonella enterica serovar Typhimurium
subsp. enterica (pathogenic bacteria), the adhesion rate was
lower in all cases, which reflects the preference
of adhesion factors for binding to probiotics.

![](https://static.igem.wiki/teams/5332/project/engineering/cmc-adhesion-factor-cmc6611.png)
![](https://static.igem.wiki/teams/5332/project/engineering/cmc-adhesion-factor-cmc6612.png)

Fig2 The adhesion rate of exogenous yeast was detected by
intragastric administration.

Based on the above characteristics, CMC was selected as a
adhesion factor in the experiment, hoping to
achieve the colonization of engineered bacteria in the intestine
and the recruitment of intestinal probiotics
through its expression in the intestine, so as to better
alleviate intestinal inflammation and regulate the
intestinal microenvironment.

Reference:

[1].NIE Shuo, WEN Zhengshun. Secretion, Structure, Synthesis
Regulation of Intestinal Mucin 2 and Its Role in
Development of Intestinal Diseases. Chinese Journal of Animal
Nutrition, 2020, 32(6): 2521-2532.

[2]. Pourjafar, Hadi et al. “Functional and health-promoting
properties of probiotics' exopolysaccharides;
isolation, characterization, and applications in the food
industry.” Critical reviews in food science and
nutrition vol. 63,26 (2023): 8194-8225.
doi:10.1080/10408398.2022.2047883

[3].Yu, Liansheng et al. “Glucansucrase Produced by Lactic Acid
Bacteria: Structure, Properties, and
Applications.” Fermentation (2022): n. pag.

[4].Chen, Ziwei et al. “Lactic acid bacteria-derived α-glucans:
From enzymatic synthesis to miscellaneous
applications.” Biotechnology advances vol. 47 (2021): 107708.
doi:10.1016/j.biotechadv.2021.107708

[5].Whitfield, Chris et al. “Lipopolysaccharide
O-antigens-bacterial glycans made to measure.” The Journal of
biological chemistry vol. 295,31 (2020): 10593-10609.
doi:10.1074/jbc.REV120.009402

[6].Park, Jeong Soon et al. “Mechanism of anchoring of OmpA
protein to the cell wall peptidoglycan of the
gram-negative bacterial outer membrane.” The FASEB Journal 26
(2012): 219 - 228.

[7]. Yin, Hongda et al. “Synthetic physical contact-remodeled
rhizosphere microbiome for enhanced
phytoremediation.” Journal of hazardous materials vol. 433
(2022): 128828. doi:10.1016/j.jhazmat.2022.128828

# idea library

---

## DL-endopeptidase

---

IBD, as an inflammatory disease, is considered to be caused by the interaction of multiple factors, among
which immune imbalance is a key factor. The gut, as a major digestive organ directly connected to the
outside
world, has a complex microbial ecology and naturally faces a complex immune environment. To maintain
intestinal immune balance, there is abundant information exchange between the gut and the microbiota to
regulate immune activity, which is often achieved through chemical signals, such as the specific recognition
of receptors and signal molecules (ligands).

---

### The NOD2 receptor receives MDP signals to regulate immune activity.

---

The nucleotide oligomerization domain 2 (NOD2) receptor is a classical pattern-recognition receptor sensing
specific microbial peptidoglycan fragments, including muramyl dipeptide (MDP), and N-acetylglucosamine
(NAG)-MDP. Activation of NOD2 is involved in a lot of physiological processes, including immune training,
epithelial regeneration, insulin resistance, vaccination response, and promoting the effects of anti-PD-L1
immunotherapy. Research shows that loss-of-function mutations in NOD2 are the strongest risk factor for CD.

![](https://static.igem.wiki/teams/5332/image/dl-idea-1548.png)

Figure 1: The NOD2 receptor can accept the signal of muramyl dipeptide (MDP) for regulation, and by
modulating pro-inflammatory cytokines, anti-inflammatory factors, antimicrobial peptides, adaptive immunity,
and autophagy, it can promote intestinal homeostasis and enhance resistance to the invasion of pathogens
that
can cause infectious colitis, thereby maintaining intestinal homeostasis by strengthening the epithelial
barrier function.

Reference: Khor B, Gardet A, Xavier RJ (2011).Genetics and pathogenesis of inflammatory bowel disease.
Nature, 474.

https://doi.org/10.1038/nature.2011.10209

In addition to mutations in the NOD2 receptor gene itself, a low level of MDP signaling may also lead to
insufficient NOD2 functional response, and the loss of NOD2 function may lead to Crohn's disease (CD).
Moreover, increasing the level of MDP signaling can also partially compensate for the reduced function of
the
NOD2 receptor caused by gene mutations, thereby alleviating CD.

![](https://static.igem.wiki/teams/5332/image/dl-idea-2696.png)

Figure 2: NOD2 mutation leading to CD mechanism diagram (upper figure) and reduction of DL-peptidase levels
causing CD mechanism diagram (lower figure).

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H
(2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe,
30.

https://doi.org/10.1016/j.chom.2022.08.002.

---

### DL-endopeptidase can increase the levels of MDP.

The main component of bacterial cell walls is peptidoglycan, and peptidoglycan hydrolases (PGHs) are the
main
enzymes responsible for cleaving peptidoglycan chains to release fragments. D-L endopeptidase is a type of
PGH
that can specifically hydrolyze special sites on bacterial peptidoglycan and produce muramyl dipeptide (MDP)
molecular structures. Since some DL-endopeptidases are secreted enzymes, they could interact with other
bacteria and enable them to release NOD2 ligands. We speculate that the DL-endopeptidase gene can be
introduced into a gene expression vector, and the engineered bacteria can be used to express and secrete it,
thereby increasing the level of MDP.

![](https://static.igem.wiki/teams/5332/image/dl-idea-4103.png)

Figure 3: A schematic diagram of the catalytic sites of the 8 PGHs, where ⑤ shows the site where
DL-peptidase
catalyzes the production of MDP.

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H
(2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe,
30.

https://doi.org/10.1016/j.chom.2022.08.002.

![](https://static.igem.wiki/teams/5332/image/dl-idea-4571.png)

Figure 4: A schematic diagram of the principle of DL-peptidase regulating intestinal homeostasis.

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H
(2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe,
30.

https://doi.org/10.1016/j.chom.2022.08.002.

---

### Experimental validation of the anti-inflammatory bowel disease (IBD) effects of DL-endopeptidase.

---

To investigate whether DL-endopeptidase deficiency could edispose mice to colitis and the role of NOD2
signaling in this process, researchers collected feces from healthy controls and CD patients, divided them
into healthy, high-endopeptidase, or low-DL-endopeptidase activity groups (indicated as healthy,
DL-endoHigh,
or DL-endoLow), and transferred the fecal microbiota to antibiotic-treated wild-type (WT) mice or their Nod2
knockout littermates (Nod2−/−) individually. After 10 days of fecal microbiota transplantation, colitis was
induced in mice by adding 3% dextran sodium sulfate (DSS). To assess the efficiency of microbiota
transplantation, researchers collected feces from all the donors and recipient mice before the DSS
challenge.

![](https://static.igem.wiki/teams/5332/image/dl-idea-5858.png)

Figure 5:Mice that received fecal microbiota from CD patients with low DL-endopeptidase activity were more
predisposed to colitis.

(A) Schematic outline of DL-endopeptidase activity stratification and the fecal microbiota transplantation
experiments.

(B) First two axes of a principal coordinate analysis (PCoA) of weighted UniFrac distances from donors in
health, \( \mathrm{DL-endo^{High}} \), or \( \mathrm{DL-endo^{Low}} \)groups. Group differences were tested by pairwise PERMANOVA. n = 3 for each group.

(C) Selected DL-endopeptidase gene-encoding microbiota from Firmicutes decreased in \( \mathrm{DL-endo^{High}} \)or \( \mathrm{DL-endo^{Low}} \)groups, compared
with the healthy controls, as revealed by t statistics of linear mixed-effects analysis: DL-endopeptidase
group (healthy,\( \mathrm{DL-endo^{High}} \) , or \( \mathrm{DL-endo^{Low}} \) and NOD2 gene status (WT, Nod2−/−) as a fixed effect and donor ID as a random
effect. Healthy and WT were used as references.

(D) NOD2 activity in HEK-Blue-NOD2 cells after incubation with the fecal supernatant of humans (n =
3/group),
or mice (n = 9–12/group) that received human fecal microbiota from indicated groups. The values are relative
ratios normalized to \( \mathrm{DL-endo^{Low}} \)group, valued as 1.

(E–H) Colitis severity of mice in different groups: body weight change (E), colon length (F),
and intestinal
barrier permeability, indicated by FITC-dextran permeability (G). Histology manifestation on
hematoxylin-eosin
(H&E) staining, scale bars represent 100 μm, and histology inflammation score (H).

(I–O) The transcriptional levels of Tnfa (I), Il-6 (J), Ccl2 (K), Cxcl1 (L), Col1a1 (M), Ripk2 (N),
and Atg16l1 (O) in colonic tissue of mice (n = 8–11/group).

Each dot indicates one sample. Data are shown as mean ± SD. Statistical comparison was performed by two-way
ANOVA followed by Bonferroni post hoc test (E), one-way ANOVA followed by Bonferroni post hoc test (D and
F–O), or Student’s t test (N and O). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant.

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou
H
(2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host
Microbe,
30.

https://doi.org/10.1016/j.chom.2022.08.002.

---

### Discussion about this idea

---

DL-endopeptidase, an enzyme secreted by probiotics in the gut, can activate the NOD2 receptor by
catalyzing the production of MDP signals to participate in intestinal immune regulation, maintain
intestinal homeostasis, and alleviate Crohn's disease (CD). Since DL-endopeptidase acts on the cell
walls
of gut bacteria and does not directly act on the human body, it can be effective without being absorbed
by
the human body, making it suitable for the expression of engineered bacteria in the gut.
DL-endopeptidase
relies on the human immune system to maintain intestinal homeostasis, has fewer side effects, and is
safer. However, due to its dependence on the NOD2 receptor pathway, it may be difficult to achieve
therapeutic effects for IBD caused by NOD2 receptor gene mutations. In addition, because the number of
engineered bacteria introduced into the patient's gut is limited, and DL-endopeptidase does not directly
exert a therapeutic effect, it may be challenging for the engineered bacteria to express enough
DL-endopeptidase to achieve the desired therapeutic effect. Therefore, we choose to first use peptides
with direct therapeutic effects as the drug molecules for this project, and then consider introducing
the
DL-endopeptidase gene to enhance its efficacy or combine it with IBD typing to create differentiated
products in subsequent product improvements.

---

## miRNA

---

Inflammatory bowel disease (IBD) is believed to be caused by the dysregulation and persistent
inflammatory response of genetically susceptible individuals to intestinal microbes, and epigenetic
factors play an important role in its pathogenesis.

microRNA (miRNA), as endogenous small non-coding RNA, can recognize target mRNA through sequence
complementation, inhibit target mRNA, and mediate post-transcriptional regulation of gene expression.
Current studies have shown that mirnas exhibit a high degree of specificity in different types of IBD,
so
mirnas can be used as biomarkers and therapeutic targets after determining the functional consequences
of
miRNA dysregulation. Based on the above ideas, we propose the application of mirNa-based logic circuits
in
the treatment of IBD.

![](https://static.igem.wiki/teams/5332/image/mirna-2-1023.png)

Fig1.miRNA

We selected specifically malregulated mirnas and protein responsive mrnas in IBD as control switches to
create a logic circuit to regulate the expression of anti-inflammatory factors or adhesion factors
associated with IBD therapy by detecting miRNA input. As shown in Fig2, in OFF state (absence of input
miRNAs), L7Ae protein represses translation of the output gene-coding mRNAs by interacting with the
kink-turn motif (Kt). In ON state (presence of input miRNAs), the L7Ae translation is repressed by the
miRNAs, which leads to output translation.

![](https://static.igem.wiki/teams/5332/image/mirna-2-1783.png)

Fig2. Schematic representation of the miRNA-responsive circuit and its
topology

At present, experiments have verified that miRNA-based circuits can control cell death signals through
logical operation. As shown in Fig3a, a 2-input (miR-21 AND miR-302a) AND circuit was designed in the
experiment, with EGFP as output. The AND circuit expresses the output only in the presence of two
miRNAs.
This experiment not only proves that miRNA-based logic loops can actually be used for expression
regulation, but also provides ideas for designing AND, OR, NAND, NOR logic loops in response to multiple
mirnas (Fig3b).

![](https://static.igem.wiki/teams/5332/image/mirna-2-2592.png)
![](https://static.igem.wiki/teams/5332/image/mirna-2-2594.png)

Fig3. (a) Schematic representation of engineered L7Ae-mediated,
miR-21-responsive circuits (b) Logic circuits can be constructed to detect cell-specific multiple miRNAs
as input

This miRNA-based logical loop treatment of IBD has higher biosafety. At the same time, due to the
highly
specific characteristics of miRNA in different IBD subtypes, miRNA can more sensitive to distinguish
between different IBDs to achieve accurate and sensitive treatment. However, this scheme still has many
problems, such as the difficulty of engineering bacteria to detect miRNA changes in the intestinal
environment in time, the difficulty of the experiment, and the high cost, so the team did not use this
idea. In the future, we will continue to pay attention to the research progress of miRNA as a
therapeutic
target, and look forward to a more optimized experimental program.

---

## SCFAs

---

Short-chain fatty acids (SCFAs) are primarily produced through the metabolism of carbohydrates,
particularly propionate and butyrate. Their health effects include inhibiting histone deacetylase (HDAC)
activity, enhancing the expression of anti-inflammatory factors, strengthening mucosal barrier function,
and reducing inflammation. Research indicates that SCFAs can improve the integrity of intestinal cells,
influence the homeostasis and function of different cell types, and promote the expression of tight
junction proteins such as claudin and occludin. Further clinical studies suggest the need for sustained
SCFA production to achieve anti-inflammatory effects, which can be delivered via oral or enema routes,
or
by utilizing specific live bacteria to enhance their production. Therefore, due to the plasticity of
yeast
fatty acid synthase (FAS) structures, we aim to use engineered yeast colonization in the gut to provide
more SCFAs for the treatment of inflammatory bowel disease (IBD). SCFAs offer a potential direction for
this project.

Short-chain fatty acids (SCFAs) are fatty acids produced by gut microbiota through the fermentation of
undigested carbohydrates, typically containing fewer than six carbon atoms. The main SCFAs include
acetate, propionate, and butyrate. Propionate and butyrate are primarily derived from the metabolism of
carbohydrates during glycolysis. The synthesis process of short-chain fatty acids is shown in Figure 1.

![](https://static.igem.wiki/teams/5332/image/1-1-1867.png)

Figure 1: Acetate is the most abundant SCFA in the gut, produced from pyruvate via acetyl-CoA. It can
also be converted into butyrate through the action of acetyl-CoA:butyryl-CoA transferase (BCoAT).
Butyrate
is synthesized by the condensation of two acetyl-CoA molecules, which are then reduced to butyryl-CoA
and
subsequently converted into butyrate. Butyrate kinase and butyryl-CoA:acetate CoA-transferase are the
two
main enzymes involved in butyrate production.

Reference:Parada Venegas, D., De la Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra,
G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated
gut
epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in
Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

Short-chain fatty acids (SCFAs) play a crucial role in protecting the intestinal mucosa and exerting
anti-inflammatory effects. They inhibit histone deacetylase (HDAC) activity, enhance the expression of
anti-inflammatory factors, and strengthen the intestinal mucosal barrier function. Additionally, SCFAs
can
reduce systemic inflammatory responses. The specific role of SCFAs is shown in Figure 2

![](https://static.igem.wiki/teams/5332/image/1-1-3200.png)

Figure 2: Health effects of SCFAs

In a healthy intestinal mucosa, SCFAs can increase the expression of anti-inflammatory factors and
inhibit pro-inflammatory factor expression, while enhancing mucosal barrier function and reducing
inflammation. In inflamed mucosa, the reduction of SCFAs leads to an increase in pro-inflammatory
factors,
a decrease in anti-inflammatory factors, compromised mucosal integrity, and abnormal cell autophagy.
Figure 3 illustrates the mechanisms of SCFAs.

![](https://static.igem.wiki/teams/5332/image/1-1-3812.png)

Figure 3:

SCFAs in healthy (A) and inflamed (B) colonic mucosa. In healthy mucosa, (1) bacterial fermentation of
dietary fiber (DF) by SCFAs-producing bacteria (e.g., F. prausnitzii), increases luminal content of
butyrate (green), propionate (blue) and acetate (purple) (2), forming a gradient along the crypt. In
lamina propria (LP) macrophages under acute inflammatory stimulus (4), butyrate inhibits histone
deacetylases (HDACs) thus; NF-κB-induced pro-inflammatory mediators (e.g. TNF-α, IL-6, IL-12 and iNOS)
expression whereas increases anti-inflammatory mediators (e.g., IL-10). In colonocytes (5), butyrate is
β-oxidized to Acetyl-CoA and constitutes the main source of energy by entering the TCA cycle.
Alternatively, butyrate initiates signaling pathway activation (or repression) by GPCRs and/or directly
inhibits HDACs, thus activating (e.g., HIF-1, STAT3 and SP1) or repressing (e.g., NF-κB) transcription
factors (TFs), increasing epithelial barrier function, antimicrobial peptides (AMPs) production, cell
proliferation and decreasing inflammation.

In inflamed mucosa as IBD, (1) a decreased fermentation of DF by low levels of SCFAs-producing bacteria
(e.g., F. prausnitzii) (2), reduces SCFAs luminal content (3). In LP inflammatory macrophages (4),
butyrate-GPCRs activation and -HDACs inhibition are downregulated, thus, there is uncontrolled
NF-κB-induced pro-inflammatory mediators’ expression (e.g. TNF-α, IL-6, IL-12 and iNOS) and decrease of
anti-inflammatory mediators (e.g., IL-10), although it appears that the inflammation increasesthe GPCRs
and transporters expression. In inflamed colonocytes (5), butyrate uptake and oxidation are decreased
and
GPCRs and transporters are also downregulated. This contributes to decreased epithelial barrier
integrity,
AMPs production, cell proliferation and increased inflammation.

Reference:Parada Venegas, D., De la Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra,
G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated
gut
epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in
Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

Table 1 describes the role and mechanism of different types of SCFAs in different mouse models.

Table 1: lmpact of SCFAs on intestinal homeostasis.

![](https://static.igem.wiki/teams/5332/image/1-1-6216.png)

Reference:Parada Venegas, D., De la Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra,
G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated
gut
epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in
Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

In current clinical treatments, butyrate or SCFA mixtures are typically administered orally or via
enema
for IBD. Studies have shown that continuous production and delivery of SCFAs to the mucosa are necessary
for anti-inflammatory effects. However, long-term medication and enemas can be burdensome for patients.
Therefore, we aim to directly introduce live bacteria that produce SCFAs into the mucosa for continuous
production and delivery. This approach requires pretreatment to enrich or restore SCFA-producing
bacteria.

The plasticity of fungal fatty acid synthase (FAS) supports this concept. Fatty acid synthesis is
carried
out by FAS in a cyclical, repetitive manner through seven distinct reaction steps, with each cycle
extending the carbon chain by two atoms. Each step is completed by an independent enzyme module within
the
FAS. The acyl carrier protein (ACP) acts as a carrier, transporting substrates and intermediates between
different enzyme modules, facilitating their transfer and reaction. We aim to incorporate additional
enzyme modules into yeast FAS to alter its activity, reducing the cycles to synthesize more short-chain
fatty acids.

![](https://static.igem.wiki/teams/5332/image/1-1-8138.png)

Figure 4: The yeast fatty acid biosynthesis cycle is depicted. ACP, acyl carrier protein (purple); AT,
acetyltransferase (blue); MPT, malonyl-palmitoyl-transferase (yellow);KS, ketosynthase (red); KR,
ketoreductase (orange); DH, dehydratase (light green); ER, enoyl reductase (green); FMN, flavin
mononucleotide.

Reference:Singh, K., Bunzel, G., Graf, B., Yip, K. M., Neumann-Schaal, M., Stark, H., & Chari, A.
(2023).
Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural
snapshots. Cell, 186(23), 5054–5067.e16. https://doi.org/10.1016/j.cell.2023.10.009

![](https://static.igem.wiki/teams/5332/image/1-1-8801.png)

Figure 5: Visualized chemical details in the 1.9 A˚ structure. One of the six asymmetric units of FAS
(indicated in cyan, top left) is extracted and depicted in the center. Individual enzymatic domains are
colored following the same scheme as in (A). The locations of zooms (i–v) in the asymmetric unit are
indicated. (i) FMN bound in the ER active site. The density indicates a 22 kink in the isoalloxazine
ring
system, consistent with its reductive role in FA biosynthesis. (ii) FMN recognition by ER active site
amino acids and their accompanying water network. (iii) NADP+ bound in the ER active site cleft with its
nicotinamide group stacked between the FMN isoalloxazine and His740, a solvent network aids in
recognition
of the ADP moiety. (iv) Recognition of NADP+ by amino acids and solvent in the KR active site cavity.
(v)
Malonyl-CoA was found in a post-hydrolysis state in the MPT active site. CoA interacts with many MPT
active sites residues, while the malonyl group is covalently attached to Ser1808 and interacts with
Arg1834.

Reference:Singh, K., Bunzel, G., Graf, B., Yip, K. M., Neumann-Schaal, M., Stark, H., & Chari, A.
(2023).
Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural
snapshots. Cell, 186(23), 5054–5067.e16. https://doi.org/10.1016/j.cell.2023.10.009

This study has detailed the synthesis process, showing that the \(\gamma\) subunit of yFAS can be used
as
a carrier to integrate other exogenous globular proteins (about 150 kDa per dome or 300 kDa per FAS)
into
the FAS dome. This discovery offers the potential to regulate the fatty acid profile of yFAS products.
Based on this, researchers reconstructed the FAS and conducted validation experiments. The results are
shown in Figure 6.

![](https://static.igem.wiki/teams/5332/image/1-1-10781.png)

Figure 6:

(A) SDS-PAGE of peak gradient fractions of FAS reconstituted with various proteins. From left to right,
these are: FAS (buffer addition only), g subunit (wild type), g-Tes V. harveyi, Tes V. harveyi (V.
harveyi
TesA alone), g-1xUnaG, or g-2xUnaG. The respective proteins reconstituted with FAS are denoted with red
asterisks.

(B) Efficiency of reconstitution can be monitored by additional fluorescence intensity. Shown are three
density gradients after sedimentation (g-1xUnaG left, g-2xUnaG middle, FAS alone right). Note that both
g-1xUnaG and g-2xUnaG increase the intrinsic FMN fluorescence of FAS alone.

(C) Relative abundance of free acyl-CoAs in the absence of protein (no protein; left), Dg-FAS (FAS; 2nd
from left), FAS bound to the g subunit (+ g subunit, middle), FAS bound to g subunit-TesA fusion protein
(+ g-Tes V. harveyi; 2nd from right), and Dg-FAS subsequently treated with TesA (+ Tes V. harveyi;
right)
as measured by LC/MS. Note that C12-CoA and C14-CoA are absent when FAS is bound to g subunit-TesA
fusion
protein; C16-CoA is markedly reduced, whereas the relative abundance of C8-CoA and C10-CoA is increased
(2nd from right). This can be attributed to thioesterase activity, as a Dg-FAS reaction subsequently
treated with TesA shows a similar product profile (right) and argues that C12, C14, and C16 are present
as
free fatty acids and therefore not detected in LC/MS. All measurements were performed in triplicate (n =
3), and the error bars for the relative abundance of the individual species are indicated.

Reference:Singh, K., Bunzel, G., Graf, B., Yip, K. M., Neumann-Schaal, M., Stark, H., & Chari, A.
(2023).
Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural
snapshots. Cell, 186(23), 5054–5067.e16. https://doi.org/10.1016/j.cell.2023.10.009

The experimental results indicate that the recombination directly modulates the configuration of FAS
products, demonstrating that TesA can alter the product profile by integrating into the γ subunit. We
can
attempt to introduce enzyme modules for short-chain fatty acid synthesis to enhance the production of
SCFAs in yeast. This improved yeast could continuously deliver SCFAs to the intestine, aiming to treat
IBD
and alleviate the discomfort associated with long-term medication and enemas in traditional therapies.

However, yeast, due to its larger size and more complex biological structure, significantly increases
the
workload in genetic engineering. In contrast, bacteria, while simpler in certain biochemical pathways,
offer advantages as experimental models due to their ease of manipulation and cost-effectiveness.
Therefore, after comprehensive evaluation, we have decided to use bacteria as the primary experimental
subject for this study. Additionally, the aforementioned research results were mainly achieved in yeast
and have not yet been validated in bacterial models. Furthermore, the redesign of yeast fatty acid
synthase is technically more challenging than in bacteria, adding to the complexity and uncertainty of
the
experiments.

Considering the urgency of the project, budget constraints, and experimental feasibility, we ultimately
decided to use engineered bacteria to explore new strategies for treating inflammatory bowel disease
(IBD). This choice is based not only on the convenience of genetic manipulation in bacterial models but
also on their potential advantages in clinical applications. Through this strategy, we hope to overcome
the limitations of existing treatments and provide more effective therapeutic options for IBD patients.

[In Vitro Experiment](#section1)

[In Vivo Experiment](#section2)

[Team Collaboration](#section3)

# Experiments

---

## In Vitro Experiment

---

Macrophage Thawing
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/macrophage-thawing.pdf)

Macrophage Passage
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/macrophage-passage.pdf)

Macrophage Plating
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/bacterial-plating.pdf)

S.Tm Liquid Culture
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/s-tm-liquid-culture.pdf)

S.Tm Subculture
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/s-tm-subculture.pdf)

Macrophages Starvation Treatment
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/macrophages-starvation-treatment.pdf)

Preparation of Culture Medium
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/preparation-of-culture-medium.pdf)

Sterilization of Culture Medium
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/sterilization-of-culture-medium.pdf)

Pouring of Culture Medium
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/pouring-of-culture-medium.pdf)

S. Tm-macrophage Infection
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/s-tm-macrophage-infection.pdf)

Macrophage Collecting
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/macrophage-collecting.pdf)

Cell Number Counting
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/cell-number-counting.pdf)

Determination of Reactive Oxygen Species (ROS) Content
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/determination-of-reactive-oxygen-species.pdf)

Cell Lysis, Bacterial Suspension Dilution
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/cell-lysis.pdf)

Bacterial Plating
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/bacterial-plating.pdf)

Bacterial Colony Number Counting
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/bacterial-colony-number-counting.pdf)

RNA Extraction
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/rna-extraction.pdf)

Reverse Transcription
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/reverse-transcription.pdf)

RT-qPCR
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/rt-qpcr.pdf)

---

## In Vivo Experiment

---

Establishment of colitis mouse model
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/establishment-of-colitis-mouse-model.pdf)

Peptide gavage
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/peptide-gavage.pdf)

Engineering bacteria gavage
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/engineering-bacteria-gavage.pdf)

Determination of Adhesion Efficiency of Engineered Bacteria
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/determination-of-adhesion-efficiency-of-engineered.pdf)

cytometry detection in in vivo experiments
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/flow-cytometry-detection-in-in-vivo-experiments.pdf)

HE staining
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/he-staining.pdf)

Immunofluorescence staining
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/immunofluorescence-staining.pdf)

lsolation of cells from intestinal lamina propria of mice
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/lsolation-of-cells-from-intestinal-lamina-propria-of-mice.pdf)

Microbial diversity
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/microbial-diversity.pdf)

Tissue embedding and sectioning
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/in-vivo/tissue-embedding-and-sectioning.pdf)

---

## Team Collaboration

---

Target gene amplification
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/engineered-bacteria/1-target-gene-amplification.pdf)

Plasmid enzyme digestion
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/engineered-bacteria/2-plasmid-enzyme-digestion.pdf)

Gel recycling
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/engineered-bacteria/3-gel-recycling.pdf)

Connection between target gene and plasmid
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/engineered-bacteria/4-connection-between-target-gene-and-plasmid.pdf)

Plasmid transformation
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/engineered-bacteria/5-plasmid-transformation.pdf)

Plasmid extraction
[![下载图标](https://static.igem.wiki/teams/5332/project/experiments/download-image.png)](https://static.igem.wiki/teams/5332/project/experiments/engineered-bacteria/6-plasmid-extraction.pdf)

---

![loading](https://static.igem.wiki/teams/5332/homeimage/loading.gif)

[In Vivo Experiments With Engineered Bacteria](#InVivo)

[Synthesis](#Synthesis)

[Mouse Model Experimentation](#Mouse-Model-Experimentation)

[Microbial Analysis](#Microbial-Analysis)

[Conclusion](#Conclusion)

[The Journey of Hope for Engineered Bacteria](#TheJourney)

## **In Vivo Experiments With Engineered Bacteria**

---

Based on the above experimental results, we ultimately selected Mel as the anti-inflammatory
factor
and synthesized the target plasmid with adhesion factor (CMC) and reactive oxygen species promoter (P [OSR]).
The
gene was cloned in pET-28a(+) by BamHI/XhoI. (Figure1) The target plasmid was transformed into Escherichia coli
strain Nissle 1917 (EcN) and the engineered strain FMK was synthesized, which was named iEraser by our team.
Next,
we will verify the anti-inflammatory effect of the synthesized strain in mice.

![图片1](https://static.igem.wiki/teams/5332/project/new-results/01.png)

Figure 1

Order a batch of female Balb/cJNju-Foxn1nu/Nju Hfh11nu mice and conduct
experiments according to the procedures
shown in the table. All liquids were administered into the mice by gavage, and the concentrations of Salmonella
and FMK were resuspended in PBS at a concentration of . The intestinal specimens taken were from the cecum to
the
colon.

![图片1](https://static.igem.wiki/teams/5332/project/results/69e35f9cfa49b230cd1bbe36a86807e.png)

The colon length of each group of mice was measured, and the results showed that the engineered bacteria
could significantly alleviate the shortened colon length caused by intestinal inflammation in mice. (Figure
2A)

![图片1](https://static.igem.wiki/teams/5332/project/new-results/02.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/03.jpg)

Figure 2A

Take some colon tissue for microscopic observation. After dehydration, embedding, and sectioning
of colon tissue, HE staining was performed, and the results are shown in Figure 3A. Compared with the PBS group,
the S.Tm group showed damage to intestinal epithelial cells and disrupted intestinal mucosal integrity, while
the
symptoms of colitis in the mouse model treated with FMK were significantly alleviated. Some tissue sections were
processed for immunofluorescence staining as shown in Figure 3B. It can be seen that the IL-6 fluorescence
signal
intensity of tissues treated with Salmonella by gavage was significantly higher than that of PBS group, while
the
IL-6 fluorescence intensity of tissues treated with engineered bacteria FMK was significantly lower than that of
the S group, indicating that the synthetic bacteria FMK has a significant anti-inflammatory effect.

![图片1](https://static.igem.wiki/teams/5332/project/new-results/04.jpeg)

Figure 3A

![图片1](https://static.igem.wiki/teams/5332/project/new-results/05.jpg)

Figure 3B

Take a portion of colon tissue, completely dissociate it, stain immune factors and immune cell marker
proteins, and then use flow cytometry to measure their expression levels. As shown in Figure 4A, after oral
administration of FMK, the levels of IL-6, IFN-γ, F4/80, and Lys6G in the colon tissue of mice with
enteritis
significantly decreased. Due to the fact that F4/80 and Lys6G are the marker proteins of immune cells, the
decrease in their levels indicates that the synthetic strain FMK can significantly reduce the aggregation of
immune cells near colitis tissue, thereby alleviating colitis in mice.

After extracting RNA from some colon tissue cells, RT-qPCR was performed to detect the
expression levels of various inflammatory factors in the cells. The results are shown in Figure 4B. Compared
with the control group, the levels of pro-inflammatory factors IL-6, IL-1 β, and IL-8 in the intestinal
tissue
treated with FMK were significantly reduced, while the levels of anti-inflammatory factors TGF-β and IL-10
were significantly increased.

![图片1](https://static.igem.wiki/teams/5332/project/new-results/06.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/07.jpg)

![图片1](https://static.igem.wiki/teams/5332/project/new-results/08.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/09.jpg)

![图片1](https://static.igem.wiki/teams/5332/project/new-results/10.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/11.jpg)

![图片1](https://static.igem.wiki/teams/5332/project/new-results/12.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/13.jpg)

![图片1](https://static.igem.wiki/teams/5332/project/new-results/14.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/15.jpg)

Figure 4A

![图片1](https://static.igem.wiki/teams/5332/project/new-results/16.jpg)
![图片1](https://static.igem.wiki/teams/5332/project/new-results/17.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/18.jpg)

![图片1](https://static.igem.wiki/teams/5332/project/new-results/19.jpg)
![图片1](https://static.igem.wiki/teams/5332/project/new-results/20.jpg)
![图片2](https://static.igem.wiki/teams/5332/project/new-results/21.jpg)

Figure 4B

In order to further investigate the effects of the engineered bacterium FMK on the gut
microbiota of IBD
mice, we conducted the following series of treatments.

Take colon tissue from mice, extract total DNA from the sample, perform PCR amplification, purify,
quantify, and homogenize the product to form a sequencing library. The constructed library is first
subjected to library quality inspection and sequencing, and the results are converted into the original
sequence through base calling analysis. Next, a series of operations such as filtering the raw data,
identifying and removing primer sequences, denoising, and concatenating double ended sequences are performed
to obtain the final valid data.

ASVs (amplicon sequence variants) represent real biological sequences that can distinguish differences
between sequences with precision in single nucleotides, enabling the identification of real species in
samples. Use the dada2 method in QIIME2 2020.6 software to denoise the sequence and obtain ASVs, determining
the presence of species in the sample.

Perform inter group ANOVA analysis at the Phylum level, as shown in Figure 5A. At the phylum level, the
engineered bacterium FMK significantly increased the abundance of Firmicutes and Verrucomicrobota in the
mouse gut, while reducing the abundance of Proteobacteria and Bacteroidota (Figure 5A). Previous studies
have shown a decrease in the number of Firmicutes and an enrichment of Proteobacteria in the intestinal
tract of IBD. However, this phenomenon was improved after the use of FMK. Therefore, FMK has a significant
function in improving the structure of intestinal microbiota, promoting the enrichment of beneficial
bacteria, and reducing the number of harmful bacteria. The heatmap analysis of sample species composition
and relative abundance, as well as the sample community distribution map of species evolutionary tree, are
shown in Figures 5B and 5C.

![图片1](https://static.igem.wiki/teams/5332/project/new-results/22.jpg)

Figure 5A Bar chart of intergroup analysis of variance

![图片1](https://static.igem.wiki/teams/5332/project/new-results/23.jpg)

Figure 5B Heat map of taxonomic relative abundance clustering

Perform species heatmap analysis based on the species composition and
relative abundance of each sample, extract species at the phylum level, use R language tools for mapping,
and perform heatmap clustering analysis. The values corresponding to the heatmap in the figure represent
the Zscore normalization of the same species across different samples using the R scale function. The
color gradient from blue to red indicates the abundance from low to high among samples, and the colors
correspond to the column in the graph.

![图片1](https://static.igem.wiki/teams/5332/project/new-results/24.jpg)

Figure 5C Sample community distribution map of Phylogenetic tree

Select the feature sequences with the top 80 abundance ratios at the OTU/ASV
taxonomic level, perform multiple sequence alignment using the QIIME command line, and construct a
phylogenetic tree. Then, combine the phylogenetic tree and species taxonomic abundance data, and visualize
them using the R package ggtreeExtra in the form of a species circular phylogenetic tree and bar chart.

Figure 6A shows the Shannon index rarefaction curve plotted using Mothur software and R language tool based
on the sequencing volume of each sample at different sequencing depths. It can be seen that when the curve
tends to flatten, except for the BCK1 group, the Shannon index in the group using the engineered bacterium
FMK is higher than that in the IBD mouse group, indicating that FMK has the effect of enriching intestinal
microbial diversity. Similar conclusions can also be drawn based on the rank-abundance curve(Figure 6B).

To describe the beta diversity of the two sets of data, we plotted the principal component analysis (PCA)
of the samples, and the results are shown in Figure 6C.

![图片1](https://static.igem.wiki/teams/5332/project/new-results/25.jpg)

Figure 6A Shannon index rarefaction curve

![图片1](https://static.igem.wiki/teams/5332/project/new-results/26.jpg)

Figure 6B Rank Abundance Curve

![图片1](https://static.igem.wiki/teams/5332/project/new-results/27.jpg)

Figure 6C PCA analysis chart

Overall, administering the engineered bacteria FMK designed by us to a mouse colitis model by
gavage
resulted in a recovery of colon length shortening, inhibition of immune cell recruitment, enrichment of
beneficial bacteria in the gut, an increase in gut microbiota diversity. The upregulation of
pro-inflammatory factors and the downregulation of anti-inflammatory factors are also inhibited accordingly.
The above results indicate that the engineered bacterium FMK synthesized by our team can effectively
alleviate the symptoms of enteritis in model mice.

---

## **The Journey of Hope for Engineered Bacteria**

---

In this era of human struggle against diseases, Inflammatory Bowel Disease (IBD) has become a significant
challenge for many patients. Countless individuals feel helpless in their battle with this condition, as the
limitations of traditional treatments erode their hope for recovery. Against this backdrop, our project emerged,
aiming to bring new hope to IBD patients through the innovative design of engineered bacteria.

Our project utilizes engineered Escherichia coli as a vector, designing plasmids capable of spatiotemporal
expression of anti-inflammatory factors. This innovative approach not only overcomes the limitations of
traditional drug therapies but also offers patients new treatment options. We employ synthetic biology
techniques,
supported by mathematical modeling, to continuously explore IBD treatment methods within the
design-build-test-learn cycle.

To design the anti-inflammatory component, we conducted extensive research and screening, ultimately selecting
Mel and Indo for our studies. We artificially melittin by connecting two bee venom peptide monomer molecules
together through a linker to form a unique hairpin structure. This modification has brought about performance
improvements in many aspects. We established cell and mouse inflammation models using Salmonella infection to
validate and compare the effectiveness of these anti-inflammatory peptides across various dimensions. Our
findings
revealed that Mel demonstrated superior anti-inflammatory performance compared to Indo across all measured
indicators, leading us to choose Mel for further experimental research.

At the same time, in order to ensure that our engineered bacteria only work at the right time and place, we
thought that the high ROS environment in the intestines of patients with enteritis could serve as a switch for
gene expression., particularly by utilizing the ROS-responsive OxyR regulatory factor. OxyR regulates multiple
genes, including katG, dps, and gorA. Based on experimental results, the promoter of the katG gene was deemed
suitable for the design of engineered bacteria.

The complex intestinal environment presents a significant challenge for the stable survival of engineered
bacteria. To address this, we enhanced bacterial adhesion by incorporating adhesive factors, allowing the
engineered bacteria to attach more effectively to the intestinal wall. This prolongs their presence in the body
and ensures the sustained release of anti-inflammatory factors. Notably, the project of adhesion protein is our
laboratory design a new protein：we use one of the main components of intestinal mucus glycoprotein and
probiotics
usually exposed the characteristics of glucan, hope to design glucan binding domain to realize the intestinal
adhesion, at the same time it can also combine probiotics, regulate the intestinal microbial environment. This
idea inspired us to design the CBMcipc domain, which is derived from the C. cellolytic scaffold protein CipC. To
ensure optimal performance, we ingeniously incorporated additional protein sequences during the design process,
creating various binding proteins. Through a series of rigorous experiments and inflammation tests, we
identified
the CMC protein with the highest adhesion rate and optimal probiotic binding ratio. This choice not only
enhances
the overall effectiveness of the project but also lays a solid foundation for future research and applications.

Finally, we cloned the anti-inflammatory factor (Mel), adhesive factor (CMC), and ROS-responsive promoter
(P[OSR]) into the plasmid pET-28a(+). The synthesized target plasmid was transformed into Escherichia coli EcN,
creating the engineered strain FMK, which we named iEraser. Our experiments further validated the excellent
anti-inflammatory effects of the engineered bacteria FMK (iEraser) in mice, successfully alleviating intestinal
inflammation. This discovery demonstrates the feasibility of FMK as a potential therapeutic approach, laying a
solid foundation for developing new treatments for Inflammatory Bowel Disease (IBD) and showing promising
clinical
application potential. The successful construction and validation of the engineered bacteria FMK provide crucial
support for the development of innovative therapies. This targeted approach enhances treatment efficacy, rapidly
alleviating patients' inflammatory symptoms.

However, the progress of the experiment hasn't always been smooth. As young project members, we are facing
significant challenges posed by complex experiments. In the first two months, the atmosphere in the lab was
somewhat heavy. Cell cultures repeatedly failed, with cells either dying prematurely or experimental data
falling
short of expectations. The language problem also creates great resistance to handling official information from
iGEM and using official tools. Every day, we worked hard to identify the root causes of these issues, always
feeling just a step away from success.

Despite these setbacks, we remained undeterred. We held multiple meetings to scrutinize each step, actively
reflecting and summarizing our experiences. We conducted daily reviews, meticulously recording every detail of
the
experiments to identify potential improvements. At the same time, we sought external assistance, reaching out to
professors and experts from other labs for advice and learning new techniques.Gradually, we adjusted
experimental
conditions and optimized techniques and methods. Through continuous trial and error, the experiments finally
showed progress. Seeing the healthy growth of cells in the culture dishes brought long-awaited smiles to our
faces. It was this persistence and effort that put our project back on track.

What we gained from the project was not only technical advancement but also the spirit of teamwork, resilience,
and the relentless pursuit of excellence. These qualities enable us to continue moving forward with confidence
in
the face of challenges.

At the initial stage of the project, we engaged in extensive discussions about the experimental subjects and
methods, ultimately leading to the engineering bacteria experiments of this project. Due to time constraints,
some
steps in our experiments remain incomplete, such as the validation of adhesion factors and the limited time
points
in microbial diversity experiments. Additionally, due to the design scale, our project components are relatively
few, currently focusing on polypeptides and simple proteins. To address this, our team conducted a brainstorming
session to introduce more interesting mechanisms into the project, aiming to enhance the experimental design.
These include miRNA components, SCFAs, and DL-endopeptidase; please refer to the Idea Library for details. In
the
future, we hope to continuously improve the project to address these problems and create more novel components.
In
terms of experiments, we did not achieve perfection. For example, in the microbial diversity experiment, we are
doing a more complete and formal supplementary experiment to make the experiment more rigorous and designed.
This
time, our experimental group will include engineered bacteria containing only adhesion factors and engineered
bacteria containing only anti-inflammatory factors to further verify their respective mechanisms of action. In
addition, the modules of the safety plasmid have not been experimentally verified, and we hope that future teams
can get inspiration from them and complete more rigorous testing.

IBD patients often experience a significant decline in quality of life. Our project aims to help them regain
normalcy and hope for the future through effective treatments. Behind this project is our team's deep care and
hope for every IBD patient. We believe that through continuous exploration and effort, engineered bacteria can
provide not only physiological improvements but also emotional comfort. In the future, we hope to extend this
innovative treatment to a broader patient population, allowing more people to benefit from scientific
advancements
and human compassion.

---