iGEM 2024 — Team whu-china

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

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WHU-China emphasizes maintaining a detailed timeline of our yearly activities and ensuring thorough documentation of experimental procedures. We aim for our notebook to serve as a valuable resource for future WHU-China members and others. Additionally, we hold academic integrity in high regard, and all raw data from the "Part," "Engineering," and "Results" sections are available within the notebook. We invite external parties to review and assess our work.

## Experimental records

●Download [Secretion(1). pdf](https://static.igem.wiki/teams/5048/ppdfsecretion-1.pdf)

●Download [Secretion(2). pdf](https://static.igem.wiki/teams/5048/ppdfsecretion-2.pdf)

●Download [Secretion(3). pdf](https://static.igem.wiki/teams/5048/ppdfsecretion-3.pdf)

●Download [Secretion(4). pdf](https://static.igem.wiki/teams/5048/ppdfsecretion-4.pdf)

●Download [Adhesion. pdf](https://static.igem.wiki/teams/5048/notebookadhesion.pdf)

●Download [Biosafety. pdf](https://static.igem.wiki/teams/5048/notebookbiosafety.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5048/til8.png)
![](https://static.igem.wiki/teams/5048/til4.png)
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- [Part 1: Secretion](#Secretion)

- [Cycle1](#Cycle11)

- [Cycle2](#Cycle12)

- [Cycle3](#Cycle13)

- [Part 2: Adhesion](#Adhesion)

- [Cycle1](#Cycle21)

- [Cycle2](#Cycle22)

- [Cycle3](#Cycle23)

- [Part 3: Biosafety](#Biosafety)

- [Cycle1](#Cycle31)

- [Cycle2](#Cycle32)

- [Cycle3](#Cycle33)

- [Cycle4](#Cycle34)

- [References](#references)

Aiming to increase the efficiency of verifying the feasibility of our project, the students in the wet lab group were divided into three subgroups and carried out three sets of experiments on different goals.

The whole engineering process is conducted under the principles of the DBTL (Design, Build, Test, Learn) cycle. The following are the engineering successes we have completed.

## Part 1: Secretion

The overall goal of the secretion module is to design a small peptide surface display system in *E. coli* and verify the enzymatic cleavage of the system.

#### Cycle 1: surface display verification

**● Design**

In order to verify the surface display system, we design two plasmids: Lpp'OmpA\_lptE and lptE, using the plasmid pET28a as the backbone.

 In lptE, full-length lptE(signal peptide included) is fused with a FLAG tag. The fusion protein is controlled by T7 promoter. Once expressed, the full-length lptE will anchor in the inner leaflet of the outer membrane, which is unable to be displayed on the surface.

 In Lpp'OmpA\_lptE, lptE whose signal peptide is cleaved with FLAG tag is fused at the C-terminus of Lpp'OmpA, activated by T7 promoter. Once expressed, Lpp'OmpA\_lptE will display on the surface of the outer membrane and be detected. T7 promoter is regulated by lac operator and terminated by double terminators(BBa\_B0015).

![](https://static.igem.wiki/teams/5048/engineering-fig1re.png)

Fig. 1  Genetic circuits of lptE and Lpp'OmpA\_lptE.

**● Build**

For lptE, lptE-FLAG was synthesized by Genscript, and subcloning was performed to get the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig2rere.webp)

Fig. 2 Map of lptE

For Lpp'OmpA\_lptE, lpp'ompA-lptE(signal peptide removed)-FLAG was synthesized by Genscript and subcloning was performed to get the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig3rerere.webp)

Fig. 3 Map of Lpp'OmpA\_lptE

**● Test**

Plasmids were transformed into *E. coli* BL21(DE3) separately. Sequencing verified transformation success.

![](https://static.igem.wiki/teams/5048/engineering-fig4subpic1re.webp)
![](https://static.igem.wiki/teams/5048/engineering-fig4subpic2re.webp)

Fig. 4 Sequencing of lptE and Lpp'OmpA\_lptE transformant

Western Blot tests were conducted to verify the protein expression in engineered bacteria. Immunofluorescence assay(IFA), FACS analysis, and Cryo-electron microscopy verify the surface display result. To see more details, visit [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#Lpp'OmpA-peptide chimaera synthesis)

**● Learn**

During our testing process, we found that there is a serious leakage of the lac operator. Parameters like temperature, concentration of IPTG, and induction time had minimal effects on the protein amount. A promoter that is more strictly regulated should be applied if we want to put our product into real use.

What's more, the induction condition described in the original literature was not satisfying for our conditions. Since we learned that the lac operator was leaking, we suspected that the high expression rate caused the formation of the inclusion body, preventing the protein from transporting into the outer membrane. After we decrease the induction temperature, we obtain satisfying results.

During our Cryo-electron microscopy experiment, we conducted our investigation with the help of the instrument-assisted manager. After knowing the goal of our experiment, she suggested that Cryo-EM might not be suitable for us. Since Cryo-EM couldn't observe the internal structure of bacteria, we were unable to confirm if the antibody entered the bacteria. She suggested that we try the ultrathin section, which can display the whole structure of Bacteria clearly.

From this cycle, the most important learning was the choice of control group. When similar results were found in the lptE and Lpp'OmpA\_lptE. We immediately communicated with our advisors Jincheng Wang and Jiaze Liu. Both Wang and Liu suggested that pretreatment like fixation might affect the polarity of the outer membrane, thereby causing unsatisfying control. To solve this problem, Liu recommended we use lipoprotein(sp removed) as a control, which will be expressed only in the cytosol. Wang advised us to conduct a blocking procedure in the experiment.

#### Cycle 2: Small peptide secretion and cleavage verification

**● Design**

In order to verify that the small peptide is displayed and can be cleaved by enterokinase, we designed two sets of plasmids. One set includes AQ with different protein tags(AQ\_GST and AQ\_6xhis), and the other set QEP(QEP\_GST and QEP\_6xhis). Take QEP\_GST as an example, small peptide QEP is fused with GST tag at the C terminal and enterokinase site at the N terminal. The fusion is linked with the C terminal of lpp’OmpA, activated by the T7 promoter.

![](https://static.igem.wiki/teams/5048/engineering-fig5re.png)

Fig. 5 Genetic circuit of AQ and QEP sets.

**● Build**

For the AQ set, Lpp'OmpA-enterokinase site-AQ-protein tag was synthesized by Genscript, and subcloning was performed to get the final products.

![](https://static.igem.wiki/teams/5048/engineering-fig6subpic1re.svg)
![](https://static.igem.wiki/teams/5048/engineering-fig6subpic2re.svg)

Fig. 6 Map of AQ set

For the QEP set, Lpp'OmpA-enterokinase site-QEP-protein tag was synthesized by Genscript, and subcloning was performed to get the final products.

![](https://static.igem.wiki/teams/5048/engineering-fig7subpic1re.svg)
![](https://static.igem.wiki/teams/5048/engineering-fig7subpic2re.svg)

Fig. 7 Map of QEP set

**● Test**

Plasmids were transformed into *E. coli* BL21(DE3) separately. Sequencing verified transformation success.

![](https://static.igem.wiki/teams/5048/engineering-fig8rere.webp)

Fig. 8 Sequencing of AQ&QEP sets transformant.

Similar to cycle 1, Western Blot tests were conducted to verify the protein expression in engineered bacteria. Immunofluorescence assay(IFA), FACS analysis, and Cryo-electron microscopy verified the success of surface display. However, different from cycle 1, the enzymatic cleavage experiment verified the availability of the whole system. To see more details, visit the
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#Enterokinase digestion).

**● Learn**

Bacteria will lyse either by prolonged induction at low temperature or by extended digestion in the nutrient-deprived buffer.

The results of the enterokinase digestion experiment (37℃-1h is more positive) is not contradict to results of FACS.

In FACS analysis, the instrument only detects intact cells, and the results show a higher positive signal in bacteria induced at 16°C for 12 hours. This suggests that the chimeras on the outer membrane are more accessible to antibodies under these conditions. However, this does not necessarily mean that the chimeras presentation is more pronounced at lower temperatures. Factors such as antibody accessibility and potential damage to the bacteria during the incubation process could influence the results.

In the enterokinase digestion experiment, bacteria induced at 16°C for 12 hours gradually lyse in the medium, releasing Lpp'OmpA-FLAG-protein-GST and some degraded protein-GST. This released protein may be involved in the digestion, leading to a stronger signal compared to bacteria induced at 37°C for 1 hour induction.

All Lpp'OmpA-FLAG signal is missing in the experiments. The reason may be that the FLAG tag is in an inherently undetectable structural domain after digestion or the antibody affinity is not good enough to generate detectable signal. To further investigate the protein we can conduct TEM experiment to detect Lpp'OmpA-FLAG which is anchored on outer membrane after digestion.

#### Cycle 3. Promoter Changing

**● Design**

Based on the Learn in cycle 1, we knew that the lac operator was leaking. Therefore, to make our induction more controllable, we decided to replace the T7 promoter and the lac operator with the PBAD promoter, which is induced by L-arabinose. We choose QEP\_GST and AQ\_GST as our targets and design pBAD\_QEP\_GST and pBAD\_AQ\_GST. The genetic circuits are similar to the QEP\_GST and AQ\_GST, except that the PBAD substitutes the T7 and the lac operator.

![](https://static.igem.wiki/teams/5048/engineering-fig9rere.png)

Fig. 9 Genetic circuit of pBAD\_AQ/QEP\_GST

**● Build**

We conducted PCR on QEP\_GST and AQ\_GST to get insertion fragment. The subcloning was performed to get the final result.

![](https://static.igem.wiki/teams/5048/engineering-fig10subpic1re.svg)
![](https://static.igem.wiki/teams/5048/engineering-fig10subpic2re.svg)

Fig. 10 Maps of pBAD\_QEP\_GST and pBAD\_AQ\_GST

**● Test**

Plasmids were transformed into *E. coli* BL21(DE3) separately. Sequencing verifies transformation success.

![](https://static.igem.wiki/teams/5048/engineering-fig11rere.webp)

Fig. 11 Sequencing of pBAD\_QEP\_GST and pBAD\_AQ\_GST
transformant.

Due to the time limitation, we were unable to test further.

## Part 2: Adhesion

In the Adhesion module, we aim to construct a cell-cell adhesion system that enables our engineered bacteria to colonize the intestine with adhesion proteins LAP and HSP60, and the surface display toolbox Neae. To evaluate the adhesion effects of LAP and HSP60 and prove the display box can work, we conducted 3 cycles.

#### Cycle 1

**● Design**

To assess the adhesion effects of LAP and HSP60, we individually inserted the corresponding genes into the pET-28a vector backbone. Additionally, we fused the FLAG tag and the 6xHis tag with HSP60 and LAP, respectively. For Neae, the FLAG tag was incorporated into the display region of the outer membrane. All of the coding sequences have gone through codon optimization to improve the expression of the proteins. See more in [Parts page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%7D%7D).

![](https://static.igem.wiki/teams/5048/engineering-adhesion-cycle1-hsp-flag.png)
![](https://static.igem.wiki/teams/5048/engineering-adhesion-cycle1-lap-his.png)
![](https://static.igem.wiki/teams/5048/engineering-adhesion-cycle1-neae-flag.png)

Fig. 12 The genetic circuits for Adhesion Constrct.

**● Build**

The plasmids were synthesized by Genscript. We transferred these plasmids into *E. coli* DH5α and BL21 (DE3) respectively and tested the presence of specific genes through PCR.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig1.webp)

Fig. 13 Overview of plasmids used in the adhesion construct group

                A. Map of pET-28a-[ LAP-His]

                B. Map of pET-28a-[ HSP60-Flag]

                C. Map of pET-28a-[Neae-Flag]

**● Test**

After strictly following the protocol, we sequentially transformed the plasmids into DH5α and BL21 (DE3) and confirmed this by sequencing afterward.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig2.webp)

Fig. 14 The confirmation of transformation respectively.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig2.webp)

Fig. 15 The sequencing results of transformation in *E. coli*
BL21(DE3).

            A: The sequencing result of pET-28a-[HSP60-FLAG]

           B: The sequencing result of pET-28a-[LAP-His]

            C: The sequencing result of pET-28a-[Neae-FLAG]

Next, we conducted expression tests on three proteins: Neae, LAP, and HSP60. First, we used IPTG-induced BL21(DE3) cells lysate to directly prepare samples for Western Blot, confirming the expression of three proteins. For the surface display system Neae, we extracted membrane proteins and confirmed the expression of Neae on the membrane using Western Blot. In addition, we used immunofluorescence further to observe its localization on the cell membrane surface. For LAP and HSP60 proteins, we extracted membrane and cytosolic proteins, lysed the bacteria with WB&IP lysate and ultrasonication, and obtained proteins in the bacterial supernatant to perform Co-IP to verify the interaction between the two proteins. For LAP, we also performed nickel column purification and Coomassie Brilliant Blue staining to verify the expression. However, although both proteins are expressed, their concentration in the bacterial lysis supernatant is extremely low, which hinders the verification of protein interactions through Co-IP. See more in [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#Confirmation of the protein).

**● Learn**

Despite attempting various induction conditions, the protein content in the bacterial supernatant remains low. This might be attributed to the low solubility of the two proteins or the excessively fast expression rate of the T7 promoter that led to the formation of a large number of inclusion bodies. In such a circumstance, it becomes challenging to obtain sufficient soluble proteins for interaction verification. Furthermore, there is a certain degree of leakage expression associated with the T7 promoter.

#### Cycle 2

**● Design**

After a conversation with Professor Fei Gan, we obtained advice on changing protein tags to enhance the solubility of LAP and HSP60. SUMO(Small Ubiquitin-like Modifier) is an N-terminus tag that has been proven to be able to enhance protein solubility and improve protein expression in prokaryote[1]. In addition, the GST tag is a good choice for optimizing the expression of soluble proteins. In light of the situation that the specificity of FLAG was better than the 6xHis tag, we decided to add a SUMO tag at the N-terminus of HSP60 and to replace the 6xHis tag with a GST tag at the C-terminus of LAP. Moreover, we would like to change the promoter T7 to araBAD to settle the issue of inclusion body formation and expression leakage.

![](https://static.igem.wiki/teams/5048/engineering-fig14-1-png.png)
![](https://static.igem.wiki/teams/5048/engineering-fig14-2-png.png)
![](https://static.igem.wiki/teams/5048/result-adhesion-fig13c-11.webp)
![](https://static.igem.wiki/teams/5048/result-adhesion-fig13d-11.webp)

Fig. 16 The genetic circuit that after modification

**● Build**

To add or change protein tags, we designed PCR primers to clone SUMO fragment from pYSW-2(pala-SUMO) (gifted by Gan Lab), and GST fragment from pBAD AQ\_GST (See our [Parts page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%7D%7D)). To change promoters, we would clone genes of LAP and HSP60 and insert them into the backbone of pBAD AQ\_GST plasmid (pBAD). See all of the primers in the Experiment.

We conducted reverse PCR to linearize the vectors and PCR to amplify the fragments. After linearization, we would ligate the PCR products to construct the plasmids by homologous recombination.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig4.webp)

Fig. 17 Plasmid construction

            A.Map of LAP-GST (pET backbone)

            B.Map of SUMO-HSP60 (pET backbone)

            C.Map of pBAD-HSP60-Flag

            D.Map of pBAD-LAP (Tag changed, LAP-GST)

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig5.webp)

Fig. 18 The target fragments.

            A.SUMO; Length: 325bp.

            B.GST; Length: 689bp.

            C.HSP60; Length: 1816bp.

            D.LAP; Length: 2654bp.

**● Test**

The fragments SUMO and HSP60-pET-backbone were successfully amplified through PCR and then joined to form plasmid SUMO-HSP60 through homologous recombination. Ultimately, we successfully transferred the plasmid into *E. coli* DH5α and BL21 (DE3) (See more in [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D)). Moreover, we successfully amplified LAP and HSP60 segments for the backbone of pBAD, yet with the change of GST tag and further optimization (mentioned below) they couldn't be employed anymore.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig6.webp)

Fig. 19 The agarose gel electrophoresis of LAP and HSP60 segments PCR products. LAP: 2654bp; HSP60: 1816bp.

**● Learn**

We found that the map of pBAD AQ\_GST that we use to design primers was wrong, for it was the unoptimized version with an original GST sequence instead of the codon-optimized one, which brought about the differences in the specific base pairs. Under this circumstance, we had to redesign the homologous arm of the primers thus leading to the changes of the segments.

Moreover, to obtain more HSP60 proteins, we inserted the SUMO tag into the pBAD-HSP60-Flag plasmid, which also changed the specific segment of HSP60.

**● Redesign**

We corrected the mistakes in the primers and changed the strategy of inserting HSP60 into the pBAD backbone——we used SUMO-HSP60 instead.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig7.webp)

Fig. 20 The new target segments.

A. GST segment. The codon optimization is employed and only the specific bases change. Length: 689bp.

B. LAP segment. Changes as the GST segment changes. Length: 2652bp.

C. HSP60 segment. Changes with the insertion of the SUMO tag to pBAD-HSP60-Flag plasmids; Length: 1760bp.

D. SUMO segment. Changes with the insertion of the SUMO tag to pBAD-HSP60-Flag plasmids; Length: 2654bp.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig8.webp)

Fig. 21 The improvement of the pBAD-HSP60-Flag gene circuit

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig9.webp)

Fig. 22 The new pBAD-SUMO-HSP60-Flag plasmid.

**● Build**

We conducted reverse PCR to linearize the vectors and PCR to amplify fragments. After which we would conduct homologous recombination to construct plasmids.

**● Test**

The target sequences HSP60(for pBAD), LAP(for pBAD), GST(for pET), and pBAD backbone(for SUMO-HSP60) were successfully amplified. See more in [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D).

The progress of our experiment is as follows after the primers change:

![](https://static.igem.wiki/teams/5048/engineering-adhesion-table1.webp)

Table. 1 The amplification and purification processes for the target segments.

**● Learn**

Next, we will continue to construct the plasmids and try to solve the problems mentioned above, finally verifying the interactions between LAP and HSP60.
We may employ agarose gel recovery to acquire purified SUMO-HSP60 pBAD backbone or modify the primers for better specificity to obtain a single band. Moreover, for segments with no
PCR success, we may improve the annealing program or change the primers.

#### Cycle 3

**● Design**

Finally, our completed adhesion systems Neae-LAP and Neae-HSP60 were constructed to verify the function of the adhesion module.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig10.webp)

Fig. 23 The completed adhesion construction, including Neae-LAP and Neae-HSP60.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig11.webp)

Fig. 24 Plasmid maps of pET-28a-[Neae-HSP60] and pET-28a-[Neae-LAP]

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig12.webp)

Fig. 25 Linearized fragment for pET-28a-[Neae-HSP60] plasmid

        A. Segment of EcoRI-Neae backbone-HindIII. Length: 7087bp.

        B. Segment of HindIII-HSP60-EcoRI. Length: 1955bp

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig13.webp)

Fig. 26 Linearized fragment for pET-28a-[Neae-LAP] plasmid

            A. Segment of Neae backbone. Length: 7259bp.

            B. Segment of LAP. Length: 2649bp

**● Build**

By employing PCR, DNA purification, restriction enzyme digestion, gel extraction and T4 ligase ligation, the fragment and the vector were linearized and then ligated to obtain the target plasmids.

**● Test**

Agarose gel electrophoresis confirmed the successful construction and transformation of the Neae-HSP60 plasmid. Further WB analysis demonstrated the successful expression of the protein. See more in [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#Protein fusion expression).

Interestingly, during the plasmid transformation, we found a variation in efficiency when inputting different backbone-to-insert fragment ratios. A ratio of 1 to 3 backbone to insert fragment showed better transformation. Furthermore, the colonies on the resistant plates that from a 1 to 3 ratio also grew larger than the other group.

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig14.webp)

Fig. 27 The colony PCR from 1:3 and 1:10 transformation ratio. The success segments are marked by the grey arrow.

For the Neae-LAP plasmid, we obtain the target band and successfully transfer the combined plasmid into *E. coli* BL21(DE3).See more in [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#Protein fusion expression).

![](https://static.igem.wiki/teams/5048/engineering-adhesion-fig15.webp)

Fig. 28 The output for DNA ligation. The reactant are marked in the red box. Segment LAP is marked in the red box below while the segment Neae backbone is marked in the upper box. Neae backbone: 7259bp; LAP: 2649bp

**● Learn**

 For Neae-HSP60, A ratio of 1:3 which corresponds to the specification of ligase contributes to a better transformation effect and growth. Moreover, although we used restriction enzymes that were overdue for two years, they can still function properly. The restriction enzymes from two different companies also can implement double enzyme digestion if the digestion time is the same.

 For Neae-LAP, the primers examined by websites or software may provide better specifications and obtain a single band. A gel extract may provide a more accurate fragment concentration when implemented DNA restriction enzyme digestion and T4 ligase.

## Part 3: Biosafety

#### Cycle 1

**● Design**

In order to verify how strong the quorum sensing promotors Plux、Plas are and to explore a proper concentration to induce them, we designed two plasmids: Plux-deGFP and PLas-deGFP, using the plasmid pET28a as the backbone.

In Plux-deGFP plasmid, full-length deGFP(green fluorescence protein) is fused with a Plux. Moreover, we use T7 promotor to express transcription factor LuxR. Then, promotor Plux is activated by LuxR which is binding with another external quorum sensing N-acyl homoserine lactones (AHLs) Lux 3OC6 Once expressed, the protein deGFP can be excited to green fluorescence and thus be detected by Fluorescence microplate reader.

The PLas-deGFP plasmid nearly works the same as PLux-deGFP plasmid, except that the promotor Plas is activated by the LasR coexisting with Las AHL.

![](https://static.igem.wiki/teams/5048/engineering-fig26re.png)

Fig. 29 genetic circuit of PLux-deGFP and PLas-deGFP

**● Build**

For PLux-deGFP, LuxR-PLux-deGFP was synthesized by Genscript and subcloning was performed to get the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig27re.svg)

Fig. 30 Map of Plux-deGFP

For PLas-deGFP, LasR-PLas-deGFP was synthesized by Genscript and subcloning was performed to get the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig28re.svg)

Fig. 31 Map of PLas-deGFP

**● Test**

Plasmids were transformed into *E. coli* BL21(DE3) separately. Sequencing verifies transformation success.

![](https://static.igem.wiki/teams/5048/engineering-fig30-11.webp)

Fig. 32 Sequencing of PLux-deGFP and PLas-deGFP transformants

Fluorescence microplate reader tests were conducted to verify the protein expression in engineered bacteria. The fluorescence intensity of induced promoter expression was measured in a bivariate between the concentration of the AHLs solution and the induction time. To see more details, visit the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#System verification).

**● Learn**

During our testing process, we found that there was a gradient of fluorescence intensity with the gradient difference in induced concentration of the AHLs and time, and the gradient was in line with our expectations.

What's more, some lptE groups showed a high level of leaking expression of deGFP, which is embodied in the fluorescence intensity. We suspect that in *E. coli*, there exist other AHLs to affect the activation of Plux and Plas.

From this cycle, we had a better understanding of the Lux system and the Las system, which will guide us in the following cycle.

#### Cycle 2: CcdA&CcdB verification

**● Design**

After verifying the functionality of the QS system, we further tested the toxicity of CcdB and the detoxification ability of CcdA. In this cycle, we chose Pet-28a as our backbone and designed one plasmid: CcdB\_CcdA. In the plasmid, CcdB is expressed by the T7 promoter, while CcdA is under the control of araBAD promoter, which is triggered by L-arabinose. All transcription processes are terminated by double terminators.

![](https://static.igem.wiki/teams/5048/engineering-fig30re.png)

Fig. 33 The genetic circuit of CcdB\_CcdA

**● Build**

In plasmid CcdB\_CcdA, the designed fragment was synthesized by Genscript, and subcloning was performed to obtain the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig31re.svg)

Fig. 34 The map of CcdB\_CcdA

**● Test**

During the test process of this cycle, we encountered various problems. The initial problem was transformation. The fact that CcdB was toxic to *E. coli* and the lac operator was leaking made it difficult to transform the plasmid into *E. coli* BL21(DE3). After a month's try, we eventually selected another strain *E. coli* Tuner(DE3)pLysS, and successfully transformed the plasmid into the strain.

![](https://static.igem.wiki/teams/5048/engineering-fig32re.svg)

Fig. 35 Sequencing success for CcdB\_CcdA transformant.

After the transformation, we conducted 2 sets of experiments: CcdB toxicity verification and CcdA detoxicaiton verification. To see more details, visit the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D#CcdB-CcdA toxicity-detoxification verification).

**● Learn**

Firstly, to express a type of protein that is toxic to the host, strain selection must be carefully considered. We chose *E. coli* Tuner(DE3)pLysS instead of *E. coli* BL21(DE3) to obtain the transformants. The protein expression level of Tuner series strains can be precisely regulated by IPTG concentration, with strict concentration dependence. The pLysS plasmid can produce T7 lysozyme, which can effectively reduce the basal expression level of the target gene.

Secondly, the IPTG gradient didn't strictly echo with the OD600 gradient. But there was a turning point between 103μM and 102μM. From 103μM to 102μM, bacteria underwent a growth inhibition state to a normal growth state. In contrast, the result of the L-arabinose gradient didn't show this phenomenon. The L-arabinose gradient was in line with the OD600 gradient well, except the final OD600 in 10g/L L-arabinose was lower than 1g/L. We hypothesized that 10g/L was too high, adding extra metabolic stress to the bacteria and leading to cell death.

Based on this cycle, we better understood the toxicity and detoxication effects of CcdB and CcdA, which will guide us in the following cycles.

#### Cycle 3

**● Design**

After verifying the functionality of basic parts
(LuxR/LuxI system, LasR/LasI system, CcdA&CcdB), we design an exogenous verification
experiment. In this cycle, we combined the QS and CcdA/B toxin-antitoxin systems.
We designed two plasmids: Exo\_LuxR and Exo\_LasR, using pBAD\_hisA as the backbone.

![](https://static.igem.wiki/teams/5048/engineering-fig33re.png)

Fig. 36 The genetic circuit of Exo\_LuxR

In Exo\_LuxR, CcdB is activated by araBAD promoter, which is induced by L-arabinose. CcdA is controlled by promoter Plux. Promoter J23106 constitutively expresses LuxR. All gene transcription processes are terminated by double terminators(BBa\_B0015). In this circuit, when we add arabinose into the medium, the transformed bacteria will be killed by the expression of CcdB. When we add 3OC6, CcdA will be expressed and prevent the toxic effect of CcdB.

In Exo\_LasR, LasR is expressed by araBAD promoter, while CcdB is expressed by Plas.
All the processes of transcription are terminated by double terminators(BBa\_B0015).
When we add 3OC12HSL and L-arabinose, the engineered bacteria will be killed by the
expression of CcdB.

![](https://static.igem.wiki/teams/5048/engineering-fig34re.png)

Fig. 37 The genetic circuit of Exo\_LasR

**● Build**

For both Exo\_LuxR and Exo\_LasR, the designed fragments were synthesized by Genscript, and subcloning was performed to obtain the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig35subpic1re.webp)

Fig. 38 Map of Exo\_LuxR and Exo\_LasR

**● Test**

The test of this cycle was not as expected.

For Exo\_LuxR, we successfully transformed the plasmid into *E. coli* Tuner(DE3)pLysS. The sequencing result was as follows.

![](https://static.igem.wiki/teams/5048/engineering-fig36re.svg)

Fig. 39 Sequencing success of Exo\_LuxR transformants

However, we tried several methods to verify the functionality of the plasmid, all gaining unsatisfying results. Initially, we tried to measure OD600 changes over time. We tried the L-arabinose gradient and 3OC6HSL gradient, but all showed no obvious difference in the growth of bacteria.

![](https://static.igem.wiki/teams/5048/engineering-fig38rerere.webp)

Fig. 40 OD600changes overtime in L-arabinose gradient and 3OC 6HSL gradient

We also tried the CFU method and the trypan blue staining method. In the CFU method, we found no obvious difference between the situation of induction and without induction. In the trypan blue staining method, we found almost all bacteria that appeared in the field of view were alive.
For the time limitation, we didn't conduct testing for the Exo\_LasR plasmid.

**● Learn**

Aiming at figuring out the problem, we discussed it with our advisors Jincheng Wang and Zixuan Ding.

We initially talked about the design of the plasmids. Since all parts were verified in the previous cycle, the sequence wasn't the source of problem. The only difference was that this time, CcdB was expressed under the control of araBAD promoter instead of the T7 promoter. We applied this change due to the finding of leakage of the T7 promoter in the previous cycle and cycle 1 in the Secretion subgroup. We assumed that the strength of araBAD promoter was too weak to express a sufficient amount of CcdB. In the following experiment, we will try to change the promoter.

What's more, since CcdB is toxic to bacteria, it might have random mutations in the sequence during the propagation. We then conducted PCR, using bacteria incubated for 2 weeks as templates. The sequencing result showed that the sequence of CcdB was complete.

![](https://static.igem.wiki/teams/5048/engineering-fig38re.svg)

Fig. 41 Sequencing result for Exo\_LuxR, using bacteria incubated 2 weeks as templates

#### Cycle 4

**● Design**

While we designed the exogenous QS verification, we also designed the endogenous verification experiment. In this cycle, We designed two plasmids: LuxR\_LasI and LasR\_LuxI, using pBAD\_hisA as the backbone.

The overall genetic circuits of these two plasmids are similar to exogenous systems. But this time, the quorum sensing substances are synthesized by bacteria. In LuxR\_LasI, LasI is expressed, which will synthesize 3OC12HSL. In LasR\_LuxI, LuxI is expressed and synthesize 3OC6HSL. Therefore, bacteria transformed with these plasmids separately will secrete quorum sensing substances and activate the genetic circuit of each other.

![](https://static.igem.wiki/teams/5048/engineering-fig40rere.webp)

Fig. 42 the genetic circuit of LuxR\_LasI and LasR\_LuxI

**● Build**

For both LuxR\_LasI and LasR\_LuxI, the designed fragments were synthesized by Genscript and subcloning was performed to obtain the final product.

![](https://static.igem.wiki/teams/5048/engineering-fig41rere.webp)

Fig. 43 The map of LuxR\_LasI and LasR\_LuxI

**● Test**

Due to the time limitation, we were unable to test the endogenous system.

## References

[1]

Panavas, T., Sanders, C., Butt, T.R. (2009). SUMO Fusion Technology for Enhanced Protein Production in Prokaryotic and Eukaryotic Expression Systems. In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4\_20

{% endblock %}

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

![](https://static.igem.wiki/teams/5048/til8.png)
![](https://static.igem.wiki/teams/5048/til4.png)
![](https://static.igem.wiki/teams/5048/til3.png)
![](https://static.igem.wiki/teams/5048/til2.png)
![](https://static.igem.wiki/teams/5048/til1.png)
![](https://static.igem.wiki/teams/5048/til6.png)
![](https://static.igem.wiki/teams/5048/til7.png)
![](https://static.igem.wiki/teams/5048/til5.png)
![](https://static.igem.wiki/teams/5048/ex.png)

- [Part 1: Fragment Obtaining
             & Subcloning](#Fragment Obtaining & Subcloning)

- [PCR](#PCR)

- [Homologous Recombination](#Homologous Recombination)

- [PCR Product Purification](#PCR Product Purification)

- [Plasmid Preparation](#Plasmid Preparation)

- [Chemically Competent Cell Transformation](#Chemically Competent Cell Transformation)

- [Part 2: Key Experiments](#Key Experiments)

- [Growth Condition](#Growth Condition)

- [Western Blot](#Western Blot)

- [Immunofluoroscene Assay](#Immunofluoroscene Assay)

- [Co-IP](#Co-IP)

- [FACS](#FACS)

- [QS Induction](#QS Induction)

## Part 1: Fragment Obtaining & Subcloning

### PCR

#### Taq PCR

Reaction system

![](https://static.igem.wiki/teams/5048/experiments-table1.png)

Program

![](https://static.igem.wiki/teams/5048/experiments-table2.png)

#### touch down PCR

Reaction system

![](https://static.igem.wiki/teams/5048/experiments-table3.png)

Program

![](https://static.igem.wiki/teams/5048/experiments-table4.png)

#### reverse PCR

**System**

ddH2O: 19 μL

2x Phanta Max Buffer: 25 μL

dNTPs: 1 μL

Forward Primer: 2 μL

Reverse Primer : 2 μL

DNA Polymerase: 1 μL

DNA template: 0.1 μL(10pg-30ng)

**Primers**

- Index
- Name
- Sequence
- 1
- F-pET-28a-verify
- GGTGTTATGAGCCATATTCAACG
- 2
- R-pET-verify
- ACGTTTACAATTTCAGGTGGC
- 3
- F-Hsp60/LAP-verify
- CGATCCCGCGAAATTAATACGA
- 4
- R-Hsp60/LAP-verify
- GGGTTATGCTAGTTATTGCTCAG
- 5
- F-EcoRI-backbone-Neae
- CGGAATTCCGCGAATGGGACGCGCCCTGTAG
- 6
- R-backbone-Neae-HindIII
- CCCAAGCTTGGGGTCATCGTCGTCCTTGTAATCCGCAC
- 7
- F-HindIII-HSP60
- CCCAAGCTTGGGTACCGCGGCCGCATAAATTA
- 8
- R-HSP60-EcoRI
- CGGAATTCCGATCCGGATATAGTTCCTCCTTT
- 9
- F sumo hsp60
- GAAGGAGATATACCatgCTGCAGGACTCAGAAG
- 10
- R sumo hsp60
- GTGGGTAGCCTTAAACCTCCAATCTGTTCG
- 11
- F hsp60
- TTAAGGCTACCCACAGTATTT
- 12
- R hsp60
- CATGGTATATCTCCTTCTTAAAGTTAAAC
- 13
- F LAP
- GATCCGGCTGCTAAC
- 14
- R LAP
- CACACCTTTGTACGCTT
- 15
- F GST LAP
- AAGCGTACAAAGGTGTGATGTCCCCTATACTA
- 16
- R GST LAP
- GTTAGCAGCCGGATCTTATTTTGGAGGATG
- 17
- F pBAD hsp60
- GGAGGAATTAACCATGGaaggagATATACCGC
- 18
- R pBAD hsp60
- ACTGTGTTAATTTAACATCACCATTACTTGTCGTCGTC
- 19
- F pBAD hsp60-B
- TGGTGATGTTAAATTAACACAGT
- 20
- F pBAD hsp60-B
- ATGTCCCCTATACTAGGTTATTG
- 21
- R pBAD lap/hsp60-B
- CCATGGTTAATTCCTCCTGT
- 22
- F lap pBAD
- ACAGGAGGAATTAACCATGGAAGGAGATATACCATGGCTATAAAAGAAAAT
- 23
- R LAP pBAD
- CAATAACCTAGTATAGGGGACATCACACCTTTGTACGCTT
- 24
- F sumo hsp60
- GAAGGAGATATACCatgCTGCAGGACTCAGAAG
- 25
- R sumo hsp60
- GTGGGTAGCCTTAAACCTCCAATCTGTTCG
- 26
- F hsp60
- TTAAGGCTACCCACAGTATTT
- 27
- R hsp60
- CATGGTATATCTCCTTCTTAAAGTTAAAC
- 28
- F LAP
- GATCCGGCTGCTAAC
- 29
- R LAP
- CACACCTTTGTACGCTT
- 30
- F GST LAP(N)
- AAGCGTACAAAGGTGTGATGTCGCCGATTCTGG
- 31
- R GST LAP(N)
- GATCCGGCTGCTAACTTACTTCGGTGGGTGATCG
- 32
- F pBAD lap-B(N)
- ATGTCGCCGATTCTGGGCTACTG
- 33
- R pBAD lap/hsp60-B(N)
- TGGTTAATTCCTCCTGTTAGCCCAAA
- 34
- R LAP pBAD(N)
- CAGTAGCCCAGAATCGGCGACATCACACCTTTGTACGCTT
- 35
- F lap pBAD(N)
- ACAGGAGGAATTAACCAAAGGAGATATACCCATGGCTATAAAAGAAAATGCG
- 36
- F SUMO-HSP60 pBAD-B(N)
- TAAGTACATCGCCTGGC
- 37
- R SUMO conju.
- CTGCAGGACTCAGAAG
- 38
- R SUMO-hsp60 pBAD(N)
- GCCAGGCGATGTACTTATTACTTGTCGTCGTCGTC
- 39
- F hsp60
- TTAAGGCTACCCACAGTATTT
- 40
- F SUMO conju.
- CTTCTGAGTCCTGCAGCATGGTATATCTCCTTCTTAAAGT
- 41
- R sumo hsp60
- GTGGGTAGCCTTAAACCTCCAATCTGTTCG
- 42
- F LAP EcoRI
- TCGGACGAATTCCGCATGGCTATAAAAGAAAATG
- 43
- R LAP XhoI
- GTGGTGCTCGAGCGGTTAGT
- 44
- F neae XhoI
- TAAGAATGCGGCCGCACTCGA
- 45
- R neae EcoRI
- GAGCTCGAATTCGTCATCGTCGTCCTTGTA

**PCR condition**

**The First batch**

- Fragment
- Ta(℃)
- Label
- Extension
- Touch down
- SUMO
- 60
- S
- 1min
- No
- GST
- 59
- G
- 1min20s
- Yes
- SUMO-HSP backbone
- 47
- SH
- 8min
- No
- GST-LAP backbone
- 44
- GL
- 8min30s
- No
- HSP
- 59
- H
- 2min30s
- No
- HSP-pBAD backbone
- 46
- HB
- 5min
- No
- LAP-pBAD backbone
- 47
- LB
- 5min20s
- Yes
- LAP
- 62
- L
- 3min20s
- No

**The Second batch**

- Fragment
- Ta(℃)
- Label
- Extension
- GST
- 64
- G
- 1min20s
- LAP-GST backbone
- 44
- GL
- 8min30s
- HSP
- 52
- H
- 2min30s
- SUMO(pBAD)
- 63
- S'
- 1min
- SUMO-HSP-pBAD backbone
- 45
- HB
- 5min
- LAP-GST-pBAD backbone
- 55
- LB
- 5min20s
- LAP
- 65
- L
- 3min20s

### Homologous Recombination

Formula for the amount of linearized vector (ng):
(Number of linearized base pairs x 0.65 x Total pmol) / (1 + 3n)

Formula for the amount of insert fragment (ng):
(Number of linearized base pairs x 0.65 x Total pmol x 3) / (1 + 3n)

Based on a total of 0.05 pmol, the calculated amount for the SUMO-HSP60 (pET) backbone is 4μL, and the amount for the insert fragment SUMO is 1.4μL.

**Reaction System:**
ddH2O (to adjust the total volume)

2x Hieff Clone Universal Enzyme Premix: 10μL

SUMO-HSP60 backbone: 4μL

SUMO fragment: 1.4μL

(Note: The total volume of the system is 20μL, without adding any enzyme directly. The enzyme mix is already included in the 2x Hieff Clone Universal Enzyme Premix.)

The reaction system is incubated in a metal bath at 50°C for 10 minutes.

### PCR Product Purification

1.Add 500 μL of Equilibration Buffer BL to the spin column and centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.

2.Add 5 volumes (250 μL) of Binding Buffer PB to the PCR product, mix well, and transfer the mixture to the spin column. Allow the mixture to stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.

3.Add 600 μL of Wash Buffer PW to the spin column and centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Repeat this step twice.

4.Place the spin column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove any residual Wash Buffer. Allow the spin column to air-dry at room temperature for 5 minutes.

5.Place the spin column into a clean, new 1.5 mL EP tube. Add 30 μL of ddH2O and centrifuge at 12,000 rpm for 2 minutes to elute the DNA. The resulting solution will contain the purified DNA.

### Plasmid Preparation

1.Pick a single colony, and cultivate in 5mL LB medium with antibiotic to saturation.

2.Put the media into tubes repetitively.

3.Centrifuge at 13000rpm for 1min.

4.Discard the medium, and make the remaining pellet as dry as possible.

5.Add 240μl SolutionBuffer I (mainly contains Glucose, NaCl) into the 1.5mL EP tube and resuspend the pellet.

6.Add 240μl Buffer II (whose key component is NaOH) into the same EP tube.

7.Mix it by gentle reversal 5 times until the solution gets clear.

8.Add 350μl SolutionBuffer III (whose key component is acetate) into the same EP tube.

9.Vortex the tube until the solution is uniform.

10.Centrifuge at 12000rpm for 10min.

11.Transfer the supernatant to a new mini-prep column.

12.Centrifuge at 12000rpm for 1min and discard the flowthrough.

13.Add 750μl SolutionBuffer PW (absolute alcohol containing) into the column.

14.Centrifuge at 12000rpm for 1min and discard the flowthrough.

15.Centrifuge again at 12000rpm for 2min to completely dry the column.

16.Put the column into a new EP tube, and add 50μl ddH2O.

17.Centrifuge at 12000rpm for 1min.

### Chemically Competent Cell Transformation

1. Thaw chemically competent cells on ice.
2. Add DNA (30ng), and pipette gently to mix.
3. Let sit for 30 minutes on ice.
4. Incubate cells for 45 seconds at 42℃.
5. Chill on ice for 2 min.
6. Add 1 ml LB at room temperature.
7. Cultivate for 1 hour at 37℃, 150rpm.
8. Centrifuge at 5000rpm for 2min, discard the supernatant to 100 μl and spread onto a plate with appropriate antibiotics.
9. Grow overnight at proper temperature.

## Part 2: Key Experiments

### Growth Condition

Bacteria were inoculated into Luria Bertani (LB) medium at 37°C, 25 °C, 20°C or 16°C, with shaking at 160 rpm.
When required, ampicillin was added to a final concentration of 100 μg/mL.
Cultures grown overnight were diluted to give OD600=0.1 and when they reached OD600=0.6 expression of the recombinant fragment was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at various final concentrations. (0.1mM, 0.5mM, 1 mM, etc.)
The time of induction was one hours.

### Western Blot

1. Inoculate the bacteria in LB medium containing kanamycin and incubate overnight at 37°C with shaking at 250 rpm.

2. Dilute the overnight culture 1:50 in fresh LB medium containing kanamycin. Continue incubating at 37°C with shaking at 250 rpm until OD600 reaches 0.6-0.8.

3. Add IPTG to a final concentration of 1 mM and incubate at 37°C or 25°C with shaking at 250 rpm for 1 hour.

4. Take 1-1.5 mL of the bacterial culture, centrifuge at 12,000 rpm for 1 minute to remove the supernatant, and resuspend the pellet in 40 µL ddH2O. Add 10 µL of 5X SDS loading buffer and mix well. Heat at 102°C in a metal bath for 10 minutes, then immediately place on ice.

5. Load the prepared samples into the wells of an SDS-PAGE gel. Run at 80V for 20 minutes until the blue dye front is compressed at the interface of the stacking and separating gels, then increase the voltage to 130V and continue running for 40 minutes.

6. Assemble the transfer sandwich in the order of Cathode - Filter paper - Gel - Nitrocellulose membrane - Filter paper - Anode, and transfer at 300 mA for 40 minutes.

7. Place the nitrocellulose membrane in an incubation box. Wash with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.

8. Add 5% non-fat milk prepared in TBST and incubate at room temperature with shaking for 1 hour.

9. Wash the membrane with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.

10. Incubate with the primary antibody (1:3000) overnight at 4°C with shaking.

11. Wash the membrane with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.

12. Incubate with the secondary antibody (1:3000) at room temperature with shaking for 1 hour.

13. Wash the membrane with 1X TBST at room temperature with shaking for 5 minutes, repeating three times.

14. Mix the developing solution by combining equal parts of Solution A and Solution B. Add 200 µL to the membrane, protect from light, and wait for 2 minutes. Then develop using the imaging system.

### Immunofluoroscene Assay

Strains were grown as described in the section “Growth conditions”.
Slide pretreatment
Coverslips were dipped in 70% ethanol and kept in the flame using forceps for approximately 15-20 s and directly put inside the well of a sterile tissue culture plate.
The slides were pre-treated with L-polylysine(50μg/mL) to encourage cell attachment. The slides were then air-dried, followed by fixation.
Approximately 108 bacterial cells were collected by centrifugation (10,000\*g for 5 min).
Bacteria were washed with 300 µL of PBS and fluorescently labelled with Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer diluted 1:1000 in PBS.
Stock solution of Oregon Green: dissolve 5mg of the dye into 0.5mL DMSO. When using the dye, dilute 1:1000 in PBS. Wrap it in tin foil to avoid light exposure.
Bacteria were washed twice with 300 µL of sterile PBS and then resuspended in 100 µL of PBS containing 2% (v/v) formaldehyde.
Samples were spotted onto the polylysine-treated slide and incubated for 10-15 min.
Bacteria were washed with 100 µL of PBS and then incubated for 40 min at room temperature with the primary antibody diluted.1:500 In PBS.
Bacteria were washed with 300 µL of PBS and incubated for 20-30 min at RT in the dark with 100 µL of PBS containing a ABflo® 594-conjugated Goat anti-Mouse IgG (H+L) diluted 1:250.
After two washes with 300 μL of PBS, a droplet of DAPI was applied. Incubate 5 min at RT in the dark.
Stock solution of DAPI: dissolve 1mg of the dye into 1mL DMSO. When using the dye, dilute 1:1000 in PBS. Wrap it in tin foil to avoid light exposure.
Analyse the samples with a fluorescence microscope.

### Co-IP

#### Day 1

Strains were grown as described in the section “Growth conditions”.

#### Day 2

1. Take out the culture and collect 40 μL of the induced culture sample.

(Sample preparation: Take 1 mL of the culture (amount can vary), centrifuge at 12000 rpm for 1min, discard the supernatant, add 50 μL of 1x SDS Loading Buffer (containing β-mercaptoethanol), heat at 102°C for 10 min until the sample becomes clear and non-viscous; avoid it being sticky.)

2. Prepare an ice bath, pre-cool the centrifuge at 4°C, and thaw the two types of magnetic beads on ice.

3. Lysis and preparation of bacterial or yeast samples.
For 1 mL of culture, centrifuge in a large centrifuge at room temperature, 8000 rpm, for 5min. If necessary, wash once with PBS. Remove all residual liquid, add 100-200 μL of lysis buffer with inhibitors, gently resuspend, and transfer to another 1.5 mL EP tube.
Lyse on ice for 2-10 min, typically 10 min (during this time, prepare the magnetic beads as described in step 4).
After thorough lysis, centrifuge at 4°C, 10,000-14,000\*g for 3-5 min, and collect the supernatant (remember to add control IgG).

4. Prepare magnetic beads.
Since magnetic beads are stored in a special protective solution, they need to be washed appropriately before adding the sample.

(1) Resuspend the magnetic beads with a pipette, prepare the appropriate amount of beads according to the ratio of 20 μL beads per 500 μL sample, and transfer the desired amount of beads to a clean centrifuge tube.
4 μL of control IgG beads is appropriate, requiring 50 + 50 μL of the supernatant from step 3.

(2) Add TBS to a final volume of approximately 500 μL.

(3) Gently resuspend the beads with a pipette. Place on a magnetic rack for 10 seconds to separate and discard the supernatant. Repeat the above steps twice.

(4) Resuspend the beads in TBS to the initial volume.

5. Immunoprecipitation (IP)

(1) Add the beads and incubate. Add the beads at a ratio of 20 μL beads per 500 μL protein sample (60 μL for 1500 μL) and incubate on a rotating mixer at room temperature for 2 hours or overnight at 4°C (tested 4°C for 7 hours).

(2) Magnetic separation. After incubation, place the supernatant on a magnetic rack for 10 seconds to separate it and discard it. Note: Some supernatants can be kept to detect the effectiveness of the immunoprecipitation. Keep 40 μL of the final Flag wash solution for best results.

(3) Washing. Add 0.5 mL of lysis buffer with inhibitors, gently resuspend the beads with a pipette, place on a magnetic rack for 10 seconds, and discard the supernatant. Repeat the washing with lysis buffer containing inhibitors three times. Note: Washing completeness can also be judged by measuring the OD280 of the wash liquid. If OD280 is greater than 0.05, increase the number of washes as needed.

(4) Elution: SDS-PAGE Sample Buffer Elution Method:

(a) For each 20 μL of original bead volume, add 100 μL of SDS-PAGE Sample Loading Buffer (1X), and heat at 95°C for 5 minutes.

(b) Place on a magnetic rack for 10 seconds to separate and collect the supernatant for SDS-PAGE electrophoresis or Western blot detection.

### FACS

1. Target strain is induced with 1mM of IPTG in 25℃ incubator shaking at 250rpm for 1h

2. Approximately 108 bacteria were collected by centrifugation (10,000 g for 5 min).

3. Bacteria were resuspended and fixed for 20 min at 4 °C with 100uL PBS containing 4% (v/v) formaldehyde following centrifugation

4. The fixed bacteria were then washed with 500uL PBS containing 1% BSA (w/v) and centrifuged

5. Bacteria were resuspended in 100 µL of a solution containing mouse anti-DDDDK antibody diluted 1:500 in PBS containing 1% BSA and incubated at approximately 100rpm for 2h at room temperature.

6. Bacteria were washed with 500 µL of PBS + 1% BSA. Bacteria were then resuspended in 100 µL of a FITC-conjugated goat anti mouse antibody diluted 1:250 in PBS + 1% BSA and incubated at approximately 100rpm for 1h at room temperature in the dark.

7. After a final washing with 500uL PBS, the cells were resuspended in 200 μL of PBS.

8. All data were collected using a CytoFLEX S by acquiring 100,000 events, and the data analysed using the Flow-Jo software.

### QS Induction

#### GFP Induction

1. Strains were grown as described in the section “Growth conditions”.

2. Induce Strains with 3OC12HSL and 3OC6HSL, measure RFU and OD600 under the following conditions, and use RFU/OD600 as the final result

3. RFU measurement requires the use of a Cytation3 multifunctional microplate reader.

(1) Incubate the bacterial solution until the OD600 reaches 0.6, then add 270 μL of bacterial solution and 30 μL of AHL solution to a 96-well plate

(2) Set up a total of 8 concentration gradients for the AHL solution, namely, 0, 10-10, 10-9, 10-8, 10-7, 10-6, 10-5, and 10-4 M, respectively

(3) Measure fluorescence using a black 96-well plate

(4) Measure OD600 and fluorescence every 10 minutes

#### CcdB Killing Verification Experiment

1.Incubate bacteria overnight (100uL into 5mL LB liquid medium with Kanamycin(100μg/mL), add chloromycetin (34μg/mL)[based on our experimental strain])
2.Dilute the bacteria solution with 200uL into 3ml LB liquid medium with Kanamycin, Additional chloromycetin is still
required. Shake the liquid for 5-6 hours till the OD600~0.6(OD~0.8 optimum)
3.Dilution of gradients-induced bacterial solution using IPTG for six samples. The first group was diluted ten times
the stock solution, the second group was diluted one hundred times the stock solution, and so on. We used a total
of six experimental groups. The specific operation was to add 270uL of bacterial solution to the wells, then add
30uL 0.1M IPTG to the first well, mix well, and then transfer 30uL from the first well to the second well, and so on.
In the control wells, we added 30uL ddH2O to 270uL of bacterial solution and aspirated it after mixing.
4.Prolonged assay using a fluorescent microplate reader: using a 96-well plate, 270ul of bacterial solution was
added to each well along with a gradient of IPTG. The machine was adjusted to measure the absorbance of
OD600 every twenty minutes, during which time the plate was incubated at 37° with shaking.

#### CcdA Detoxification Experiment

1.Incubate bacteria overnight (100uL into 5mL LB liquid medium with Kanamycin(100μg/mL), add chloromycetin (34μg/mL)[based on our experimental strain])
2.Dilute the bacteria solution with 200uL into 3ml LB liquid medium with Kanamycin, Additional chloromycetin is still required. Shake the liquid
for 5-6 hours till the OD600~0.6(OD~0.8 optimum)
3.Dilution of gradients-induced bacterial solution using L-arabinose for six samples.
The first group was diluted ten times the stock solution, the second group was diluted
one hundred times the stock solution, and so on. We used a total of six experimental groups.
The specific operation was to add 0.1M IPTG to the original solution to reach the concentration of 0.1 mM IPTG.
Then add 270uL of bacterial solution to the wells, then add 30uL 100g/L L-arabinose(with 0.1mM IPTG) to the first well,
mix well, and then transfer 30uL from the first well to the second well, and so on. In the control wells,
we added 30uL ddH2O to 270uL of bacterial solution and aspirated it after mixing.
4.Prolonged assay using a fluorescent microplate reader: using a 96-well plate, 270ul of bacterial solution was added to each well along with a gradient of IPTG. The machine was adjusted to measure the absorbance of OD600 every twenty minutes, during which time the plate was incubated at 37°C with shaking.

#### Cryo-EM

**1.Bacterial Culture and Induction:**
Inoculate 30 μL of an overnight bacterial culture into 3 ml of medium containing antibiotics. Incubate at 37°C in a shaker at 220 rpm until the OD600 reaches 0.6-0.8.

Add IPTG or arabinose to the desired concentration and induce at the target temperature in a shaker at 220 rpm for the desired duration. 

Take 1 ml of the induced bacterial culture and centrifuge at 6000 rpm for 10 minutes.

**2.Preparation of Cell Suspension:**

Resuspend bacterial cells in 1 ml of 4% paraformaldehyde solution.

**3.Incubation:**

Incubate the cell suspension at room temperature (approximately 20-25°C) for 30 minutes. Ensure the temperature remains stable throughout the incubation process and avoid vigorous shaking or disturbing the suspension.

**4.Washing:**

Preparation of Washing Buffer: Use 1X PBS as the washing buffer, pre-prepare it, and keep it at room temperature.

First Wash: Centrifuge the cell suspension at 5000 rpm (or an appropriate speed) for 5 minutes to pellet the cells. Carefully remove the supernatant without disturbing the pellet. Add an appropriate amount of 1X PBS (typically 1-2 times the initial volume), resuspend the pelleted cells, and gently mix.

Second and Third Washes: Repeat the centrifugation and resuspension process from the first wash, washing the cells a total of three times. During each wash, ensure the cells are thoroughly mixed to remove excess fixative. After the final wash, resuspend the cells in 1 ml of PBS for subsequent experiments.

Note: Handle the cells gently throughout the procedure to avoid damaging their structure. Select an appropriate centrifugation speed and duration to ensure the cells are fully pelleted without being damaged.

Sample Loading:

(1).Preparation of Nickel Grids:
Use 200 or 300 mesh nickel grids.

(2).Cell Coating:
Take 5 μL of the fixed and washed cell suspension and apply it onto the nickel grid. Allow it to sit for a few minutes to let the cells adhere to the grid.

(3).Antibody Incubation:
Blocking: Apply 100 μL of PBS solution containing 1% BSA to the nickel grid and incubate at room temperature for 30 minutes to block nonspecific binding sites.
Primary Antibody Incubation: Prepare the primary antibody (e.g., mouse antibody) by diluting it with PBS at the recommended ratio. Transfer the blocked nickel grid to 50 μL of the primary antibody solution and incubate at room temperature for 1 hour. Wash the grid three times with PBS, using 100 μL each time, for 5 minutes.
Secondary Antibody Incubation: Prepare the gold-labeled secondary antibody (e.g., gold-labeled anti-mouse IgG) by diluting it with 1% BSA-PBS at the recommended ratio. Transfer the washed nickel grid to 50 μL of the secondary antibody solution and incubate at room temperature for 1 hour. Wash the grid three times with PBS, using 100 μL each time, for 5 minutes.

(4).Washing and Drying:
Final Wash: Wash the nickel grid three times with 100 μL of distilled water for 5 minutes each to remove PBS and BSA.
Drying: Place the nickel grid on clean filter paper and allow it to air dry naturally or dry it in a dust-free environment.

(5).Negative Staining:
Add 5 μL of 2% uranyl acetate stain and stain the sample for 30 seconds to 1 minute.

(6).Observation:
Examine the sample under a cryo-electron microscope (cryo-EM).

#### Enzymatic Cleavage Experiment

1.Activation: In *E. coli* BL21(DE3), spread on LB agar plates with antibiotic resistance, incubate overnight at 37°C, pick a single colony, and culture in LB broth until OD600 reaches 0.6-0.8.

2.Induction: Add 1mM final concentration IPTG.

    Induction at 37°C, 16°C, all for 1h.

3.Collection: Centrifuge at 5000rpm for 10min, wash the pellet with PBS twice.

4.Cleavage: Add 25mM pH 8.0 Tris-HCl and 1-3U enterokinase (Enterokinase, Recombinant), and incubate overnight at 25°C (reaction volume according to the enzyme's instructions).

 (1). Adjust the cleavage conditions according to the experimental arrangement.

 (2). Note: The definition of activity units: At 25°C, in a reaction buffer of 50 mM Tris-HCl (pH 8.0), the amount of enzyme required to cleave 50ug (1mg/ml) of fusion protein by more than 95% in 16 hours is defined as one unit of activity U.

5.Detection: Centrifuge at 5000rpm for 10 minutes and take the supernatant.

     Perform WB detection on proteins with GST/6xHis tags to check for the presence of proteins with GST/6xhis tags in the supernatant and check for proteins with FLAG tags on the bacterial surface proteins.

#### Protein Purification

1. Shake a bacterial culture of more than 10mL. Centrifuge at 5000rpm for 5 minutes to collect the bacteria. Following the protocol of the Beyotime P2226 His-Tagged Protein Purification Kit (Redox-Resistant Chelating Type), add non-denaturing lysis buffer at a ratio of 4mL per gram of wet bacterial weight. Resuspend the bacterial cells thoroughly by pipetting.

2. Add 24μL of 100mg/mL lysozyme. Mix well and place on ice for 30 minutes. Pre-cool a centrifuge to 4°C.

3. Transfer the bacterial suspension to a 10mL EP tube. Sonicate the bacteria on ice using a power setting of 40% (total power 650w), with each sonication pulse lasting 10s followed by a 10s interval, for a total of 6 pulses. After sonication, transfer the lysate to two 1.5mL EP tubes, approximately 1.0mL in each. Centrifuge at 4°C and 10,000g for 25 minutes. Collect the supernatant in a 10mL centrifuge tube. Reserve 20μL as a sample, labeled "Supernatant".

4. Take 1mL of well-mixed 50% BeyoGoldTM Purification Resin. Centrifuge at 4°C (1000gx10s) and discard the supernatant. Add 0.5mL of non-denaturing lysis buffer to the resin, mix well to equilibrate, and centrifuge again at 4°C (1000gx10s) before discarding the supernatant. Repeat this equilibration step twice. Since the bacterial lysate volume exceeds 1mL, mix approximately 500μL of the lysate supernatant with all the resin and inject the mixture into a 10mL EP tube containing the remaining lysate supernatant. Place the 10mL EP tube horizontally on a shaker at 4°C and shake gently for 60 minutes.
Load the mixture of lysate and resin into an empty affinity chromatography column provided with the kit.

5. Open the bottom cap of the purification column, then the top cap, and collect 1mL of flow-through. For subsequent operations, always close the top cap before the bottom cap, and when draining liquid, open the bottom cap first, followed by the top cap.

6. Wash the column 5 times, adding 700μL of non-denaturing wash buffer each time, and collecting approximately 0.5mL of wash buffer after each wash.

7. Elute the target protein 6 times, using 0.5mL of non-denaturing elution buffer for each elution.

8. Detect the protein by WB or Coomassie brilliant blue stain.

#### Membrane and Cytoplasm Protein Extraction

1. Thoroughly dissolve and mix the Membrane Protein Extraction Reagents A and B at room temperature, and immediately place them on ice after dissolution. Before use, add PMSF to the appropriate amount of A and B a few minutes prior, to achieve a final concentration of 1mM for PMSF.

 2.Culture approximately 20 to 50 million cells, centrifuge to collect the cells, remove the supernatant, and retain the precipitate.

3.Wash the cells with an appropriate amount of ice-cold PBS (gently resuspend the precipitate), followed by centrifugation at 600g for 5 minutes at 4°C to sediment the cells.

4.Discard the supernatant, then centrifuge again at 600g for 1 minute at 4°C to sediment any residual liquid on the walls of the centrifuge tube and further sediment the cells. Try to aspirate as much residual liquid as possible.

5.Add 1mL of Membrane Protein Extraction Reagent A, with PMSF added immediately before use, to the cell precipitate from step 3. Gently and thoroughly resuspend the cells, and place on ice for 10-15 minutes.

6.Sonicate the cells to disrupt them.

7.Centrifuge at 700g for 10 minutes at 4°C, carefully collect the supernatant into a new centrifuge tube to remove cell nuclei and unbroken cells.

8.Centrifuge at 14,000g for 30 minutes at 4°C to sediment cell membrane fragments. Try to aspirate as much supernatant as possible. It is acceptable to gently touch the precipitate or even aspirate a very small amount of it.

9.Add 200μL of Membrane Protein Extraction Reagent B, vortex at the highest speed for 5 seconds to resuspend the precipitate, and place on ice for 5-10 minutes. Repeat the vortexing and ice incubation steps 1-2 times to thoroughly extract membrane proteins.

10.Centrifuge at 14,000g for 5 minutes at 4°C, and collect the supernatant, which is now the cell membrane protein solution. Using this product to lyse 50 million cells can yield 0.3-3mg of cell membrane proteins, with variations depending on the cell type.

#### Coomassie Brilliant Blue staining

1.After SDS-PAGE,transfer the gel into a container, soak the gel in Coomassie brilliant blue and shake it slowly on s shaker for 25min.

2. Wash the gel with ddH2O for several times. Photograph the gel under white light.

{% endblock %}

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

![](https://static.igem.wiki/teams/5048/til8.png)
![](https://static.igem.wiki/teams/5048/til4.png)
![](https://static.igem.wiki/teams/5048/til3.png)
![](https://static.igem.wiki/teams/5048/til2.png)
![](https://static.igem.wiki/teams/5048/til1.png)
![](https://static.igem.wiki/teams/5048/til6.png)
![](https://static.igem.wiki/teams/5048/til7.png)
![](https://static.igem.wiki/teams/5048/til5.png)
![](https://static.igem.wiki/teams/5048/result.png)

- [Part 1: Secretion](#Secretion)

- [Lpp'OmpA-peptide
  chimaera synthesis](#Lpp'OmpA-peptide chimaera synthesis)

- [Surface detection for
  chimaera transportation](#Surface detection for chimaera transportation)

- [Enterokinase digestion](#Enterokinase digestion)

- [Part 2: Adhesion](#Adhesion)

- [Transformation of the plasmids](#Transformation of the plasmids)

- [Confirmation of the protein](#Confirmation of the protein)

- [Protein transport
  sites exploring](#Protein transport sites exploring)

- [Protein fusion expression](#Protein fusion expression)

- [Improvement in
  protein expression](#Improvement in protein expression)

- [Part 3: Biosafety](#Biosafety)

- [LuxI/LuxR system and LasI/LasR system verification](#System verification)

- [CcdB-CcdA toxicity-
  detoxification verification](#CcdB-CcdA toxicity-detoxification verification)

- [References](#references)

[##### Part 1: Secretion](#Secretion)
[##### Part 2: Adhesion](#Adhesion)
[##### Part 3: Biosafety](#Biosafety)

## Part 1: Secretion

To facilitate the secretion of functional peptides from *Escherichia coli*, we engineered a chimeric construct combining peptide sequences with Lpp'OmpA. Lpp'OmpA functions as a transport protein. This protein spans the outer membrane approximately five times. Peptides linked via an enterokinase cleavage site are appended to the C-terminus of Lpp'OmpA. The arrangement enables peptide transport across the bacterial outer membrane and exposure within the intestinal milieu. Subsequently, intestinal enterokinase cleaves at the designated site, allowing functional peptides to be absorbed by epithelial cells. To evaluate the system's efficiency, we constructed six variants of the Lpp'OmpA chimeric protein: Lpp'OmpA-lptE, LptE (no signal negative control lacking Lpp'OmpA), Lpp'OmpA-QEP-GST (QEP-GST), Lpp'OmpA-AQ-GST (AQ-GST), Lpp'OmpA-AQ-6xhis (AQ-6xhis) and Lpp'OmpA-QEP-6xhis (QEP-6xhis). We will present our findings in four dislevertinct sections.

#### 1.Lpp'OmpA-peptide chimaera synthesis

First, we performed transformation experiments to establish our secretion system.
We selected BL21(DE3) as the expression host, utilizing the T7-lac promoter in our plasmids.
Following successful transformation, bacterial colonies were subjected to PCR,
gel electrophoresis(Fig. 1), and verified by Sanger sequencing(Fig. 2).

![](https://static.igem.wiki/teams/5048/secretion-fig1-2-0.webp)

Fig. 1 Gel electrophoresis results of PCR products from transformed bacterial colonies with the corresponding plasmids.(Lpp'OmpA-lptE:1321bp, lptE:961bp, AQ-GST:1465bp, QEP-GST:1468bp, AQ-6xhis:826bp and QEP-6xhis:829bp constructs)

![](https://static.igem.wiki/teams/5048/secretion-fig2-2-0.webp)

Fig. 2 Successful sequencing of PCR products from transformed bacterial colonies with the corresponding plasmids.
Panels: (1) Lpp'OmpA-lptE (2) lptE (3) AQ-GST (4) QEP-GST (5) AQ-6xhis (6) QEP-6xhis.

To confirm the expression of Lpp'OmpA-peptides in bacteria from the transformed plasmids, we performed Western blot on bacterial lysates. We observed that induction temperature affects the stability of Lpp'OmpA-peptide expression under the T7-lac promoter. At temperatures of 37°C, 30°C, 25°C, and 16°C, the negative groups (0 mM IPTG) exhibited significant promoter leakage. Notably, lower temperatures resulted in more stable expression of the Lpp'OmpA-peptide system. At 37°C, which is close to human intestinal temperature (36.5-37.7°C), all six chimeras were successfully expressed (Fig. 3).

![](https://static.igem.wiki/teams/5048/secretion-fig3-3-0.png)

Fig. 3 Western blot analysis of six chimeras. Lower temperatures correlate with reduced leakage.
Panels: (1) Induction of Lpp'OmpA-lptE and lptE at 37°C, 30°C, or 16°C (2) Induction of QEP-GST at 37°C and 25°C (3) Induction of AQ-GST at 37°C and 16°C (4) Induction of QEP-6xhis at 37°C and 16°C (5) Induction of AQ-6xhis at 37°C and 16°C. (Protein sizes: Lpp'OmpA-lptE: 35.07kDa, lptE: 22.35kDa, QEP-GST: 41.65kDa, AQ-GST: 41.49kDa, QEP-6xhis: 16.8kDa, AQ-6xhis: 16.8kDa)

**● Discussion:**

 After analysis, we hypothesized that lactose and its analogues in LB culture medium might induce expression even in the absence of IPTG. To address this problem, we replaced LB medium with a basic medium (without yeast extract) during the induction phases. However, comparing the induction results from basic medium with those from LB medium indicated that the yeast extract in LB medium may not be the primary cause of promoter leakage (Fig. 4).

![](https://static.igem.wiki/teams/5048/secretion-fig4.webp)

Fig. 4 Western blot results of QEP-GST at different temperatures. Bacteria induced at 16°C in basic medium exhibit minimal differences compared to samples induced in LB medium even at higher temperatures.

#### 2.Surface detection for chimaera transportation

The surface exposure of all constructs was verified by FACS analysis. Bacteria were incubated first with corresponding Mouse anti-tag antibodies and subsequently incubated with the FITC-conjugated Goat anti-Mouse IgG (H+L). No detergent was used in the staining process. Initially, we induced for 1h at 16°C, which showed display success in Lpp'OmpA\_lptE, but not in the QEP and AQ sets. (Fig. 5)

![](https://static.igem.wiki/teams/5048/result-fig5-re.webp)

Fig. 5 Lpp'OmpA\_lptE surface display verification , induced at 16°C for 1h

We suspected that the induction time was not enough for the bacteria to complete the surface display. Therefore, we extended the induction time to overnight. This time we observed out that not only QEP and AQ sets had positive results, but also the lptE.

Lpp'OmpA\_lptE incubated with mouse anti-6xhis tag and mouse anti-GST tag were used to confirm the effect of endogenous binding.

![](https://static.igem.wiki/teams/5048/result-secretion-facs-flag-gst-his.webp)

Fig. 6
FACS analysis of Lpp'OmpA\_lptE and lptE, induced at 16°C overnight. L1: Lpp'OmpA\_lptE; N1:lptE

![](https://static.igem.wiki/teams/5048/result-secretion-facs-l-n-37-overnight.webp)

Fig. 7
FACS analysis of QEP sets and AQ sets, induced at 16°C overnight. L1: Lpp'OmpA\_lptE; N1:lptE; QH:QEP\_6xhis; AH:AQ\_6xhis; QG:QEP\_GST; AG:AQ\_GST

In contrast with Lpp'OmpA\_lptE incubated with 6xhis tag, AQ\_6xhis and QEP\_6xhis showed higher fluorescence intensity. QEP\_GST and AQ\_GST showed similar results. Moreover, Lpp'OmpA\_lptE incubated with the FLAG tag showed higher fluorescence intensity than the sample incubated with the 6xhis tag and GST tag. (Fig. 7)

To observe the surface display more clearly, we performed fluorescence microscopy on bacteria expressing the Lpp'OmpA chimeras. Bacteria were incubated first with corresponding Mouse anti-tag antibodies and subsequently incubated with the ABflo*®* 594-conjugated Goat anti-Mouse IgG (H+L) without detergent. The ABflo*®* 594 can be visualized in red, the DNA in blue (DAPI), and the membranes in green (Oregon green).

Similar results were observed:not only QEP and AQ sets had positive results, but also the lptE.

![](https://static.igem.wiki/teams/5048/result-secretion-ifa-16.webp)
![](https://static.igem.wiki/teams/5048/result-secretion-ifa-37.webp)
![](https://static.igem.wiki/teams/5048/result-fig8subpic3re.webp)

Fig. 8
Fluorescence microscopy of Lpp'OmpA chimera expressed in *E. coli*, induced overnight at 16°C or 37°C

To obtain a better resolution ratio, we performed cryo-electron microscopy. Bacteria were incubated first with corresponding Mouse anti-tag antibodies and subsequently incubated with the 12nm Colloidal Gold-AffiniPure™ Goat Anti-Mouse lgG (H+L). Similar results were obtained. Both lptE and chimeras showed positive results.

![](https://static.igem.wiki/teams/5048/result-fig9re.webp)

Fig. 9
Cryo-EM of Lpp'OmpA chimeras, induced overnight at 37°C

From our results, we could confirm that Lpp'OmpA\_lptE had been successfully displayed
on the surface because when we induced for 1h at 37°C, FACS analysis showed a clear contrast
between Lpp'OmpA\_lptE and lptE. However, the surface display of QEP and AQ sets might
not be verified, since lptE showed positive results in the same experiment.
We hypothesized that during the long-term induction or pretreatment process, the polarity
of the outer membrane wasn't maintained, which caused the LptE expressed in the negative
control to move from the inner leaflet to the outer leaflet. Another hypothesis was that during
the induction, the permeability of bacteria was affected. The antibody moved into the cells
and interacted with the non-displaying proteins. More discussions can be seen on the engineering
page.

To validate the QEP sets and AQ sets, enzymatic cleavage experiment was performed.

#### 3.Enterokinase digestion

To confirm that the enterokinase can target the DDDDK site and successfully cleave off the peptide into the environment, a series of enterokinase digestion experiments and western bolt experiments were conducted. We first designed experiments to investigate the minimum digestion concentration of enterokinase, digestion temperature, pH, and duration. Then, we expressed AQ-GST, QEP-GST, and lptE-GST under the optimal environmental factors that we explored before.

There are six possible circumstances we found in the following experiments: (1) bacteria membranes are intact and the proteins are presented on the surface, (2) bacteria membranes are not intact (bacteria are broken during induction or digestion), (3) bacteria membranes are intact and the proteins are not present on the surface, and the mixture of (1) and (2), (2) and (3), or (1) and (3) (Fig. 10). The bacteria culture was centrifugated after induction and digestion. The supernatant and precipitation are incubated with two different antibodies. To get a clearer picture, the hypothesized western blot results of different circumstances are demonstrated below (Fig. 11).

![](https://static.igem.wiki/teams/5048/secretion-fig10.webp)

Fig. 10 The schematics of deductions of the first three circumstances. (1) bacteria membranes are intact and the proteins are presented on the surface (2) bacteria membranes are not intact. (bacteria are broken during induction or digestion) (3) bacteria membrane are intact and the proteins are not present on the surface.

![](https://static.igem.wiki/teams/5048/secretion-fig11-1-11.webp)
![](https://static.igem.wiki/teams/5048/secretion-fig11-2-11.webp)
![](https://static.igem.wiki/teams/5048/secretion-fig11-3-11.webp)
![](https://static.igem.wiki/teams/5048/secretion-fig11-4-11.webp)

Fig. 11
The deduction of western blot results of supernatant and precipitation incubated with two different antibodies. (1) precipitation incubated with anti-FLAG antibody (2) precipitation incubated with anti-GST antibody (3) supernatant incubated with anti-GST antibody (4) supernatant incubated with anti-FLAG antibody

**Phase 1: the exploration of digestion factors of enterokinase**

To investigate the environmental factors affecting digestion—specifically the minimum enterokinase concentration, digestion temperature, time, and pH—we conducted a series of experiments. In each experiment, subsequent tests were adjusted based on the optimal conditions identified in the previous stage. By progressively aligning the experimental conditions with those of the intestinal environment, successful in vitro digestion may suggest similar effectiveness in vivo.

First, we tested the enterokinase concentration from 0 to 2U/50 μL in bacterial medium and digested the mixture at 25°C for 12 hours in pH 8.0, 25 mM Tris-HCl buffer (Fig. 12).

**● Analysis**

In the exploration of the minimum concentration of enterokinase, we first induced bacteria under 16℃ overnight with 1mM IPTG as our previous results indicated that the chimeras expressed and presented in a more stable way. To get a more specific picture of the digestion results, we incubated precipitation and supernatant with two different antibodies and repeated the experiment twice.

In the precipitation-FLAG blots, no signal was observed at 15KDa in both results, corresponding to Lpp'OmpA-FLAG. The degradation protein in precipitation-GST and supernatant-FLAG of two results indicate bacterial lysis. Although the results of the supernatant-GST show the successful digestion of Lpp'OmpA-FLAG-QEP-GST, the digestion occurring in the lysed bacteria did not confirm that the process was targeted to the bacterial surface. Although the two result have different signals, they both shows protein degradation. The reason for the differences may be because of the different condition of bacteria storage before induction (in 4℃ and -30℃) or the operation differences.

![](https://static.igem.wiki/teams/5048/arrrow.png)
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Although the bacterial lysis happened, there is still positive signal of QEP-GST which may means the 2U, 1.5U and 1U all can digest the protein. From the signal intensity at the three enzyme concentrations, we regard they all have similar and ability to digest when bacteria lysed. However, we cannot draw the inference about which concentration is optimal in the condition that the bacteria are intact.

![](https://static.igem.wiki/teams/5048/secretion-fig12-2-0.webp)

Fig. 12
Digestion of Lpp'OmpA-FLAG-QEP-GST to explore the minimum enterokinase concentration. Bacteria is induced by 1mM IPTG under 16℃ 12h. (1) the first experiment (2) the second repeat

Next, we tested the effect of digestion temperature (25°C and 37°C) and digestion time (3 and 5 hours) using 2U of enterokinase in the same buffer (Fig. 13).

**● Analysis**

In our exploration of optimal digestion temperature and time, we induced bacterial expression at 16°C overnight and at 37°C for 1 hour using 1mM IPTG, as bacterial lysis was observed in previous experiments. We hypothesized that the lysis was caused either by prolonged induction at low temperatures or by extended digestion in the nutrient-deprived buffer.
In the precipitation-FLAG sample, no signal was observed at 15 kDa in plus enzyme group, corresponding to Lpp'OmpA-FLAG. Additionally, the absence of degraded proteins in the precipitation-GST and supernatant-FLAG samples suggests no bacterial lysis occurred. In the supernatant-GST sample, a comparison of plus and no enzyme group at 37°C for 1 hour showed successful digestion of Lpp’OmpA-FLAG-QEP-GST. However, the presence of degraded protein in the supernatant-GST sample from the 16°C, 12-hour induction with negative enzyme concentration supports the hypothesis that bacterial lysis was caused by prolonged low-temperature induction.

![](https://static.igem.wiki/teams/5048/arrrow.png)
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From the results, we can tell that the group with 37℃-1h induction and 25℃-5h and 37℃-3h digestion can not only keep the bacteria intact but also suceessfully digest chimeras as the AQ-GST signals are positive in 2 groups.

![](https://static.igem.wiki/teams/5048/secretion-fig13-2-0.webp)

Fig. 13
Digestion of Lpp'OmpA-FLAG-AQ-GST to explore digestion temperature and time. Bacteria is induced by 1mM IPTG under 16℃ 12h and 37℃ 1h.

Following that, we varied the pH of the 25 mM Tris-HCl buffer to 6.5, 7.5, and 8.0 (the pH range of the intestine) and digested the mixture at 37°C for 3 hours with 2U of enterokinase (Fig. 14).

**● Analysis**

In our exploration of optimal digestion pH, we induced bacterial expression at 16°C overnight and at 37°C for 3 hours using 1mM IPTG, as the 37℃ 1h induction signal is quite weak. We hypothesized that the weak signal is due to the incomplete presentation of chimeras on the outer membrane.

In the precipitation-FLAG sample, no signal was observed at 15 kDa in plus enzyme group, corresponding to Lpp'OmpA-FLAG. Additionally, the absence of degraded proteins in the precipitation-FLAG and supernatant-GST samples suggests bacterial lysis occurred. Although the results of the supernatant-GST in positive enzyme group in both induction group showed the successful digestion of Lpp'OmpA-FLAG-QEP-GST, the digestion occurring in the lysed bacteria in both groups did not confirm that the process was targeted to the bacterial surface. However, the presence of weak signals in the supernatant-GST sample from the 37°C, 3-hour induction does not support the hypothesis that the weak signal is due to incomplete presentation because of short induction time.

![](https://static.igem.wiki/teams/5048/arrrow.png)
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From the results of group with 37℃-3h induction and pH 7.5 digestion and group with 16℃-12h induction and pH 6.5 digestion shows that positive pH digestion range can cover the pH range of intestine environment.

![](https://static.igem.wiki/teams/5048/secretion-fig14-2-0.webp)

Fig. 14
Digestion of Lpp'OmpA-FLAG-AQ-GST to explore digestion temperature and time. Bacteria is induced by 1mM IPTG under 16℃ 12h and 37℃ 3h.

**Phase 2: expression of AQ-GST, QEP-GST, and lptE-GST under optimal digestion conditions**

For lptE-GST and QEP-GST, the degradation protein signal in supernatant-GST and in supernatant-FLAG indicates bacterial lysis. In supernatant-GST, without enterokinase, the Lpp'OmpA-FLAG-lptE-GST signal indicates the incomplete digestion of lptE-GST. While QEP-GST only shows degradation protein signal and Lpp'OmpA-FLAG-QEP-GST signals, it may indicate the inclusion body formation.

For AQ-GST, no degradation protein signal indicates no bacterial lysis. In supernatant-GST, without enterokinase, the Lpp'OmpA-FLAG-AQ-GST signal also indicates the incomplete digestion.

![](https://static.igem.wiki/teams/5048/secretion-fig15-3-0.webp)

Fig. 15
 Digestion of Lpp'OmpA-FLAG-AQ-GST, Lpp'OmpA-FLAG-QEP-GST and Lpp'OmpA-FLAG-lptE-GST. Bacteria is induced by 1mM IPTG under 37℃ 3h.

**● Discussion:**

We also conducted an experiment to investigate whether there will be some bacteria resuspended in the supernatant, which may cause the same results as bacterial lysis. In this experiment, there is no difference in filter used and no filter used sample preparation. It may mean that there is no bacteria in the supernatant without the filter(Fig. 16).

![](https://static.igem.wiki/teams/5048/secretion-fig16.webp)

Fig. 16
Digestion of Lpp'OmpA-FLAG-AQ-GST with or without filter used. Bacteria is induced by 1mM IPTG under 37℃ 3h.

**● Future:**

All Lpp'OmpA-FLAG signal is missing in the experiments. The reason may be that the FLAG tag is in an inherently undetectable structural domain after digestion or the antibody affinity is not good enough to generate detectable signal. To further investigate the protein we can conduct TEM experiment to detect Lpp'OmpA-FLAG which is anchored on outer membrane after digestion.

## Part 2: Adhesion

#### 1. Transformation of the plasmids

To evaluate the functions of the target proteins, we separately transformed the pET-28a-[HSP60-Flag],
pET-28a-[LAP-His], pET-28a-[Neae-Flag] into strain *E. coli* BL21 (DE3). We conducted colony PCR to verify the transformation(Fig. 17).
Moreover, The Sanger sequencing conducted by Tsingke ensureed the specific sequences (Fig. 18).

![](https://static.igem.wiki/teams/5048/result-adhesion-fig1.webp)

Fig. 17 The confirmation of transformation. LAP 1, HSP60 1-3, and Neae 3 are successful transfromation samples

![](https://static.igem.wiki/teams/5048/result-adhesion-fig2.webp)

Fig. 18  The sequencing results of transformation in *E. coli*
BL21(DE3).

            A: The sequencing result of pET-28a-[HSP60-FLAG]

            B: The sequencing result of pET-28a-[LAP-His]

            C: The sequencing result of pET-28a-[Neae-FLAG]

#### 2. Confirmation of the protein

To ensure the correct expressions of target proteins, we utilized protein purification resin(Fig. 19) and Western Blot(WB)(Fig. 20) to detect the cellular products.

It's demonstrated that our engineered bacteria underwent cellular metabolism properly and synthesize desired proteins.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig3.webp)

Fig. 19  Coomassie Brilliant Blue stained gel image for LAP protein after protein purification resin treatment. LAP protein was expressed in *E. coli* BL21(DE3), under the induction of 0.1mM IPTG, 25 ℃, and 200rpm for 6 hours. LAP protein can be seen in samples of elution buffer2-5, which are marked by dark arrows, with a size between 100kDa and 130kDa. Samples of non-denaturing wash buffer 1-4 are collected corresponding to the first through fourth pre-treatments of the purification resin. Sample supernatant was collected after the lysate of the non-denaturing wash buffer. Flow-through was collected after the supernatant flows through the purification resin. Samples of elution buffer 1-6 were collected corresponding to the first through sixth washes of elution buffer through the purification resin.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig4.webp)

Fig. 20 Western blot analysis of HSP60, LAP, and Neae proteins.
A: WB analysis of HSP60 protein in *E. coli* BL21(DE3) under induction of 0.5mM IPTG and 250rpm for 3 hours. The differences in induction temperatures are marked on the image. The exposure time is 50 seconds.
B: WB analysis of LAP protein in *E. coli* BL21(DE3) under induction of 0.5mM IPTG, 25℃, and 250rpm for 1 hour. The exposure time is 30 seconds.
C: WB analysis of Nese protein in *E. coli* BL21(DE3) under induction of 0.5mM IPTG and 200rpm for 6.5 hours. The differences in induction temperatures are marked on the image. The exposure time is 20 seconds.

The expression levels of HSP60, LAP, and Neae were better at higher temperatures, which may correspond with the optimal growth temperature of *E. coli* and indicate that our proteins can function better at a temperature closer to the human body.

However, the expression level of HSP60 has been consistently concerning, and its content in the lysate supernatant is relatively low. The same situation happened to protein LAP as well. Both of the circumstances pose challenges for subsequent experiments like co-immunoprecipitation (Co-IP).

#### 3. Protein transport sites exploring

To determine whether the surface display protein, Neae, anchors on the outer membrane or not, we conducted a range of experiments.

Firstly, we employed indirect immunofluorescence(IF) to confirm the appearance of Neae. The result demonstrated that Neae presented a stronger fluorescence at 30℃(Fig. 21).

![](https://static.igem.wiki/teams/5048/result-adhesion-fig5.webp)

Fig. 21 Indirect immunofluorescence for Neae on the outer membrane of *E. coli* BL21(DE3). Primary antibody: Mouse anti DDDDK-Tag (mAb); secondary antibody: 594-conjugated Goat anti-Mouse lgG(H+L). The Neae can be visualized in red at an excitation wavelength of 585nm, and the DNA in blue (DAPI) at an excitation wavelength of 358nm. The cells were under an induction of 0.5mM IPTG for 2 hours.

Furthermore, we also conducted WB to ensure the expression level of Neae on the outer membrane(Fig. 22). The result indicates that Neae is successfully present on the outer membrane as well and yields more at a higher temperature, which corresponds to the IF result.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig6.webp)

Fig. 22 WB analysis of Neae protein of E. coli BL21(DE3) under the induction of 0.5mM IPTG at 250rpm for 3 hours. The differences in induction temperatures are marked on the image. The exposure time is 10 seconds.

Apart from Neae, we also examined the expression level of LAP in cell supernatant and outer membrane(Fig. 23). Both of the component contained LAP though the concentration of the protein remained low.

**As a cell membrane protein that enables *Listeria* to adhere to the intestine epithelium, the LAP we chose from non-pathogenic *Listeria* (*L. innocua*) also presents itself on the cell membrane of our engineered *E. coli*. *L. innocua* is able to take advantage of LAP and binds with HSP60 in the small intestine, occupying the binding site of pathogenic Listeria and avoiding intestinal infection induced by pathogenic *Listeria*[1].**

![](https://static.igem.wiki/teams/5048/result-adhesion-fig7.webp)

Fig. 23 WB analysis of LAP protein in *E. coli* BL21(DE3) under induction of 0.5mM IPTG at 250rpm for 3 hours. The differences in induction temperatures are marked on the image. The exposure time is 100 seconds.

#### 4. Protein fusion expression

We constructed pET-28a-[Neae-HSP60-FLAG] and pET-28a-[Neae-LAP] after the confirmation of the target proteins(Fig. 24). Moreover, we detected the expression levels of the fusion proteins.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig8.webp)

Fig. 24 The genetic circuit respectively for pET-28a-[Neae-HSP60-FLAG] and pET-28a-[Neae-LAP]

For pET-28a-[Neae-HSP60-FLAG], we employed PCR to introduce restriction enzyme cleavage sites for EcoRI and HindIII. After performing DNA purification, restriction enzyme digestion, and T4 ligation, we successfully constructed the target plasmid within *E. coli* DH5α and BL21(DE3)(Fig. 25)(Fig. 26).

![](https://static.igem.wiki/teams/5048/result-adhesion-fig9-11.webp)

Fig. 25

The construction and verification of pET-28a-[Neae-HSP60-FLAG]

A: The PCR product of Neae backbone and HSP60 segments. Both ends of the DNA fragment were introduced to restriction enzyme cleavage sites for EcoRI and HindIII. Neae Backbone 1-2 and HSP60 1-2 all have segments corresponding to the estimated DNA length. EcoRI-Neae backbone-HindIII: 7087bp; HindIII-HSP60-EcoRI: 1955bp

B: The colony PCR for *E. coli* BL21(DE3) . Neae-HSP60 3-5 and 7 all have segments corresponding to the estimated DNA length. Neae-HSP60: 3983bp

![](https://static.igem.wiki/teams/5048/result-adhesion-fig10.webp)

Fig. 26

The sequencing results of Neae-HSP60, indicates that we have successfully transferred the plasmid into *E. coli* BL21(DE3) without mutations

The expression level of Neae-HSP60 was detected by WB (Fig. 27). The expression characteristics of protein Neae-HSP60 were quite similar to protein Neae and HSP60. All of them exhibited a better yield at a higher temperature.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig11.webp)

Fig. 27

WB analysis of Neae-HSP60 protein in *E. coli* BL21(DE3) under 250rpm induction for 1.3 hours. The uninduced group shows slight protein leakage while the induced group demonstrates an abundant protein expression. Neae-HSP60 is estimated at around 130kDa. The exposure time is 10 seconds.

For pET-28a-[Neae-LAP], we constructed plasmids using similar methods, except using EcoRI and XhoI as restriction enzymes. We successfully transferred the plasmid into *E. coli* BL21(DE3) and received expected sequencing result(Fig. 28).

![](https://static.igem.wiki/teams/5048/result-adhesion-fig12-11.webp)

Fig. 28

Neae-LAP was successfully transferred into *E. coli* BL21(DE3)

A. The colony PCR of *E. coli* BL21(DE3). Neae-LAP 3 has the segment corresponding to the estimated DNA length. Neae-LAP: 4831bp

B. The sequencing result of Neae-LAP in *E. coli* BL21(DE3), demonstrates the successful transformation.

#### 5. Improvement in protein expression

The lack of soluble proteins in the supernatant and non-specific binding of the tag has always been a persistent challenge for us. Under this circumstance, we tried to replace the promoter and protein tag for WB and add a solubilizing protein tag for the original part (See more in [Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%7D%7D)).

Due to time constraints, not all genetic circuits were completed. We successfully built up SUMO-HSP60 circuits in E. coli BL21 and other three fruitful circuits are presented below (Fig29).

We introduced the soluble protein tag SUMO into the HSP60 construct to increase the amount of soluble protein in the supernatant, and we replaced the 6xHis tag with GST to improve antibody binding specificity. Furthermore, we tried to introduce SUMO-HSP60 and LAP fragment into plasmid backbone pBAD to change the promoter, and to replace LAP-6x His with LAP-GST at the same time.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig13a-11.webp)
![](https://static.igem.wiki/teams/5048/result-adhesion-fig13b-11.webp)
![](https://static.igem.wiki/teams/5048/result-adhesion-fig13c-11.webp)
![](https://static.igem.wiki/teams/5048/result-adhesion-fig13d-11.webp)

Fig. 29

The genetic circuits, SUMO-HSP60, LAP-GST, araBAD-SUMO-HSP60-FLAG and araBAD-LAP-GST

For SUMO-HSP60, we added the soluble protein tag SUMO into the circuit of HSP60 to enhance soluble protein expression in the supernatant. This is also a successfully constructed genetic circuit;

For LAP-GST, we replace the 6xHis with GST to achieve better antibody binding specificity and protein solubility.

Gene segments SUMO, HSP60 and LAP are cloned and inserted into the pBAD backbone, forming pBAD-SUMO-HSP60-FLAG and pBAD-LAP.

For the genetic circuit SUMO-HSP60, DNA segments SUMO and HSP-pET-backbone were amplified successfully (Fig. 30). Then, we ligated the two DNA segments and constructed plasmid SUMO-HSP60 in *E. coli* BL21(DE3) with single colonies containing the target band (Fig 31).

![](https://static.igem.wiki/teams/5048/result-adhesion-fig14-11.webp)

Fig. 30

The construction of SUMO-HSP60

A. The agarose gel electrophoresis of SUMO segment for SUMO-HSP60. SUMO: 325bp.

B. The agarose gel electrophoresis of HSP60-pET-backbone segment for SUMO-HSP60. HSP60-pET-backbone: 6982bp.

![](https://static.igem.wiki/teams/5048/result-adhesion-fig15-11.webp)

Fig. 31

SUMO-HSP60 was successfully transferred into *E. coli* BL21(DE3)

A. The plate of *E. coli* DH5α harboring the transferred plasmid SUMO-HSP60, which contains abundant single colonies. Resistance: Kanamycin (Kan).

B. The colony PCR for *E. coli* BL21(DE3). SUMO-HSP60 1 and 3-5 all have segments corresponding to the estimated DNA length. SUMO-HSP60: 2055bp

C. The sequencing result of SUMO-HSP60 in *E. coli* BL21(DE3), demonstrates the successful transformation.

Apart from the SUMO-HSP60 genetic circuit, we also amplified segments GST, LAP, HSP60, and pBAD backbone for other genetic circuits. However, they haven't been ligated to form complete plasmids yet due to time constraints(Fig. 32).

![](https://static.igem.wiki/teams/5048/result-adhesion-fig16-11.webp)

Fig. 32

The segments that have been amplified successfully for other genetic circuits

A. The agarose gel electrophoresis of GST for plasmid LAP-GST and LAP segment for plasmid pBAD-LAP. GST: 689bp; LAP: 2652bp.

B. The agarose gel electrophoresis of HSP60 segment for plasmid pBAD-SUMO-HSP60-FLAG

C. The agarose gel electrophoresis of pBAD backbone segment for pBAD-SUMO-HSP60-FLAG. The specificity of primers was not very well so we acquired two bands. The band with a longer length is the target segment. pBAD backbone: 4266bp.

## Part 3: Biosafety

#### 1. LuxI/LuxR system and LasI/LasR system verification

We first verified the quorum sensing system. For more details on the plasmid, visit the [Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%7D%7D#Cycle31)
PLux-deGFP and PLas-deGFP were transformed into *E. coli* BL21 separately. Sequencing verifies transformation success.

![](https://static.igem.wiki/teams/5048/result-fig33.jpg)

Fig. 33

Sequencing of PLux-deGFP and PLas-deGFP transformants

To confirm that pLux is induced by the combination of LuxR and 3OC6HSL, and pLas is induced by the combination of LasR and 3OC12HSL, we conducted induction experiments to measure fluorescence intensity at different AHL concentrations. The results show a clear dose-dependent pattern, with fluorescence intensity increasing as the concentration of AHL rises, indicating a well-functioning system (Fig. 34).

![](https://static.igem.wiki/teams/5048/secretion-fig34.webp)

Fig. 34

The fluorescence intensity changes over different AHL induction concentrations within a 2-hour induction period.

#### 2. CcdB-CcdA toxicity-detoxification verification

Initially, we performed transformation experiments to establish the CcdB-CcdA system. We selected *E. coli* Tuner(DE3)pLysS as the expression host, utilizing the T7-lac promoter in our plasmids. Following successful transformation, bacterial colonies were subjected to PCR, gel electrophoresis, and verified by Sanger sequencing. The sequencing results confirmed the correct sequence (Fig. 35).

![](https://static.igem.wiki/teams/5048/result-fig35.webp)

Fig. 35

Sequencing success for CcdB\_CcdA transformant.

After the transformation, we conducted 2 sets of experiments: CcdB toxicity verification and CcdA antioxidation verification. In the former set, different concentrations of IPTG were added to the transformant, which would induce the expression of CcdB and kill bacteria. In the latter set, the same concentration of IPTG and different concentrations of L-arabinose were added to the transformant. L-arabinose would trigger the expression of CcdA and detoxify CcdB. We measured OD600 changes over time using the plate reader as an indication of the viability of bacteria. (Fig. 36)

![](https://static.igem.wiki/teams/5048/result-fig36rere.webp)

Fig. 36

OD600 changes over time under IPTG gradient and L-arabinose gradient

Compared with the no-induction group, the cells in the L-arabinose gradient showed relatively high density in the LB medium, which indicated that the L-arabinose triggered the expression of CcdA and rescued the cells. In the IPTG gradient, Growth was significantly inhibited from the concentration of 104 μM to the 100μM, which fully induced CcdB expression. However, when the concentration reached 10μM and below, growth was back to normal. Overall, the toxicity and detoxification of the CcdB-CcdA set were verified.

## References

[1]

Drolia, R., Amalaradjou, M. A. R., Ryan, V., Tenguria, S., Liu, D., Bai, X., Bhunia, A. K. (2020). Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nature Communications, 11(1), 6344. doi:10.1038/s41467-020-20200-5

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