iGEM 2024 — Team szpu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block bgimg %}notebook.jpg{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

## Notebook

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

## Engineering

{% include 'pages/wikimenu.html' %}

## Overview

For the consideration of future application scenarios of our engineered bacteria, we will construct a hybrid
colony, which will start from the two modules of defense and offense respectively. At the defense level, we will
improve the defense ability of the colony by increasing the thickness of pod membrane polysaccharide and
knocking out genes to make the hairs longer; at the offense level, we plan to overexpress remote
offensive-related genes (mcmA, mchB and mcmI, mchI), enhance the aggressiveness of the CDI system, and we also
try to activate the T6SS of EcN by introducing the tagh genes of Escherichia coli 042 into EcN to see if the
T6SS of EcN can be activated. Escherichia coli 042 by introducing the tagh gene into EcN to see whether the
aggressiveness of EcN was improved, and we also designed two lethal gene circuits to ensure the safety of our
engineering experiments.

## Engineering Success

### Cycle 1: Overexpression of podoplanin genes

Click to Expand

### Design

Click to Expand

In podocarp studies, the 2023 XJTU-iGEM team has used pgmA and gal U gene overexpression to
enable
increased extracellular polysaccharide (EPS) production, based on which we additionally introduced
overexpression of KpsE, KpsT, and FliC genes, which are capable of increasing the rate of podocarp
polysaccharide translocation from intracellular to extracellular, thus optimizing our podocarp
level
of Defense.

### Build

Click to Expand

Two expression vectors were constructed pETDuet-1-gal U-pgmA , pACYCDuet-1-KpsE-KpsT
(inserthyperlinks), and the constructed plasmids and their empty vectors were introduced into
Nissle
1917 (DE3) respectively.

![...](https://static.igem.wiki/teams/5248/experiment/description/engineering1.png)

Figure 1: Mapping of pETDuet-1-gal U-pgmA plasmid

![...](https://static.igem.wiki/teams/5248/experiment/description/engineering2.png)

Figure 2: Mapping of pACYCDuet-1-KpsE-KpsT plasmid

![...](https://static.igem.wiki/teams/5248/part/build1-1.webp)
![...](https://static.igem.wiki/teams/5248/part/build2.webp)

Figure 3: pETDuet-1-galU pgmA plasmid electrophoresis
image;
Figure 4: pACYCDuet-1-KpsE-KpsT plasmid electrophoresis image;
Lane M: DNA Marker ;
Lane 1: Plasmid digested by EcoRV and XhoI ;
Lane 2: Uncut plasmid DNA;

### Test

Click to Expand

We carried out bacterial pod polysaccharide extraction on the above modified bacteria and
measured the content of the extracted polysaccharides, using the empty vector-imported bacteria as
the
control, as shown in Fig. 1, our constructed KpsE-KpsT genetically engineered bacterium
showed a significant increase in pod production compared with the empty vector-imported bacterium.
This is some placeholder content for a horizontal collapse. It's hidden by default and shown when
triggered.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/experiment/description/engineering3.png)![WikiBreeze Image](https://static.igem.wiki/teams/5248/experiment/description/engineering4.png)

Figure 3 and 4: Pod polysaccharide production of
pACYCDuet- 1- 1917 (DE3), pACYCDuet-1-KpsE-KpsT, and pETDuet-1-gal U-pgmA when cultured to
OD=0.6,
expression was induced by the addition of 0.5 mM IPTG for 3 h, and the OD of the bacterial
solution was diluted to 0.4

### Learn

Click to Expand

As mentioned above, KpsE and KpsT genes can effectively increase the pod yield, in the future, we
will
introduce pgmA and gal U together with KpsE and KpsT genes into EcN, meanwhile, considering the
possible influence of expression vectors on the expression of exogenous genes by the chassis
bacteria,
we propose the idea of integrating the exogenous genes genetically into EcN genome in order to
eliminate the influence of the expression vectors on the strain.

### Cycle 2: Biofilm System

Click to Expand

### Design

Click to Expand

FliC protein overexpression can increase the amount of podoplanin polysaccharide attachment on
the surface of the bacterium, and we took biofilm as a branch, from which we introduced FimH, NirC
can all increase the bacterial biofilm area as a way of providing enough membrane sites for
overexpressed FliC protein.

### Build

Click to Expand

We constructed four plasmids, pACYCDuet-1-NirC, pACYCDuet-1-FimH, pETDuet-1-FimH,
pETDuet-1-NirC, which were imported into Nissle 1917 and Nissle 1917(DE3), respectively, and
tested.

### Test

Click to Expand

Biofilm production was quantified by washing and crystal violet staining, and absorbance values
were detected after solubilization with 95% ethanol under the same initial OD600 of 0.05 and
incubation of the six-well plate for 12h.

![...](https://static.igem.wiki/teams/5248/part/cycle2-1.webp)
![...](https://static.igem.wiki/teams/5248/part/cycle2-2.webp)

Figure3 : Nissle 1917 biofilm formation; Figure 4: Nissle 1917 (DE3) biofilm formation

### Learn

Click to Expand

The experiments related to biofilm systems were not carried out in depth as we failed to validate the function of FliC and we reflected and considered, but our preliminary experiments did provide some valuable conclusions.

### Cycle 3: Knockout systems

Click to Expand

### Design：

Click to Expand

Based on the fact that bacterial motility is affected by increased pod thickness, we designed the ΔfimE mutant to achieve gene knockout by homologous recombination method to overexpress the type 1 bacterial hairs of EcN , which in turn improves the motility and defense of the engineered bacteria.

### Build

Click to Expand

The targeting vector (pCVD442- ΔfimE::Kn) was constructed, and the targeting vector was introduced into E. coli β 2155 strain by electrotransformation, and the β2155/pCVD442- Δ fimE::Kn bacterial fluid was mixed with the bacterial fluid of the recipient bacteria (Nissle1917) to carry out the splicing experiments, and after completion, the knockout strains were screened by using the kanal-resistant plate.

### Test

Click to Expand

The upstream and downstream homologous recombination arms of the fimE gene were amplified and cloned from the genome of Escherichia coli (Nissle1917) using ultra-fidelity DNA polymerase, the Kn resistance gene was amplified from the pKD4 plasmid by a high-fidelity PCR reaction, and the complete targeting fragment Δ fimE ::Kn was obtained by connecting the fimE gene to the Kn resistance gene using a fusion PCR technique by referring to the interface (insert hyperlink).After performing splicing experiments, fimE knockout strains were screened and verified by PCR electrophoresis.

![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle3-1.png)

Electropherogram:
M: DNA molecular weight standard.
From top to bottom the molecular weights are: 1200, 900, 700, 500, 300, 100bp, with 700bp brightened.
1-14: Internal primer amplification results for clones 1- 14;
15: Internal primer amplification result of the original strain, product length: 268bp;
16: Negative control amplification results.

### Learn

Click to Expand

The homologous recombination method to construct the ΔfimE mutant strain was successful, and we screened the knockout bacteria for bacterial preservation.

### Cycle 4: Antimicrobial peptide gene overexpression

Click to Expand

### Design

Click to Expand

E. coli Nissle 1917 (EcN) was utilized as a host and engineered to enable efficient
production of micromycin,
thereby enhancing its ability to fight pathogens. To this end, we focused on the overexpression of
the endogenous mcmA
and mchB genes of EcN, which encode key enzymes for micromycin synthesis. Also, to avoid the
phenomenon of autotoxicity,
whereby antimicrobial peptides may cause damage to their host cells, we also considered the
overexpression of the
corresponding immune proteins, mcmI and mchI, to protect the host cells from self-produced
antimicrobial peptides. In
addition, we verified the overexpression of EcN mcmA and mchB, as well as the related immunity
proteins mcmI and mchI by
bacterial antagonism assay to determine whether the tele-aggressiveness of EcN is enhanced.

### Build

Click to Expand

Two antimicrobial peptide expression plasmids, pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB(strep)
and
pETduet-1-mcmI-2(6his)-mcmA(strep), were constructed and introduced into the corresponding chassis
bacteria.

![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-1.png)

Figure 1: Mapping of pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB (strep) plasmid

![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-2.png)

Figure 2: Plasmid mapping of pETduet-1-mcmI-2(6his)-mcmA(strep) plasmid

![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-3.png)
![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-4.png)

Figure 3: Electropherogram of pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB (strep) plasmid
Figure 4: Electropherogram of pETduet-1-mcmI-2(6his)-mcmA (strep) plasmid

### Test

Click to Expand

To test whether our designed antimicrobial peptide expression plasmids functioned as expected, we
performed a zone of
inhibition (ZOI) assay. As detailed on the Experiments page, we inoculated 100 μL of concentrated
overnight cultures of
each predator strain on LB agar plates that were covered with a layer of soft LB agar containing
the prey strains. For
the mcmA and mchB antimicrobial peptide expression plasmids, the prey strains were E. coli DH5α.
the predator strains
were E. coli 1917 DE3 and BL21 DE3. in this experiment, the points of the first two plates ①
predators expressed mcmA,
mchB, respectively, and the prey were sensitive to this antimicrobial peptide.
![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-5.png)

(i) E. coli 1917 DE3 containing an expression plasmid for the mcmA gene, (ii) LB blank validation,
(iii) Fusobacterium
bezoarum ADP1 without an expression plasmid for the mcmA gene, (iv) E. coli 1917 without an
expression plasmid for the
mcmA gene, (v) E. coli 1917 DE3 without an expression plasmid for the mcmA gene
![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-6.png)

(ii) E. coli BL21 DE3 containing the expression plasmid of the mchB gene, (ii) LB blank validation,
(iii) Fusobacterium
berghei ADP1 without the expression plasmid of the mchB gene, (iv) E. coli BL21 without the
expression plasmid of the
mchB gene, and (v) E. coli BL21 DE3 without the expression plasmid of the mchB gene.
After overnight incubation, we observed that the strains containing our designed mcmA or mchB
expression plasmids showed
visible zones of inhibition. In addition, we verified the growth of these two antimicrobial
peptide expression plasmid
strains.

![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle4-7.png)

### Learn

Click to Expand

After overnight incubation, we observed that significant repressive regions formed around the
strains containing our
designed mcmA and mchB expression plasmids. The discovery of these repressive regions not only
confirmed the success of
our plasmid constructs, but also showed that our strategy was effective. The results of these
experiments demonstrate
that our engineered strains are able to produce antimicrobial peptides as expected and that these
antimicrobial peptides
have the ability to inhibit the growth of other bacteria.

### Cycle 5: Bacterial antagonism test

Click to Expand

### Design

Click to Expand

The modified engineered bacteria have both defensive and offensive capabilities, so we planned to
design an
inter-bacterial antagonism test to analyze the strength of bacterial antagonism. We compare the
antagonistic strains to
bactericidal cells and prey bacteria, and use the different resistance genes carried by the
bacteria to design the
experiment, and distinguish the strength of the inter-bacterial antagonism after antagonism by the
antibiotic plate
standoff method.

### Build

Click to Expand

We constructed the following sets of inter-bacterial antagonism experiments as a way to verify
whether the defense and
offense ability of the engineered bacteria was improved.

| Bactericidal cells | Prey bacteria |
| --- | --- |
| Acinetobacter baylyi ADP1 | pACYCDuet-1-1917（DE3） |
| Acinetobacter baylyi ADP1 | E. coli strain Nissle 1917 (DE3)：pACYCDuet-1-kpsE-kpsT |
| Acinetobacter baylyi ADP1 | pETDuet-1-1917（DE3） |
| Acinetobacter baylyi ADP1 | pETDuet-1-galU-pgmA |
| Acinetobacter baylyi ADP1 | E. coli strain Nissle 1917 |
| Acinetobacter baylyi ADP1 | Knockout fimE of E. coli strain Nissle 1917 |
| pETDuet-1-1917（DE3） | Acinetobacter baylyi ADP1 |
| 1917（DE3）：pETduet-1-mcmI-2（6his）-mcmA(strep) | Acinetobacter baylyi ADP1 |

### Test

Click to Expand

Bactericidal cells and prey bacteria bacterial fluids were inoculated into LB liquid medium and
cultured in a certain
proportion, respectively; after adding inducers and mixing them in a certain proportion, they
were
cultured; the mixed
bacterial fluids were pipetted and coated on plates containing different resistances,
respectively, and the survival
rate of the strains was calculated. The survival rate of the strains was calculated and plotted
as
an ordinary one-way
ANOVA algorithm (you can go to “Experiment” to see the detailed steps). The results of the
antagonism experiments
between several groups of bacteria in the table are shown below:

![...](https://static.igem.wiki/teams/5248/part/cycle5-1.webp)

### Learn

Click to Expand

Through the above four groups of ordinary one-way ANOVA analysis, in the inter-bacterial
antagonism experiment, compared
with the control group, the modified strains have a significant increase in the defense and
offense ability than the
pre-modified strains.
Figure 1 and 2: Increased pod production, which plays a big role in interbacterial antagonism,
can
improve the defense
ability of the strain itself, thus resisting the attack of other bacteria.
Figure 3: Higher survival of strains with knockdown of fimE, the ΔfimE mutant was able to lead
to
the formation of
microcolonies, where target cells were protected from contact-dependent killing mediated by e.g.
T6SS, leading to the
survival of the engineered bacteria.
Figure 4: The modified EcN producing micromycin enhances the long-range aggressiveness of the
strain itself compared to
the control, and the strain's own offensive system is thus greatly enhanced.

### Cycle 6: Lethal Gene Circuit

Click to Expand

### Design

Click to Expand

The ccdB gene was utilized to make suicide switches through synonymous codon mutations and the
LapB/LpxC system in hopes
of finding the genetic switch that kills our engineered bacteria within 20-24h.
CONSTRUCTION: We introduced the involved lethal gene into Nissle 1917 (DE3) and screened it
using
resistance plates.

### Build

Click to Expand

Nissle 1917 (DE3) was cultured using SOC medium and its growth was monitored by detecting the
OD600 value, when the
OD600 value reached 0.6, the lethal gene was induced to be overexpressed by adding 0.5mM IPTG,
and
the time of death of
the engineered bacteria was determined by measuring the OD value at regular intervals.
Figure: Growth curves of several ccdB synonymous codon lethal records

### Test

Click to Expand

Due to the limited experimental time, we were not able to complete all the tests of the lethal
gene circuits, but our
experiments will not stop here, in the future we will continue to test and verify, so as to draw
a
perfect conclusion
for our work.

![...](https://static.igem.wiki/teams/5248/wet-lab/engineering/cycle6-1.png)

### Learn

Click to Expand

Due to the limited experimental time, we were not able to complete all the tests of the lethal gene circuits, but our experiments will not stop here, in the future we will continue to test and verify, so as to draw a perfect conclusion for our work.

## References

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block bgimg %}experiment.jpg{% endblock %}
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## Experiments

{% include 'pages/wikimenu.html' %}

## Capsular Polysaccharide Yield Test

#### I. Materials:

•Bacterial Polysaccharide Extraction Kit (Bolson Bioscience)

• Anhydrous Ethanol

• 80% Ethanol

• Polysaccharide Assay Kit (Grace Biosciences)

• Concentrated Sulfuric Acid

#### II. Sample Processing

1. Cultivate the engineered bacteria in LB liquid medium to an OD600 of 0.6, then add 0.5mM IPTG to induce gene
overexpression for 3 hours, and finally measure and record the OD600.

2. Dilute the aforementioned treated bacterial cultures to an OD600 of 0.6 with LB medium.

3. Take 2ml of bacterial culture in a microcentrifuge tube and centrifuge at 8000×g for 3 minutes, discard the
supernatant, and collect bacterial cells.

4. Add 500μl of Reagent A to resuspend the cells, centrifuge at 8000×g for 3 minutes, and discard the
supernatant.

5. Collect the pellet and repeat step 4.

6. Collect the pellet, add 500μl of Reagent B to resuspend the cell pellet, and incubate in a water bath at
80°C
for
6
hours.

7. Centrifuge at 10000×g for 10 minutes.

8. Transfer the supernatant to another clean centrifuge tube to obtain the extracellular polysaccharide sample
for
testing.

### III. Differential Determination of Capsular Polysaccharide Production

1. Take 0.3mL of extracellular polysaccharide sample into a new EP tube, add 1.2mL of ethanol, mix well, and
place
at
4°C for 1 hour. After centrifugation at 8000rpm for 5 minutes, discard the supernatant and retain the pellet.

2. Add 1mL of 80% ethanol to the pellet from the previous step, mix briefly, centrifuge at 8000rpm for 5
minutes,
discard the supernatant, and retain the pellet.

3. Add 2mL of distilled water to the pellet from the previous step and heat in a boiling water bath (95-100°C)
until
the pellet is completely dissolved (approximately 5 minutes) to obtain the bacterial polysaccharide sample for
testing.

4. For each sample, follow the instructions in the table:

| Reagents (μL) | Test Tube |
| --- | --- |
| Sample for Testing | 100 |
| Reagent One | 50 |
| Concentrated Sulfuric Acid | 250 |

5. Mix the solution well, seal tightly with a membrane, place in a 95°C water bath for 20 minutes, cool to room
temperature, transfer 200μL to a 96-well plate, and read the absorbance at 488nm.

## Biofilm Detection

1. Add 2ml of liquid culture medium to each well of a 6-well polystyrene microplate, inoculate the bacterial
culture
diluted to an OD of 0.05 in the total system, add 0.5mM IPTG, and incubate statically at 37°C for 6 hours.

2. Aspirate the culture medium, add 2ml of sterile PBS buffer to each well to wash the plate wells once
(washing
must
be gentle, as the biofilm is at the bottom of the wells).

3. Add 2ml of 10% methanol to each well to fix for 15 minutes, then aspirate the methanol and air dry.

4. Add 2ml of 0.1% crystal violet solution to each well and stain at room temperature for 5 minutes (the
concentration
of crystal violet solution can vary depending on the thickness of the biofilm, generally controlled within an OD
of
0.2-1 for accuracy).

5. Aspirate the crystal violet staining solution from the culture wells and rinse off excess dye with water.

6. Invert the culture plate on filter paper to remove residual water and dry in a 37°C oven or at room
temperature.

7. After complete drying, add 2ml of 95% ethanol solution to each well and let it act for 30 minutes at 37°C
to
dissolve the crystal violet.

8. Measure the OD of the solution in the culture wells under 590nm (570nm/595nm) conditions using a microplate
reader.

9. Use uninoculated culture medium as a negative control, with all other operations consistent with the
experimental
group.

10. Perform the experiment with three replicates for each bacterial strain in each trial, and take the average
of
three trials for the experimental values.

## The Impact of Gene Overexpression on Bacterial Growth

1. First, spray the laminar flow hood with alcohol, and then spray the relevant experimental equipment with
alcohol before placing them into the laminar flow hood. Sterilize with UV light for 20 minutes.

2. Add bacterial culture with an OD600 of 0.6-0.8 to 0.5mM IPTG induction medium and cultivate for 3 hours.

3. Control group: Inoculate 10% of the culture into 5ml of LB liquid medium, mix well; Experimental group:
Inoculate 10% of the culture into 5ml of LB liquid medium containing 0.5mM IPTG, mix well.

4. At regular intervals (every 2 hours), take a small amount of bacterial culture, mix it evenly with a vortex
mixer, and measure the OD600 value of the culture (take the average of three parallel experimental data) to plot
the growth curve graph.

## Heparin Adsorption Experiment Protocol

### I. Materials

• Sodium Heparin

• 0.45μm Sterile Microporous Membrane

• Sterile Syringe

• Anhydrous Ethanol

• 80% Ethanol

• Glycoprotein Assay Kit (Grace Biosciences)

• Concentrated Sulfuric Acid

### II. Verification of Flic Protein's Heparin Adsorption Capacity (Under Similar Growth Conditions)

1. Cultivate the modified strain, the wild-type strain, and the empty vector to an OD600 of 0.6. Add 20mg/ml
sodium
heparin solution (filtered and sterilized) to each bacterial culture, and add 0.5mM IPTG inducer to the modified
strain, continue to culture for 3 hours, and finally measure and record the OD600.

2. Take 3ml of each bacterial culture and filter through a microporous membrane to remove bacteria.

3. Transfer 0.3mL of the filtrate into a new EP tube, use LB liquid medium as the blank group, add 1.2mL of
ethanol
to
each, mix well, and place at 4°C for 1 hour. After centrifugation at 8000rpm for 5 minutes, discard the
supernatant
and retain the pellet.

4. Add 1mL of 80% ethanol to the pellet from the previous step, mix briefly, centrifuge at 8000rpm for 5
minutes,
discard the supernatant, and retain the pellet.

5. Add 2mL of distilled water to the pellet from the previous step, heat in a boiling water bath (95-100°C)
until
the
pellet is completely dissolved to obtain the supernatant sodium heparin solution for testing.

6. Perform three parallel measurements, and follow the instructions of the microplate method glycoprotein assay
kit
(phenol-sulfuric acid method):

| Reagents (μL) | Test Tube |
| --- | --- |
| Sample for Testing | 100 |
| Reagent One | 50 |
| Concentrated Sulfuric Acid | 250 |

7.Mix the solution well, seal tightly with a membrane, place in a 95°C water bath for 20 minutes, cool to room
temperature, transfer 200μL to a 96-well plate, and read the absorbance at 488nm.

### III. Exploration of the Optimal Amount of Sodium

Heparin to Add Design a gradient of sodium heparin addition at
concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6mg/ml. After cultivation, take 3ml of bacterial
culture, filter through a microporous membrane to remove bacteria, and perform heparin content determination;
adjust
the gradient addition to 0, 0.4, 0.5, 0.6, 0.7, and 0.8mg/ml, with other operations remaining the same.

### IV. Detection of Remaining Sodium Heparin in the Supernatant

1. Glucose Standard Solution: Weigh 2mg of glucose standard into a new EP tube, add 2mL of distilled water to
dissolve and mix to make a 1mg/mL glucose solution. Dilute the mother liquor with distilled water to make six
concentration gradients of standards: 0, 0.02, 0.04, 0.06, 0.08, and 0.1mg/mL, and create a standard curve.

2. Glycoprotein Content Calculation Formula:

(mg/mL liquid) = [(ΔA-0.0065) ÷ 86.454] ÷ (V2×V1÷V) × D = 0.2313 ×
(ΔA-0.0065) ÷ V2 × D

V---Total volume of sample extraction liquid, 2mL;

V1---Volume of sample solution for testing, 0.1mL;

V2---Volume of liquid sampling, 0.3mL.

## Bacterial Motility Test

1. Prepare semi-solid agar medium (0.5% agar), sterilize, and pour into plates in a laminar flow hood. Use a
pipette tip to pick a single colony of the target bacterial strain from the plate, and inoculate it into the
semi-solid medium by stabbing. Incubate at 37°C.

2. Observe under transmitted light; if the growth is confined to the stab line with a clear edge, it indicates
that
the bacteria are non-motile. If the growth spreads from the stab line in all directions with a cloudy edge, it
indicates that the bacteria are motile. The extent of the spread from the stab line can be observed to assess
bacterial motility, indirectly indicating changes in bacterial flagella.

3. For fast-growing bacteria, observe after 1 day of incubation, and if necessary, extend the observation to
2-3
days or 5-6 days.

## Agar Well Diffusion Assay

1. Inoculate the predator and prey bacterial cultures into LB liquid medium at a defined volume and cultivate
at
a
constant temperature until the OD600 reaches 0.6 (the concentrations of both cultures should be similar). Add
0.5mM
IPTG and induce at 37°C with 220rpm for 3 hours.

2. Preliminary experiment: Mix the predator and prey cells at certain ratios (1:1, 1:100, 1:10000, 10000:1,
100:1)
and cultivate for 3 hours. Perform serial dilutions (dilution factors of 1, 100, 10^4, and 20^4), then spread
20μl
of the mixed culture on plates with varying antibiotic resistances, and include a blank control.

3. Based on the preliminary experiment, the best results were obtained when the predator and prey cells were
mixed at a ratio of 1:100 or 100:1 and cultivated for 3 hours, then diluted to 20,000-fold.

| Media and Bacteria Volume Calculator: | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Strain | Final OD600 | Final Volume(ml) | Measured OD600 | Volume to Add(ml) |
| Prey | 0.1 | 5 | Prey OD600 | 0.1\*5/Prey OD600 |
| Predator | 50 | 0.1 | Predator OD600 | 50\*0.1/Predator OD600 |

4. Then pour it onto the regular LB agar plates and allow it to cool and solidify.

5. Transfer the calculated volume of the predator strain to a centrifuge tube, centrifuge at 13000 RPM for 1
minute, discard the supernatant, and resuspend in 100μl of LB liquid medium.

6. Make holes in the solidified mixed-strain agar plate, add 100μl of the centrifuged and resuspended predator
strain to one hole, and add 100μl of LB liquid medium to another hole as a blank sample.

7.Incubate at the appropriate temperature for 12-24 hours and record the size of the inhibitory zone around the
predator strain.

## Bacterial Antagonism Assay

1. Inoculate the predator and prey bacterial cultures into LB liquid medium at a defined volume and cultivate
at
a constant temperature until the OD600 reaches 0.6 (the concentrations of both cultures should be similar). Add
0.5mM IPTG and induce at 37°C with 220rpm for 3 hours.

2. Preliminary experiment: Mix the predator and prey cells at certain ratios (1:1, 1:100, 1:10000, 10000:1,
100:1) and cultivate for 3 hours. Perform serial dilutions (dilution factors of 1, 100, 10^4, and 20^4), then
spread 20μl of the mixed culture on plates with varying antibiotic resistances, and include a blank control.

3. Based on the preliminary experiment, the best results were obtained when the predator and prey cells were
mixed at a ratio of 1:100 or 100:1 and cultivated for 3 hours, then diluted to 20,000-fold.

4. Formal experiment: ① Inoculate the predator and prey bacterial cultures into LB liquid medium at a defined
volume and cultivate at a constant temperature until the OD600 reaches 0.6 (the concentrations of both cultures
should be similar). Add 0.5mM IPTG and induce at 37°C with 220rpm for 3 hours. ② Mix the predator and prey cells
at a certain ratio (1:100 or 100:1) and cultivate for 3 hours, then dilute the culture to 20,000-fold. ③ Spread
20μl of the mixed culture on plates with varying antibiotic resistances, and include a blank control.

5. After overnight incubation in an incubator, calculate the CFU values for the predator and prey cell plates.
[CFU = (number of colonies counted) / (volume of sample applied × dilution factor)]

## Bacterial Self-Destruction Time Recording

1. Take 100μL of the killing bacteria into 5mL of SOC medium and incubate in a 37°C constant temperature shaker
until the OD reaches 0.6.

2. After cultivation, add 5μL of IPTG inducer to the bacterial culture, then incubate in a 37°C constant
temperature shaker for 3 hours.

3. Record the OD value after the 3-hour incubation, then continue to record the OD value every 2 hours for a
period of 12 hours.

{% endblock %}Heparin Adsorption

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

## Results

{% include 'pages/wikimenu.html' %}

## Overview

Considering the future application scenarios of our engineered bacteria, we will construct mixed microbial
communities,
focusing on two main modules: defense and offense.
In the defense aspect, we will enhance the microbial community's defensive capabilities by increasing the
thickness of
the capsule polysaccharide and elongating the pili through gene knockout. In the offensive aspect, we plan to
overexpress genes related to remote aggression (mcmA, mchB, mcmI, and mchI) and enhance the competitiveness of
the CDI
system. Additionally, we will attempt to activate the T6SS in EcN by introducing the tagh gene from Escherichia
coli 042
to observe any improvements in EcN's aggressiveness. We have also designed two lethal gene circuits to ensure
the safety
of our engineering experiments.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/48.webp)
![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/49.webp)

## Capsule Overexpression System:

### 1、Capsule Yield:

To enable co-expression with other genes, we selected pACYCDuet-1 and pETDuet-1 as our plasmid vectors. Through
extracellular polysaccharide extraction experiments, both genes showed significant differences, successfully
validating
that our genes can promote the production of extracellular polysaccharides.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/50.webp)
![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/51.webp)

Figure 1 and 2: Pod polysaccharide production of pACYCDuet-1-1917
(DE3), pACYCDuet-1-KpsE-KpsT, and pETDuet-1-galU-pgmA
when cultured to OD=0.6, expression was induced by the addition of 0.5 mM IPTG for 3 h, and the OD of the
bacterial
solution was diluted to 0.4.

In addition, we performed qPCR detection on the constructed pACYCDuet-1-KpsE-KpsT, and found that the mRNA
overexpression levels of KpsE and KpsT genes were high, suggesting the possibility of false positives. Currently,
we are
optimizing primer design and conducting further protein level detection. At the characterization level,
overexpression
of KspT and KspE has been shown to increase capsule yield.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/52.webp)
![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/53.webp)

### 2、Growth Curve Monitoring:

**Conclusion:**  At a 1% inoculation rate and a 5ml culture volume, incubated at 37°C and 220rpm for
12 hours, the
genetically modified strains exhibited slower growth compared to unmodified strains, though no significant
differences
were observed.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/54.webp)

Figure: Growth curves of transformed bacteria associated with
chassis
bacterium 1917 (DE3)

## Biofilm Formation Detection:

In our biofilm validation, we tested the yield of the NirC and FimH genes in Nissle 1917 and Nissle 1917(DE3).
With the
same initial OD600 of 0.05 and a 12-hour incubation in a six-well plate, we analyzed the data using standard
one-way
ANOVA. In Nissle 1917, FimH expression was higher than in the original strain but showed no significant
difference. In
Nissle 1917(DE3), the pACYCDuet-1-NirC strain showed a significant difference, with biofilm production much higher
than
the other two strains.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/55.webp)
![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/56.webp)

Figure 3: Nissle 1917 biofilm formation;
Figure 4: Nissle 1917
(DE3) biofilm formation

## Gene Knockout Module:

We validated the defensive capability of the fimE knockout strain through bacterial antagonism tests. Data
analysis
showed that the ΔfimE mutant had a higher survival rate, indicating that knocking out the fimE gene leads to
micro-colony formation, protecting target cells from T6SS-mediated contact-dependent killing, thus enhancing the
survival of the engineered bacteria. Therefore, knocking out the fimE gene can improve the strain's resistance to
bacterial attacks.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/improvement/4.webp)

## Inhibition Zone Experiment:

We inoculated 100 μL of each predator strain's overnight concentrated culture on LB agar plates, covered with
soft LB
agar containing prey strains. For the mcmA and mchB antimicrobial peptide expression plasmids, the prey strain was
Escherichia coli DH5α, and the predator strains were E. coli Nissle 1917 DE3 and BL21 DE3. In this experiment, the
first
two plates showed predators expressing mcmA and mchB, with the prey sensitive to these antimicrobial peptides.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/58.webp)

(i)E. coli 1917 DE3 containing an expression plasmid for the mcmA
gene, (ii) LB blank validation, (iii) Fusobacterium
bezoarum ADP1 without an expression plasmid for the mcmA gene, (iv) E. coli 1917 without an expression plasmid
for the
mcmA gene, and (v) E. coli 1917 DE3 without an expression plasmid for the mcmA gene.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/59.webp)

(i)E. coli BL21 DE3 containing the expression plasmid of the mchB
gene, (ii) LB blank validation, (iii) Fusobacterium
bezoarum ADP1 without the expression plasmid of the mchB gene, (iv) E. coli BL21 without the expression plasmid
of the
mchB gene, and (v) E. coli BL21 DE3 without the expression plasmid of the mchB gene.

After overnight incubation, we observed that the strains containing our designed mcmA or mchB expression plasmids
showed
visible zones of inhibition. In addition, we verified the growth of these two antimicrobial peptide expression
plasmid
strains.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/60.webp)

We also extracted the supernatant for fluorescence detection using the green fluorescence labeling gene, and the
results
were as follows.

![WikiBreeze Image](https://static.igem.wiki/teams/5248/wet-lab/results/61.webp)

Figure: pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB(strep) + IPTG,
BL21(DE3),
pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB(strep) fluorescence analysis of detected signals.

Based on these results, we concluded that our designed mcmA and mchB expression plasmids were successful, and the
overexpression of the corresponding immune proteins mcmI and mchI effectively protected host cells from the
antimicrobial peptides produced, enhancing the aggressiveness of our engineered bacteria.

## Inter-species Antagonism Verification:

For safety considerations, we temporarily did not use actual pathogenic bacteria but chose Acinetobacter baylyi
ADP1,
which has both close-range and remote aggression through T6SS, as a substitute for the bacterial antagonism tests.
The
results indicated that compared to the unmodified strains, the engineered bacteria exhibited significant
improvements in
both defense and offense capabilities, with increased capsule polysaccharide yield, the fimE knockout mutant
showing
higher survival rates, and the secretion of antimicrobial substances.

## Lethal Gene Circuit Module:

We reviewed relevant literature and proposed two types of lethal switches: one through ccdB using synonymous
codon
mutations, designing 43 lethal switch tests; the other through intermembrane induction of lethality. We
preliminarily
explored the interaction between LapB and LpxC, where increased LapB promotes the degradation of LpxC, which
negatively
regulates lipopolysaccharide synthesis. YejM prevents LpxC degradation and can transfer substances from the inner
membrane to the outer membrane. By mutating the LapB gene to increase its levels, LpxC levels decrease, leading to
cell
death at very low concentrations. Although we did not complete the lethal switch testing due to the project's full
schedule, our experimental design is ongoing, and we will continue our journey in synthetic biology!

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