iGEM 2023 — Team scau-china

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![](https://static.igem.wiki/teams/4632/wiki/wiki/cover/notebook.jpg)

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{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead %}{% endblock %} {% block cover %} {% endblock %} {% block page\_content %}

01
**Cry3A-like Toxin Section**

First Cycle:

After reviewing the literature, we discovered a synthetically engineered cyclic nonapeptide with specific toxicity towards *S. invicta*. This cyclic peptide can irreversibly alter the conformation of G-protein-coupled receptors (GPCRs)
within the *S. invicta*, disrupting signal transduction pathways, ultimately leading to the death of *S. invicta* (Choi and Vander Meer, 2021). We cloned the nucleotide sequence encoding this peptide into the pET-28(b) vector
using ΩPCR and fused it with multiple tags for expression.

However, upon completing the vector construction, we discovered the absence of a signal peptide sequence. Even if it successfully expressed, it could not assume the correct conformation. Given the limited available time and no literature
support for the prokaryotic expression of this peptide, we deemed it imprudent to proceed with this work. Instead, the best course of action was to search for another suitable candidate protein. This candidate protein should not only
exhibit toxicity to *S. invicta* but should also have literature support for successful expression in prokaryotes.

Second Cycle:

Through further literature research, we selected three additional toxin proteins: Snowdrop lectin (Galanthus nivalis agglutinin) GNA, neurotoxin Txp-I, and crystal protein Cry3A-like toxin.

While GNA has been successfully expressedin *E. coli* BL21 (DE3), previous reports suggest its toxicity is limited to piercing-sucking insects and some chewing insects, with unclear toxicity against *S. invicta* (Longstaff et
al., 1998, Martínez-Alarcón et al., 2018, Khareedu et al., 1998, Gatehouse et al., 1984).

Txp-I neurotoxin is derived from the wheat curl mite, which parasitizes insects belonging to the Membracoidea and Heteroptera. It is highly toxic to its hosts but safe for mammals, however, it has not been expressed in prokaryotic suceefully
(Tomalski et al., 1989, Tomalski and Miller, 1991).

In comparison to two other toxins, Cry3A-like toxin originates from prokaryotes, specifically a novel strain of *Bacillus thuringiensis*, UTD001. The original toxin has a size of 72.9 kDa, which, upon papain proteolytic cleavage in
vitro, forms an active protein of 66.6 kDa with demonstrated clear toxicity against *S. invicta* (Carroll et al., 1997).

We screened for signal peptide OmpA-mediated secretion expression of the candidate proteins. Using Gibson Assembly, we cloned the “OmpA-candidate protein encoding sequence” into pET-30a, then transformed to *E. coli* BL21(DE3), with
IPTG addition, only Cry3A-like toxin protein was successfully expressed and extracellular secreted.

![Fig.1](https://static.igem.wiki/teams/4632/wiki/wiki/engineering/eng-01.png)

Figure 1. SDS-PAGE Electrophoresis Detection of Cry3A-like Toxin Expression

Lane 1: Concentrated supernatant of pET-30a (+IPTG), Lane 2: Concentrated supernatant of pET30a-OmpA-Cry3A-like toxin (+IPTG), Lane 3: Concentrated supernatant of pET30a-OmpA-Cry3A-like toxin (-IPTG)

Third Cycle:

We will use enzymatic digestion/ΩPCR methods to add a 6×His tag to pET-30a-OmpA-Cry3A-like toxin. Subsequently, we will conduct western blot experiments to further confirm the secretion expression of Cry3A-like toxin.

02
**Cysteine Protease Trypsin Inhibitor (CPTI) Section**

The main problems encountered in this section are protein expression and whether the protein can be transported to extracellular. The modification process is mainly divided into three cycles.

First Cycle:

In this phase, we attempted to express a trypsin inhibitor subtype A (TIA) protein with a ZnuA signal peptide. We selected this gene because previous research had already confirmed its successful expression in *E. coli* (Sultana et
al., 2023), and it had shown a significant effect on *S. invicta*. However, after two months of attempts, we found that the TIA gene, whether with or without the ZnuA signal peptide, could not be expressed in *E. coli*. Therefore,
we concluded that *E. coli* was not suitable for expressing the TIA gene, although previous research has reported the expression. Consequently, we had to select a new protease inhibitor, initiating the second cycle.

[TIA expression section](#accordion1)
:   The first experiment conducted for our TIA involved selecting a suitable signal peptide. The signal peptide selection was carried out using the SignalP website (https://services.healthtech.dtu.dk/services/SignalP-5.0/)
    to identify the signal peptide with the highest compatibility with *E. coli*.

    Construction:

    The ZnuA-TIA fragment was synthesized (ordered from Guangzhou IGE Biotechnology Co. Ltd), and inserted into the pET-28b vector, then transformed into BL21 (DE3). Over night culture was dilute by 1/1000 to fresh LB medium,
    the IPTG was added when the OD600 reach 0.6. The medium was incubated another 4 hours, then centrifuged at 12,000 rpm 10 mins to obtain the supernatant and the precipitate. The supernatant was subjected to concentration
    using ultrafiltration, while the precipitate was treated with lysis buffer (300mM NaCl, 10mM imidazole, 50mM PBS, pH 8.0). SDS-PAGE and Western Blot analyses were performed on these samples, revealing that TIA protein
    was not expressed in *E. coli*.

    ![Fig.2](https://static.igem.wiki/teams/4632/wiki/wiki/engineering/eng-02.png)

    Figure 2. SDS-PAGE analysis of TIA expression

    Lane 1: whole cell extract of pET-28(b) with IPTG; Lane 2 -9 were ZnuA-TIA. Lane 2: precipitate without IPTG; Lane 3: precipitate with IPTG; Lane 4: supernatant without IPTG; Lane 5: supernatant with IPTG; Lane 6: concentrated
    supernatant without IPTG; Lane 7: concentrated supernatant with IPTG; Lane 8: whole cell extract without IPTG; Lane 9: whole cell extract of pET-28(b)-ZnuA-TIA with IPTG.

    ![Fig.3](https://static.igem.wiki/teams/4632/wiki/wiki/engineering/eng-03.png)

    Figure 3. Western blot analysis of TIA expression

    Lane1: concentrated supernatant of TIA with IPTG; Lane2: concentrated supernatant of TIA without IPTG; Lane3: supernatant of TIA with IPTG; Lane4: supernatant of TIA without IPTG; Lane5: precipitate of TIA with IPTG; Lane6:
    precipitate of TIA without IPTG; Lane7: supernatant of pht254-LpfA(positive control); Lane8: supernatant of pET28(b) with IPTG

Second Cycle:

We decided to select a different protease inhibitor for our project, namely, Cowpea Trypsin Inhibitor (CPTI). CPTI is a plant-derived protein known for its broad-spectrum insecticidal properties. The reason for choosing this gene is reported
that CPTI is successful expressed in *E. coli* (Zhu-jun et al., 2007, Yang et al., 2003). This provided us with confidence in achieving successful protein expression.

In contrast to the original literature, we opted not to add a GST purification tag to the CPTI gene. Our goal was to use CPTI directly for the eradication of *S. invicta*, rather than purifying the protein and subsequently removing
the GST tag. Notably, the CPTI protein, when devoid of the GST tag, processed a molecular weight of only 10.4 kDa. As a result, 12% SDS-PAGE was not sufficient to visualize the protein effectively. To address this, Tricine-PAGE and
SDS-PAGE with 17.5% acrylamide were conducted.

However, the results indicated that the CPTI gene without the GST tag could not express properly, as previous report. Consequently, we decided to follow the literature and add the GST tag to the vector, starting the third cycle.

Third Cycle:

We successfully add the GST tag to the CPTI gene, and protein expression induction is currently in progress. This modification ensures that the protein can be expressed to a significant extent. Literature has previously demonstrated the
successful expression of CPTI with the GST tag (Yang et al., 2003). Our unique approach in this cycle is to assess the activity of CPTI without removing the GST tag.

03
**Regulation of Drug Expression by Orthogonal Quorum Sensing Systems Section**

First Cycle:

Initially, we set the project goal to modify a symbiotic bacterium inside *S. invicta* to secrete toxins for eradicating them. The experimental strategy was designed as follows: the toxin protein was continuous expressed within the
engineered bacteria, when the population of the engineered bacteria reach a certain concentration in the ant's gut, the lysis genes will be activated, allowing the toxin protein to rapidly reach high concentrations, effectively exterminating
the ants.

However, through the study of *S. invicta* behavior and expert interviews, we learned that *S. invicta* are highly social organisms.  [(see human-practices)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) Their food-sharing
mechanisms, coupled with the queen's prolific reproduction and high toxicity, hinder the spread of toxins within the ant colony. Rapidly and efficiently killing a portion of ants within a short time could not completely destroy the
ant colony and would deter ants from consuming bait containing the engineered bacteria.

Second Cycle:

Based on this understanding, the release mechanism of the toxin protein was modified from lysis to secretion, allowing the engineered bacteria to maintain continuous expression of the toxin protein. Since food passes through the digestion
of fourth-instar larvae first, we introduced protease inhibitors and two orthogonal quorum-sensing systems. This ensured that the protease inhibitor was released into the gut first, protecting the subsequent activity of the toxin protein.
To control the expression level of the toxin protein, we utilized a binary oscillation system with negative feedback, which allowed the expression of the toxin protein to inhibit the expression of upstream genes, reducing the expression
of the toxin protein itself and thereby stabilizing the expression levels of both the protease inhibitor and the toxin protein. The Dry team also demonstrated the feasibility of oscillatory expression through mathematical modeling.
However, in the subsequent experiment design, we discovered that the oscillatory pathway itself posed significant complexity in adjusting various parameters of the genetic circuit. Moreover, the internal environment within *S. invicta* is highly intricate and challenging to predict, making it potentially difficult to replicate the results obtained in the laboratory. This May when we attend the iGEM Southern China Regional Meeting, experienced iGEM advisors pointed
out that our oscillatory pathway might not be the most optimal approach to achieve our intended goals.  [(see human-practices)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

Third Cycle:

Ultimately, we quite the intriguing concept of oscillatory expression and optimized the pathway to its final version. The oscillation-sustaining module was removed and lysis genes was introduced. When the quorum-sensing signal reaches
the threshold, the engineered bacteria start lysing and terminate product expression. We use the quorum-sensing threshold to control the maximum expression level of the product. Through food-sharing within the ant colony, the toxin
protein will be disseminated throughout the entire colony and accumulates within the queen's body, achieving the complete eradication of *S. invicta* society.

04
**Biological Safety Device Section**

Firstly, the pnirB promoter was planned to digest and ligate to the promoterless eGFP in the plasmid of pET-28b-singal-eGFP. Then the difference in promoter expression strength was measured by the fluorescence intensity under aerobic or
anaerobic environments.

However, multiple attempts was conducted, but the target plasmid was not obtained. The short length of the pnirB promoter fragment (78 bp after adding the ribosome binding site) may have led to a relatively low success rate in digestion
and ligation. After several unsuccessful attempts, ΩPCR was employed to construct the plasmid. Ultimately, the pnirB was firstly added to eGFP by overlap PCR, then the whole fragment was ligated to pET-28b.

We try to measure the strength of promoter expression under aerobic or anaerobic environments by the fluorescence intensity of eGFP. However, another report showed the eGFP could not work at anaerobic environment. Therefore, eGFP was replaced
with a gentamicin (Gm) resistance gene by the same method. The promoter's activity was validated by the growth performance of strain containing the plasmid pET-28b-pnirB-Gmr under aerobic or anaerobic with gradient Gm concentrations.

In addition to the anaerobic promoter, we explored methods for constructing auxotrophic strains to enhance the feasibility of our biosafety device.

First period:

The *fabA* was linked to the anaerobic promoter pnirB and then inserted into the genome to replace the original *fabA* gene in the chromosome by λ Red recombination. So that, the engineered bacteria could only survive by expressing
*fabA* in the anaerobic environment or with the exogenous oleic acid. Several attempts had been conducted, but no colony was obtained with the oleic acid addition. One possible reason is the *fabA* is the essential gene for
E.coil, it may not only important in the unsaturated fatty acid synthesis, but also important to other metabolism. Another thought is that the regulation of *fabA* is vital to determine the ratio of saturated fatty acid and unsaturated
fatty acid, because the downstream gene fabB was not only play a role in the unsaturated fatty acid synthesis but also in the saturated fatty acid synthesis.

Second period:

pnirB-dapA was cloned into the pUC18T-mini Tn7T plasmid and then transformed to mw3064 (dapA mutant strain). The growth of resulting strain is strictly dependent on the supply of exogenous DAP or DAP expressed in low-oxygen environment.
The growth of dapA- pUC18T-mini-Tn7T/△dapA and △dapA was detected under aerobic and anaerobic conditions, respectively. The results showed that △dapA could only grow under anaerobic and aerobic conditions with the addition of DAP.
And dapA- pUC18T-mini-Tn7T/△dapA can grow under both conditions.

Future prospects:

The dapA gene in the Top10 strain will be knockout to make the safety device stable in expression. In addition, the random mutation to the sequence of pnirB or the RBS will be conduct to further improve the accuracy of pnirB expression
between the anaerobic and aerobic condition.

[Reference](#accordion1)
:   |  |  |
    | --- | --- |
    | [1] | CARROLL, J., CONVENTS, D., VAN DAMME, J., BOETS, A., VAN RIE, J. & ELLAR, D. J. 1997. Intramolecular proteolytic cleavage of *Bacillus thuringiensis* Cry3A delta-endotoxin may facilitate its coleopteran toxicity. J Invertebr Pathol, 70, 41-9. |
    | [2] | CHOI, M. Y. & VANDER MEER, R. K. 2021. GPCR-Based Bioactive Peptide Screening Using Phage-Displayed Peptides and an Insect Cell System for Insecticide Discovery. Biomolecules, 11. |
    | [3] | GATEHOUSE, A. M. R., DEWEY, F. M., DOVE, J., FENTON, K. A. & PUSZTAI, A. 1984. Effect of seed lectins from Phaseolus vulgaris on the development of larvae of Callosobruchus maculatus; mechanism of toxicity. Journal of the Science of Food and Agriculture, 35, 373-380. |
    | [4] | KHAREEDU, V., RATHORE, K., HODGES, T., FU, X., STOGER, E., SUDHAKAR, D., WILLIAMS, S., CHRISTOU, P., BHARATHI, M., BOWN, D., POWELL, K., SPENCE, J., GATEHOUSE, A. & GATEHOUSE, J. 1998. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. 15, 4, 469-477. |
    | [5] | LONGSTAFF, M., POWELL, K. S., GATEHOUSE, J. A., RAEMAEKERS, R., NEWELL, C. A. & HAMILTON, W. D. 1998. Production and purification of active snowdrop lectin in Escherichia coli. Eur J Biochem, 252, 59-65. |
    | [6] | MARTÍNEZ-ALARCÓN, D., BLANCO-LABRA, A. & GARCÍA-GASCA, T. 2018. Expression of Lectins in Heterologous Systems. Int J Mol Sci, 19. |
    | [7] | SULTANA, M. S., MAZAREI, M., JURAT-FUENTES, J. L., HEWEZI, T., MILLWOOD, R. J. & STEWART, C. N. 2023. Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana. Frontiers in Plant Science, 14. |
    | [8] | TOMALSKI, M. D., KUTNEY, R., BRUCE, W. A., BROWN, M. R., BLUM, M. S. & TRAVIS, J. 1989. Purification and characterization of insect toxins derived from the mite, Pyemotes tritici. Toxicon, 27, 1151-1167. |
    | [9] | TOMALSKI, M. D. & MILLER, L. K. 1991. Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene. Nature, 352, 82-85. |
    | [10] | YANG, L. C., CHANG, T. J., CHEN, W. X., YANG, X. G. & ZHU, Z. 2003. [Fusion expression, purification and bioactivity assay of CpTI in Escherichia coli]. Sheng Wu Gong Cheng Xue Bao, 19, 63-8. |
    | [11] | ZHU-JUN, T., MING-CAI, S., FENG, Z. & HUA-PING, H. 2007. Reseach Status of the Trypsin Inhibitor. Journal of Guangdong University of Technology, 24, 6-9. |

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[Cry3A-like Toxin Section](#item-1)

[CPTI Section](#item-2)

[Drug Modulation Circuits Section](#item-3)

[Biological Safety Device Section](#item-4)

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{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block lead %}{% endblock %} {% block cover %}{% endblock %} {% block page\_content %}

[1 Plasmid prep](#accordion)
:   All plasmids were extracted by the HiPure Plasmid Micro Kit from Magen Co., LTD.

    1) Overnight culture was centrifuge at 13,000rpm for 1 min at room temperature. Decant or aspirate and discard the culture media.

    2) Resuspend pelleted bacterial cells in 250 μL Buffer P1 and transfer to a microcentrifuge tube.

    3) Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 8–10 times.

    4) Add 350 μL Buffer P3. Mix immediately and thoroughly by inverting the tube 8–10 times.

    5) Centrifuge for 10 mins at 13,000 rpm in a table-top microcentrifuge.

    6) Insert a HiPure DNA Mini Column II into a 2.0 mL Collection Tube(provided). Apply 800 μL of the supernatant from step 5 to the column by pipetting. Centrifuge for 30~60 s. Discard the flow through.

    7) Recommended: Wash the Column by adding 0.5 mL Buffer PW1 and centrifuging for 30–60 s. Discard the flow through.

    8) Wash the column by adding 0.75 mL Buffer PW2 and centrifuging for 30–60 s.

    9) Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.

    10)Place the Column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50~100 μL Elution Buffer or water to the center of each column, let stand for 1 min, and centrifuge for 1 min.

[2 Plasmid or DNA fragment digestion](#accordion)
:   1) The concentration of plasmid or DNA fragment were confirmed by the Nano Drop 2000C (Thermofisher), then the amount of enzyme will be calculated by the amount of plasmid or DNA fragment added to the digestion;

    2) Certain amount of plasmid or DNA fragment were added to the 200 μL microcentrifuge tube;

    3) The Restriction enzyme (NEB or Takara) and buffer were added based on the calculation on 1) and mixed;

    4) The microcentrifuge tube was incubated in 37 ℃ water bath for 2-3 h.

[3 Gel purification](#accordion)
:   All DNA fragments (DNA digestion or PCR purification) were pumicated by the HiPure Gel DNA Mini Kit from Magen Co., LTD.

    Protocol 1: Pure DNA from agarose gel

    1) Excise the DNA fragment from the agarose gel with a clean♯ scalpel. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5mL microcentrifuge tube.

    2) Add 2 volume of Buffer GDP to 1 volume of the gel (100 mg gel approximately 100μL). Incubate at 50~55°C for 10 mins or until the gel has completely melted. Vortex or shake the tube every 2-3 mins during the incubation.

    3) Insert a HiPure DNA Mini Column II in a 2mL Collection Tube.

    4) Add no more than 750μL DNA/agarose solution from step 2 to the Column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

    5) Repeat Step 4 until all of the sample has been transferred to the column.

    6) Add 300μL Buffer GDP to the column and incubate for 1 min. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

    7) Add 650μl Buffer DW2 to the column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

    8) Add 650μl Buffer DW2 to the column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

    9) Centrifuge the empty Column at 12,000 g for 2 mins at room temperature to dry the column matrix.

    10) Transfer the Column to a clean 1.5mL microcentrifuge tube. Add 25~50 μL Elution Buffer or deionized water directly to the center of the column membrane. Let it sit at room temperature for 2 mins.

    11) Centrifuge at 12,000 g for 1 min at room temperature. Store DNA at -20°C.

    Protocol 2: Pure DNA from PCR Product or Enzyme Reaction

    1) Determine the volume of your sample. Transfer the sample into a clean 1.5 mL microcentrifuge tube.

    2) Add 2 volumes Buffer GDP to the sample and mix well.

    3) Insert a HiPure DNA Mini Column II in a 2 mL Collection Tube.

    4) Add no more than 700 μL of the Mixture from Step 2 to the Column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

    5) Repeat Step 4 until all of the sample has been transferred to the column.

    6) Add 450μL Buffer DW2 to the column. Centrifuge at 12,000g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

    7) Repeat Steps 6 for a second Buffer DW2 wash step.

    8) Centrifuge the empty Column at 12,000g for 2 mins at room temperature to dry the column matrix.

    9) Transfer the Column to a clean 1.5 mL microcentrifuge tube. Add 25-50 μL Elution Buffer or deionized water directly to the center of the column membrane. Let sit at room temperature for 2 mins. Centrifuge at 12,000g for 1 min
    and store DNA at -20°C.

[4 Polymerase Chain Reaction（PCR）](#accordion)
:   The PCR For DNA fragment was amplified by 1.1×S4 Fidelity PCR Mix (Genesand) with the following reaction volume: 2 μL for each primers, 2 μL template DNA (10 pg to 10 ng), 44 μL 1.1×S4 Fidelity PCR Mix. For colony PCR, the volume
    could adjust to 20 μL.

    PCR program:

    | Step | Temperature | Time | Cycles |
    | --- | --- | --- | --- |
    | Pre denaturation | 98℃ | 2 mins | 1 |
    | Denaturation | 98℃ | 10 s | 30 |
    | Annealing | Tm | 10~15 s | 30 |
    | Extension | 72℃ | 10~15 s/kb | 30 |
    | Final extension | 72℃ | 3 mins | 1 |

[5 Gibson assembly](#accordion)
:   Gibson assembly reaction was conducted by the 2X MultiF Seamless kit (ABclonal)

    1) The primers were designed in the snapgene based on the sequence of vector and insert fragment;

    2) The insert fragment was prepared by PCR as described in experiment 4;

    3) Linearized vector was prepared by digestion as described in experiment 2;

    4) The reaction was prepared on the ice with the following volume: linearized vector X μL, insert fragment YμL, 2X MultiF Seamless Assembly Mix 10 μL, ddH2O to 20 μL;

    (Note: the amount of linearized vector was recommended to be 0.03 pmol, the ration of insert fragment/ vector is 1:2 (Best recommendation) to 1:3.)

    5) Mixed the sample, then incubated at 50℃ for 15mins;

    6) Transformation (see experiment 7) and use colony PCR to screen the transformants.

[6 Competent cell preparation](#accordion)
:   1) Single colony of *E.coli* strain was selected to LB medium, incubated in the shaker at 37℃, 220 rpm till the OD600 reached 0.3;

    2) The cell was transferred to 50 mL centrifuge tube and incubated on ice for 10 mins;

    3) Centrifuge 10 mins at 4,000 rpm, 4℃;

    3) Linearized vector was prepared by digestion as described in experiment 2;

    4) The reaction was prepared on the ice with the following volume: linearized vector X μL, insert fragment YμL, 2X MultiF Seamless Assembly Mix 10 μL, ddH2O to 20 μL;

    5) 50 uL/ tube stock at -80 ℃ freezer.

[7 Ligation](#accordion)
:   Insert fragment and vector was added at the ration from 3:1-10:1, with at least 0.01 pmol DNA for each, 2 uL T4 ligase buffe, 0.2 uL ligase 4 (i science) and ddH2O added to 20 uL. The sample were incubated at the heating block
    for 20 mins at 22 ℃.

[8 Transformation](#accordion)
:   1) The sample was prepared as follows: 50 μL competent cell prepared as experiment 6, 10 μL ligation sample from experiment 7, 10 μL 5\* KCM buffer (0.5M KCl, 0.15M CaCl2, 0.25M MgCl2), ddH2O 20 uL;

    2) Incubated on ice for 30 mins;

    3) Place at room temperature for 5 mins;

    4) Add 1mL LB;

    5) Recoved at 37℃ shaker for 45- 60mins, 200-220 rpm;

    6) Centrifuge 5 mins at 4,000 rpm, 4℃ and spread the sample with 100 μL sample at suitable plate;

    7) Incubate at 37℃ for overnight.

[9 Strain stock](#accordion)
:   1.4 mL overnight culture and 0.6 mL DMSO were mixed and stocked at -80 ℃ freezer.

    Expression of toxic protein and protease inhibitors

[10 Protein concentration](#accordion)
:   The protein was concentrated as the protocol of Millipore ® Filtration Selection Guide.

[11 SDS-PAGE](#accordion)
:   1) The buffer for SDS-PAGE were prepared as follows:

    SDS separating buffer: 18.165 g Tris, 4 mL10%SDS, adjust pH to 8.8 by NaOH, add ddH2O to 100 ml.

    SDS stocking buffer: 5.98 g Tris, 4 mL10%SDS, adjust pH to 6.8 by NaOH，add ddH2O to 100 ml.

    10 X running buffer (pH8.3): 72 g glycine, 15.15g Tris, 5g SDS, add ddH2O to 500mL, dilute to 1 X running buffer before use.

    4 X loading buffer: 0.5 M Tris-HCl (pH6.8) 4.5mL, 10 mL glycerol, 2.3g SDS, 0.004 g bromophenol blue, add ddH2O to 25mL.

    Coomassie brilliant blue straining solution: Coomassie brilliant blue 0.25g, methanol 227 mL, acetic acid 46 mL, ddH2O added to 500 mL.

    Destraining solution: ethanol 90 mL, acetic acid 20 mL, ddH2O added to 1L.

    2) Gel form

    |  | Spreading Gel (8 mL) | Stocking Gel (4mL) |
    | --- | --- | --- |
    | Concentration | 12% | 5% |
    | Methylenebisacrylamide/Acrylamide (1:29)(mL) | 2.4 | 0.5 |
    | Buffer (mL) | 2 | 1 |
    | ddH2O (mL) | 3.6 | 2.5 |
    | APS (uL) | 40 | 20 |
    | TEMED (uL) | 16 | 8 |

    3) Gel preparation

    Make the separating gel:

    Set the casting frames (clamp two glass plates in the casting frames) on the casting stands. Prepare the gel solution (as described above) in a separate small beaker. Wirl the solution gently but thoroughly. Pipet appropriate amount
    of separating gel solution (about 7-7.5 mL) into the gap between the glass plates. To make the top of the separating gel be horizontal, fill in the gap with 1 mL 75% ethanol. Wait for 20-30min to let it gelate.

    Make the stacking gel:

    Discard the water and you can see separating gel left. Pipet in stacking gel until an overflow. Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate.

    Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber
    and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber.

    4) Samples preparation

    Mix your samples with loading buffer. Heat them in boiling water for 5-10 mins. Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect
    the anodes.

    5) Electrophoresis

    As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 1.5 hour for an
    80V voltage of a 12% separating gel.

    6) Straining and destraining

    After electrophoresis, stain with Coomassie brilliant blue staining solution for 2 hours. Discard the staining solution, rinse several times with distilled water, and then add the destaining solution until the protein bands are
    clear.

[12 Western Blot](#accordion)
:   Material:

    10 X transfer buffer: 144.15g glycine, 30.285g Tris, ddH2O was added to 1 L, and store at 4℃. 1 X transfer buffer: 100 mL 10X transfer buffer, 200 mL methanol, ddH2O was added to 1 L, prepared before use.

    10 X TBS buffer (pH7.6): 24.22g Tris, 80g NaCl, adjust pH with 6 mL 12N hydrochloric acid, ddH2O was added to 1 L and store at room temperature.

    1 X TTBS buffer (pH7.6): 100 mL 10 X TBS buffer, 1mL Tween-20, dH2O was added to 1 L and store at room temperature.

    Blocking solution: 5g skimmed milk powder, 1 X TBST buffer was added to 100 mL and store at 4C.

    Experiment Steps:

    1) Activate PVDF membrane in methanol for 15 minutes (use a tweezers at all times—do not touch with gloves). Cut membrane and paper almost same size as gel, then soak SDS-PAGE gel (already run) and PVDF membrane and 4 whatman filter
    papers (cut to size) in Blotting buffer (15 minutes, in fact no time requirement, just wet gel, membrane, filter paper with blot buffer).

    2) Assemble sandwich submerged in blot buffer (no bubbles). Black-sponge-two layer of filter paper-SDS/No-SDS PAGE (without stacking gel)- membrane- two layer of filter paper – sponge- white.

    3) Quickly transfer sandwich to full assembly filled blot buffer (this buffer should be fresh and cold). Run in the cold room at 90V 350mA for 1 hour or 30V 90mA for overnight.

    4) Briefly wash membrane with TTBS to remove methanol.

    5) Block the membrane (facing up) with 5% non-fat dry milk in TTBS (could be without Tween 20, since background might high with Tween 20) for 1 hour (or overnight at cold room). Shake well at room temperature for all steps.

    6) Incubate with (1:10,000; 1 uL in 10 mL) primary antibody in 5% non-fat dry milk in TTBS for 1 hour.

    7) Rinse with TTBS: one quick rinse, 4\*10 minutes.

    8) Incubate with (1:5,000; 2uL in 10mL) secondary antibody in 5% non-fat dry milk in TTBS.

    9) Rinse with TTBS: one quick rinse, 4\*10 minutes.

**Gene route functional verification**

[13 Arabinose-induced expression experiment](#accordion)
:   1) The single colony of the engineered bacteria was transferred to 5 mL LB with antibiotic in the working concentration and incubate at the shaker overnight at 37℃ 220 rpm.

    2) 2% overnight culture was transferred to 5 mL fresh LB with antibiotic in the working concentration. Arabinose (ara) was added to experimental group with the final concentration of 0.02%. The seed culture was added to control
    group, but no ara was added. The blank group was set as follows: ara was added at final concentration of 0.02% but no seed culture was added to LB medium. All groups were incubated in a shaker at 37℃, 220 rpm for 6 hours and
    the OD600 were observed for the results.

[14 eGFP, RFP, OD 600 Test](#accordion)
:   1) 200 uL sample and blank control (LB medium) were added to the 96-well plate, and tested by the microplate reader. For eGFP, excitation wavelength was set at 488 nm, while the emission wavelength was 506 nm. For RFP, excitation
    wavelength was set at 587 nm, while the emission wavelength was 610 nm

    2) The OD 600 values of the experimental group, control group, and blank control group were tested in the spectrophotometer, with LB medium as the blank. All experiments were repeat 3 time.

[15 Determination of growth curve](#accordion)
:   1) The single colony of the engineered bacteria was transferred to 5 mL LB with antibiotic in the working concentration and incubate at the shaker overnight at 37℃ 220 rpm.

    2) All overnight culture were measured with a spectrophotometer, and the cell density were adjusted by LB to OD600 = 0.6. All experiments were repeat 3 time.

    3) 200 uL samples from the experimental group, control group, and blank control group were added to a sterile 96-well plate, and 3 replicates were set for each group. Control group: wild type TOP10 strain. Experimental group: Engineered
    bacterial with the corresponding antibiotic at a working concentration and 0.02% ara at the final concentration. Blank group: LB medium without any strain.

    4) The 96-well plate were placed into the bioscreen (Log Phase 600, Agilent), with continuous shake at 37℃, 500 rpm. The cell density was tested at OD600, and value was read every hour, and continuous measurement for 20 hours.

    5) After the measurement, the results were exported from the computer, and the repeated results of each group were averaged to draw a growth curve.

[16 Extraction of Quorum Sensing Signaling Molecules](#accordion)
:   Strains were grown in LB medium for required time with or without the ara. 20 mL of bacterial supernatant were collected by centrifugation and extracted with an equal volume of ethyl acetate mixed the incubated for 4 h. The organic
    phase was evaporated, and the residue was dissolved in 1 ml of ethyl acetate and transferred to 1.5 mL microcentrifuge tube. The mixture was dried by Termovap Sample Concentrator and stock at -20℃.

[17 Determination of signaling molecules](#accordion)
:   1) The treated sample was dissolved with 200 uL acetonitrile and loaded to HPLC.

    2) Using HPLC, the excitation wavelength was set to 210nm, and the measurement time was 20 minutes. The peak time and wave peak of the sample were measured.

**Validation of biosafety devices**

[18 Characterization of anaerobic promoter effects](#accordion)
:   1) Different volume of 30 mg/mL Gm stock was added to LB agarose medium to get the LB medium with gradient Gm concentrations of 30/15/10 /5/2/1/0.5/0 μg/mL.

    2) The test strains were streaked to the LBA medium with gradient Gm concentrations mentioned above.

    3) One group is placed in an anaerobic box (Mitsubishi Gas Chemical Company, INC.), while another group is placed under the aerobic environment. All plates were placed in 37°C incubator for 48 hours.

[19 Validation of the effectiveness of biosafety devices](#accordion)
:   △dapA/dapA-pUC18T-mini-Tn7T and △dapA were streaked on solid LB medium with or without 200 μM DAP. The plates were tested under aerobic and anaerobic conditions as described above.

{% endblock %}

{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead %}{% endblock %} {% block cover %} {% endblock %} {% block page\_content %}

01
**Cry3A-like Toxin Section**

Circuit Design

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-1-1.png)

Figure 1. Diagram of Cry3A-like Toxin circuit design

In our design, the coding gene fragment for the active Cry3A-like toxin will be transformed into *E.coli* by the pET-30a vector, to confer the ability to produce Cry3A-like toxin. To enable its secretion, a signal peptide sequence,
OmpA, was added to the N-terminal of the Cry3A-like toxin. OmpA is a well-studied signal peptide in *E.coli* for the secretion of foreign proteins.

Experimental Results

The OmpA-Cry3A-like toxin fragment on the plasmid (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) was amplified using PCR and cloned into the pET-30a plasmid using the Gibson Assembly method (2× MultiF Seamless Assembly Mix kit, ABclonal)
to obtain the plasmid pET-30a-OmpA-Cry3A-like toxin. Subsequently, this plasmid was transformed into *E.coli* BL21(DE3) to test the secretion expression of the toxin. The pET30a-OmpA-Cry3A-like toxin was cultured overnight in
LB broth, and the culture was induced with IPTG for 3 hours. The culture was then centrifuged at 6,000 rpm for 10 mins to separate the bacterial cells and the supernatant, and the expression results were analyzed by SDS-PAGE.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-2.png)

Figure 2. SDS-PAGE Electrophoresis Detection of Cry3A-like Toxin Expression
 Lane 1: Concentrated protein supernatant of pET-30a (+IPTG);
 Lane 2: Concentrated protein supernatant of pET-30a-OmpA-Cry3A-like toxin (+IPTG);
 Lane
3: Concentrated protein supernatant of pET-30a-OmpA-Cry3A-like toxin (-IPTG)

The supernatant of the induced culture showed a 66.6 kDa band corresponding to Cry3A-like toxin, which was absent in the supernatant without IPTG induction and the wild-type control (Fig 2). This indicates the successful secretion expression
of Cry3A-like toxin.

Next, a 6×His tag will be added to pET30a-OmpA-Cry3A-like toxin using the enzyme cutting connection/ΩPCR method and Western blot will be performed to further confirm the secretion expression of Cry3A-like toxin.

02
**Cysteine Protease Trypsin Inhibitor (CPTI) Section**

Circuit Design

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-3.png)

Figure 3. Diagram of CPTI circuit design

The TIA (trypsin inhibitor subtype A) gene was not successfully expressed (see Engineering Success Section). The proteinase inhibitor CPTI was chosen, and OmpA was selected as the signal peptide. OmpA has been successfully employed for
extracellular expression in prokaryotic systems during laboratory work (Pechsrichuang et al., 2016, Movva et al., 1980). We also obtained information from the Peking University iGEM team, indicating their successful experience using
OmpA as a signal peptide (see HP Section). Therefore, using OmpA as a signal peptide for CPTI expression appeared to be a more promising approach (Fig 3).

Experimental Results

The OmpA -CPTI fragment (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) was inserted into the pET-30a vector to generate the plasmid pET-30a-OmpA-CPTI. This plasmid was then transformed into
*E.coli* BL21 and cultured overnight in LB medium. Overnight culture was dilute by 1/1000 to fresh LB medium, the IPTG was added when the
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600
 reach 0.6. The medium was incubated another 3 hours after the ITPG addition, then centrifuged at 12,000 rpm 10 mins to obtain the supernatant and the precipitate. The supernatant was concentrated using ultrafiltration centrifuge
tubes, while the precipitate was subjected to cell lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the supernatant from the post-lysis precipitate. The post-lysis precipitate was resuspended in
lysis buffer. Tricine-PAGE (Fig 4) and Western Blot analyses (Fig 5) were performed on the above samples, and the results indicated that the CPTI protein was not expressed in *E.coli*.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-4.png)

Figure 4. Tricine-PAGE analysis of CPTI expression

Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate
of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysateof of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-5.png)

Figure 5. Western blot analysis of CPTI expression

Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate
of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysate of of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

With IPTG the cell could produce the detectable CPTI (Lane 4, Fig 5), however, the concentrated supernatant of CPTI could not been seen (Lane 1, Fig 5). This might because of the poor solution of CPTI with OmpA singlal peptide. To get
a extracellular expressed CPTI, the GST tag, which could improve the solution of certain protein, was successfully added at the C-terminal of the CPTI gene. The protein induction expression experiment is currently underway.

This modification ensures that the protein can be expressed to a significant extent, as similar experiments have been reported (Yang et al., 2003). Our unique approach involves testing the activity of CPTI without removing the GST tag,
which distinguishes our study from previous ones.

03
**Regulation of Drug Expression by Orthogonal Quorum Sensing Systems
Section**

Circuit Design

**Sequential Expression of Products for Delayed Toxin Release**

The expression of Cry3A-Like toxin requires the activation of the first set of quorum sensing signal molecules. When engineered bacteria colonized in the gut, their population density is initially low, preventing the concentration of the
first set of quorum sensing signal molecules from reaching the required threshold. Only CPTI is expressed and secreted to the gut environment (Fig 6).

As the engineered bacteria continue to be introduced and proliferate in the gut, the concentration of the first set of quorum sensing signal molecules gradually accumulates to the threshold level. This triggers the expression of Cry3A-like
toxin and the onset of the second set of quorum sensing signal molecules. Upon reaching the threshold, the expression of the lysis gene is activated, leading to cell lysis, thereby releasing all Cry3A-like toxins into the environment.

The following gene circuit (first set of verification systems) is designed as follows

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-6.png)

Figure 6. Diagram of first set of verification systems circuit design

To validate the sequential expression of products, the first set of verification systems were transformed to Top10 cells. Induction experiments were conducted using arabinose (Ara). The
O

D

600
 values, eGFP, and RFP fluorescence intensity were monitored at regular intervals using a microplate reader and plotted a curve with
O

D

600
 as the vertical axis and induction time as the horizontal axis. If the RFP signal appears with a delay after the eGFP signal, it indicates that the pathway can achieve sequential expression, confirming the success of the verification.

**Implementation of Product Expression Control**

Cry3A-like toxin and the second set of quorum sensing systems work with the same module, sharing the same promoter and the RBS of the same strength, and their expression levels are positively correlated. When the concentration of the second
set of quorum sensing signal molecules has not reached the threshold level, both accumulate in the engineered bacteria. Once the threshold is reached, the lysis gene is actived, leading to cell lysis. In such a scenario, newly introduced
engineered bacteria into the gut will promptly trigger the lysis gene, preventing the high-level expression of Cry3A-like toxin., which could significantly reduce the accumulation rate of Cry3A-like toxin in the environment, creating
a situation similar to a "threshold" for its accumulation. A similar circumstance occurs for the accumulation of CPTI in the gut.

The following gene circuit (second set of verification systems) is designed as follows

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-7.png)

Figure 7. Diagram of second set of verification systems circuit design

The second set of verification systems were transformed into Top10 cells and conducted induction experiments using Ara. At regular intervals, the
O

D

600
 values and eGFP fluorescence intensity were measured using a microplate reader. A curve with
O

D

600
 were plotted as the vertical axis and induction time were plotted as the horizontal axis. If the
O

D

600
 value were observed a significantly decreases after the initially increaseing, the expression of the lysis gene could be confrimed. We assessed whether the pathway could limit the maximum expression level based on the trend
in eGFP signal changes. If the eGFP expression rate significantly decreases or ceases to increase after the appearance of lysis, the verification is successful. The limitation of the maximum expression level was assessed by the trend
of eGFP signal change.

Experimental Results

The complete sequences, synthesized (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) and assembled onto respective plasmids, have been validated through sequencing. Both sets of plasmids were simultaneously introduced into *E.coli* Top10 using the KCM ice method. Successful transformation was confirmed by clone PCR.

Universal primers for the pBAD series plasmids were employed to confime the transformation of pBAD24M-paraBAD-esaⅠ-traR(W) or pBAD24M-paraBAD-lasⅠ-lasR. Amplification of bands corresponding to the size in pBAD24M for both sets of transformation
plasmids resulted in bands of 1712 bp and 1845 bp (Fig 8), respectively. This confirms the successful introduction of the pBAD24M plasmids into both sets of plasmid systems.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-8.png)

Figure 8. Colony PCR of co-transformation by universal primers

Use pBAD24M forward primer and pBAD24M reverse primer as universal primers. Lane 1-2: Co-transformation of pBAD24M-paraBAD-esaⅠ-traR(W) and pBAD33-paraBAD-eGFP-ptra\*-mCherry; Lane 3: Co-transformation of pBAD24M-paraBAD-lasⅠ-lasR and pBAD33-paraBAD-eGFP-plas-T4
holin-T4 endolysin

The PCR was also performed using specific primers to obtain target-sized fragments to confirms the successful introduction of pBAD33 plasmids into both sets of plasmid systems. The plasmid containing -paraBAD-eGFP-ptra\*-mCherry yielded
a 1224 bp fragment by using primers GCAGTGCTTCTCCCGTTAC/TTCCCAGCCCATAGTTTTC, while the plasmid containing paraBAD-eGFP-plas-T4 holin-T4 endolysin produced a 1296 bp fragment by using primers AGGCGGTCGTTGCTAATA/ AAACTCGTGCGGAGGTAA (Fig
9).

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-9.png)

Figure. 9 Colony PCR of co-transformation by specific primers

Lane 1: pBAD33-paraBAD-eGFP-ptra\*-mCherry; lane 2: Co-transformation of pBAD24M-paraBAD-esaⅠ-traR(W) and pBAD33-paraBAD-eGFP-ptra\*-mCherry; lane 3: pBAD33-paraBAD-eGFP-plas-T4 holin-T4 endolysin; lane 4: Co-transformation of pBAD24M-paraBAD-lasⅠ-lasR
and pBAD33-paraBAD-eGFP-plas-T4 holin-T4 endolysin

**Validation of Product Expression Level Control Using the Second Verification
System**

Pre-experiment for Induced Expression of Lysis Effect

The successfully transformed engineered bacteria were streaked on plates and grown at 37°C. Single colonies were picked and inoculated into liquid LB medium, followed by the addition of inducers. The cultures were incubated for 6 hours.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-10-2.png)

Figure.10 Verification of lysis effect

The blank control was LB with 20% Ara. The control group consisted of the second verification system engineered bacteria without inducer, while the experimental group consisted of the second verification system engineered bacteria with
a final concentration of 0.02% Ara. Each group had 3 replicates.

After zeroing with the blank control, it was observed that the optical density in the experimental group decreased to 34.3% of that in the control group (Fig 10), indicating a significant lysis effect. This confirms the successful expression
of quorum sensing and initiation of downstream lysis gene expression. Lysis gene expression was successful without leakage.

**Growth Curve Testing of the Second Verification System**

Single clones were selected and inoculated into LB medium. After overnight incubation, the
O

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600
 was adjusted to 0.6, and arabinose was added to a final concentration of 0.02%. The cultures were shaken for 21 hours in a sterile 96-well plate, and a growth curve was plotted.

The blank control group was LB broth, the control group was wild Top10, and the experimental group was the second set of engineering bacteria for verification system. There were 6 replicates per group.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-11.png)

Figure 11. Growth curve testing of the second set of verification system

The growth curve revealed that the bacterial density continued to rise in the first 4 hours and began to decrease after 4 hours, stabilizing around the 9th hour. In contrast, the control group's bacterial density continued to rise. At
the 4th hour, the quorum sensing signal reached the threshold, initiating lysis gene expression. The engineered bacteria lysed, resulting in a significant decrease in bacterial density, which stabilized around the 9th hour (Fig 11).

**Characterization of Reporter Gene eGFP**

Single colonies of engineered bacteria were selected and inoculated into LB broth. After overnight growth, the
O

D

600
 was adjusted to 0.6. Ara at a final concentration of 0.02% was added, and the cultures were incubated for 6 hours. eGFP fluorescence intensity was measured using a microplate reader (excitation wavelength 488 nm, emission wavelength
506 nm).

Blank control group: sterile LB, control group: engineered bacteria without 20% ara added, experimental group: engineered bacteria with inducer added, 3 replicates per group.

There was no significant difference in fluorescence intensity between the control group and the experimental group, indicating no expression of eGFP (data not shown).

**eGFP Protein Expression Experiment**

Single colonies of engineered bacteria were selected and inoculated into LB medium with inducer for overnight incubation. Top10 subjected to the same procedure served as the control group. Bacterial pellets were collected, and SDS-PAGE
experiments were conducted, revealing no expression of eGFP at the protein level(data not shown).

**q-RT-PCR Experiment**

q-RT-PCR experiments were conducted under various conditions, confirming no expression at the mRNA level.

The reporter genes could not be expressed. The characterization of reporter genes eGFP and RFP was performed simultaneously with the second verification system. Consistent results were obtained from SDS-PAGE experiments and q-RT-PCR experiments
(data not shown).

We hypothesize several possible reasons:
 1. The selected reporter genes eGFP and RFP may not be suitable for expression in Top10.
 2. Errors may have occurred in the design of the gene expression module.
 3. Interactions between
genes may have occurred.

**Extraction and Detection of Quorum Sensing Signal Molecules:**

According to the literature, High-performance liquid chromatography (HPLC) can be used to detect
C4-HSL(Ma et al., 2017). HPLC were utilized to determine the retention times and signals of 3-oxo-
C6-HSL and 3-oxo-C12-HSL standards, demonstrating the applicability of
HPLC for detecting both substances (Fig12-13). Standard curves correlating concentration and peak area were constructed for both 3-oxo-C6-HSL and 3-oxo-

C

12
-HSL (Fig 17).

Construction of the 3-oxo-C12-HSL Standard Curve:

Standard samples with concentrations of 0.3125 nm, 0.625 nm, 1.25 nm, 2.5 nm, and 5 nm were used for three replicates at each concentration. The mean values were then used to generate the standard curve.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-12.png)

Figure 12. Chromatographic diagram of molecule 3-oxo-C6-HSL

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-13.png)

Figure 13. Chromatographic diagram of molecule 3-oxo-C12-HSL

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res14.png)

Figure 14. Standard curve for molecule 3-oxo-C12-HSL

y=4173346.303x+341501.737

R

2
=0.997

**Extraction and Detection of *E.coli* Signal Molecules**

Inoculate seed culture with an
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D

600
 of 0.6 into 24 conical flasks containing 100 mL LB each. 1% ara was added to induce expression and cultures were shaked at 37°C and 220 rpm. At 1-hour intervals, samples were collected from 3 flasks, labeled as Sample 1, Sample
2, and Sample 3. 20 mL of bacterial culture were extracted from each flask and centrifuged at 4000 rpm for 10 minutes. The supernatant were treated with an equal volume of ethyl acetate for 4 hours. The supernatant was centrifuged
and evaporated to dry. The samples were stored at -20°C for subsequent HPLC analysis.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-15.png)

Figure 15. Chromatographic diagram of molecule 3-oxo-C6-HSL from *Pseudomonas
syringae*

The quorum sensing signal molecule 3-oxo-C6-HSL from *Pseudomonas syringae* was detected using this method (Fig 15).

However, when the same approach was applied to the engineered bacteria, we encountered issues with multiple impurities in the chromatograms and the inability to detect the signal (Fig 20-21). Since the second validation system successfully
lysed without leakage, it is certain that the quorum sensing signal molecule 3-oxo-C12-HSL was produced and reached the threshold in the extracellular environment.
After two rounds of induction and extraction experiments, this method for detecting signal molecule production were abandoned.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-16.png)

Figure 16. Result of the first set of validation system induced for 8 hours

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-17.png)

Figure 17. Result of the second set of validation system induced for 8 hours

**Validation of Sequential Expression of Products Using the First Set of
Validation System**

The characterization of reporter genes eGFP and RFP was performed simultaneously with the second validation system. The results were consistent, showing no expression in SDS-PAGE and q-RT-PCR experiments.

We will continue to attempt changing the expression vector or reporter genes to complete the functional validation of the secretion gene pathway. We will replace eGFP and RFP with the validated protease inhibitor and toxin proteins, assemble
the complete gene pathway, and perform testing. Finally, the complete gene pathway will be introduce into the chromosome of Top10 for further evaluation.

04
**Biological Safety Device Section**

Circuit Design

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-18.png)

Figure 18. Diagram of biological safety device circuit design

**Experimental Results**

The pnirB-Gm fragment was cloned to pBAD24M through digestion and ligation. Three rounds of PCR amplification were employed to introduce the pnirB promoter and restriction sites in front of the gentamicin (Gm) resistance gene. Successful
transformation of this construct into bacterial strains was confirmed through Gm resistance selection. The colonies that could grow on the Amp+Gm plate were further checked by colony PCR (Fig 19). Subsequently, the expression strength
of pnirB were assayed under aerobic or anaerobic conditions with a gradient concentration of Gm.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-19.png)

Figure 19. Colony PCR of pBAD24M-pnirB-Gm

Lane1-3: PCR of pBAD24M-pnirB-Gm

The successfully transformed strains were streaked onto eight different Gm concentration plates, ranging from 0/0.5/1/2/5/10/15/30 μg/mL. These plates were incubated for 48 hours under aerobic or anaerobic conditions (anaerobic conditions
were achieved by placing the plates inside an anaerobic chamber).

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-20-1.png)

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-20-2.png)

Figure 20. Growth of *E.coli* Top 10 containing anaerobic promoter and Gm-resistant under different conditions

Under non-resistant and anaerobic conditions, bacterial growth is expected to be less favorable compared to aerobic conditions. As observed from the results, up to 2 μg/mL resistance concentration, there was no difference in bacterial
growth between aerobic and anaerobic environments. Starting from 5 μg/mL, , only a few colonies were present in the initial streaked area under the aerobic environment. In contrast, bacterial growth was still extensive even at 10 μg/mL
under anaerobic conditions. This suggests that pnirB is specifically expressed under anaerobic conditions. However, when the Gm increased to 15 μg/mL, strain could not grow well neither under aerobic nor anaerobic conditions (Fig 20).

In conclusion, the expression of pnirB under anaerobic condition is at least two times higher than the aerobic conditions. This could help us to construct the Biological Safety Device.

*E.coli* wm3064 is a *dapA* mutant strain with diaminopimelic acid (DAP) nutritional defect. The *dapA* gene were ligated to the downstream of pnirB and cloned to pUC18T-mini-Tn7T plasmid, then transformed to wm3064. The growth of Δ*dapA*/*dapA*-
pUC18T-mini-Tn7T and Δ*dapA* strains were tested under aerobic or anaerobic conditions. The results showed that Δ*dapA* could only grow in the presence of DAP no matter in aerobic or anaerobic conditions, while Δ*dapA*/*dapA*- pUC18T-mini-Tn7T
could grow under anaerobic conditions without the supplementation for DAP. These results indicate that this strain can only grow under anaerobic conditions and possesses biological safety (Fig 21). During production, growth can be
ensured by adding DAP.

![](https://static.igem.wiki/teams/4632/wiki/wiki/result/res-21.png)

Figure 21. Results of culture experiment of *dapA* gene defective strain

This work qualitatively confirms that the pnirB element can indeed exhibit specific expression under anaerobic conditions. Furthermore, by utilizing pnirB to drive the essential diaminopimelic acid gene *dapA*, it restricts bacterial growth
to anaerobic environments, such as the gut of the *S. invicta*. These findings provide a theoretical foundation for the next steps in the development of the biological safety device, specifically the recombination of pnirB into
the *E.coli* chromosome.

[Reference](#accordion1)
:   |  |  |
    | --- | --- |
    | [1] | MA, J. C., WU, Y. Q., CAO, D., ZHANG, W. B. & WANG, H. H. 2017. Only Acyl Carrier Protein 1 (AcpP1) Functions in Pseudomonas aeruginosa Fatty Acid Synthesis. *Front Microbiol*, 8, 2186. |
    | [2] | MOVVA, N. R., NAKAMURA, K. & INOUYE, M. 1980. Amino acid sequence of the signal peptide of ompA protein, a major outer membrane protein of Escherichia coli. *J Biol Chem*, 255, 27-9. |
    | [3] | PECHSRICHUANG, P., SONGSIRIRITTHIGUL, C., HALTRICH, D., ROYTRAKUL, S., NAMVIJTR, P., BONAPARTE, N. & YAMABHAI, M. 2016. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from *E.coli* expression system. *Springerplus*, 5, 1200. |
    | [4] | YANG, L. C., CHANG, T. J., CHEN, W. X., YANG, X. G. & ZHU, Z. 2003. [Fusion expression, purification and bioactivity assay of CpTI in Escherichia coli]. *Sheng Wu Gong Cheng Xue Bao*, 19, 63-8. |

{% endblock %} {% block sidenav %}

[Cry3A-like Toxin](#item-1)
[CPTI](#item-2)
 [QS Systems
Section](#item-3)
[Biosafety Device](#item-4)

{% endblock %}