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Results
Results
project | SMU-Union-China-iGEM 2025
  • LGG GROWTH CURVE
  • PLASMID TARGET
               FRAGMENT AND
               PRIMERINFORMATION
  • RESPONSE MODULE
  • ELISA
  • FUNCTION PLASMID
               EXPRESSION IN BACTERIA
  • SAFETY MODULE

    • LGG Growth Curve

    Growth Curve of Wild-Type Lactobacillus casei rhamnosus

    fig.1 Growth Curve of Wild-Type Lactobacillus casei rhamnosus

    The experimental results of the growth curve determination of wild-type Lactobacillus casei rhamnosus show that after 24 hours of incubation at 37°C in a shaking incubator and under static culture conditions, the bacterial growth curves are generally similar. During the lag phase (0–3 hours), the bacteria adapt to the new environment and prepare for division, with the population remaining essentially unchanged. During the logarithmic phase (4–16 hours), the bacteria divide rapidly in an exponential manner, resulting in a sharp rise in the curve. In the stationary phase (17–24 hours), nutrient depletion and metabolite accumulation lead to a balance between the number of new and dead bacterial cells, which is consistent with expectations.


    Growth Curve of Recombinant Lactobacillus casei rhamnosus under Antibiotic Resistance Conditions

    fig.2 Growth Curve of Recombinant Lactobacillus casei rhamnosus under Antibiotic Resistance Conditions

    The experimental results of the growth curve determination of recombinant Lactobacillus casei rhamnosus under antibiotic resistance conditions show a deviation from the trend observed in the wild-type strain. Specifically, the lag phase of the recombinant strain is significantly prolonged under resistance conditions, likely due to the inhibition of bacterial proliferation by erythromycin during this phase. As a result, the time required for the bacterial population to reach the mid-logarithmic phase is extended. Nevertheless, the overall trend still conforms to the general pattern of bacterial growth curves.

    Growth Curve of Recombinant Lactobacillus casei rhamnosus under Non-Resistance Conditions

    fig.3 Growth Curve of Recombinant Lactobacillus casei rhamnosus under Non-Resistance Conditions

    The experimental results of the growth curve determination of recombinant Lactobacillus casei rhamnosus under non-resistance conditions indicate that the trend is generally consistent with that of the wild-type strain, which aligns with expectations.

    Plasmid Target Fragment and Primer Information


    Module Serial Number Resistance expression Plasmid Size Primer Name Sequence (5' to 3')
    Sensing Module 1 Erythromycin 8852-eGFP 352bp Pnps-F
    Pnps-R     		CGGATTTTACGCCGTGTACTGG
    GACTAACGGCAACCCACTGTCC
    2 Erythromycin 8852-eGFP 728bp medi8852-F
    medi8852-R     		CTGTCTTCCTACACTCACTG
    TGGCAGGGTAAAGTCAGTA
    Response Module 3 Erythromycin 8852-mCherry 714bp
    839bp     		8852-F
    8852-R
    ZT-pho-F
    pho-R     		CAGGTCTGGTTAAACCGTCTC
    CTTTGATTTCTACCTTGGTGCC
    CACGTGCTGTAATTTGAAGC
    CTGTCGAAGTATTGCTGGTAC
    4 Erythromycin 1G01-mCherry 822bp
    839bp     		1G01'-F
    1G01'-R
    ZT-pho-F
    pho-R     		GTTCAGCTGGTCGAATCTGG
    GATGGCCATGTTATCCTCCTC
    CACGTGCTGTAATTTGAAGC
    CTGTCGAAGTATTGCTGGTAC
    Security Module 5 Erythromycin MazF 618bp ZT-MazF'-F
    MazF'-R     		TGTCAGATAGGCCAATGACTG
    CCAATCAGTACGTAAAATTTGGC
    6 Erythromycin CI-qR-GFP-srrA 867bp CI-R
    ZT-CI-F     		TATATTACAGCTCCAGATCTACCG
    CACTGACTAGCGATAACTTTCC
    6 Erythromycin YF1-FixJ-FixK2-GFP 835bp FixJ-YF1-F
    ZT-FixJ-YF1-R     		TTTCTTCACCACCAAGGACAC
    GGTCGACAATGAGTGAGCTAAC
    7 Erythromycin GFP 1072bp ZT-GFP-F
    GFP-R     		CAACACGTGCTGTAATTTGAAGC
    CACTTGTACAGCTCGTCCATG
    8 Erythromycin RNAthermo-GFP 1110bp ZT-GFP-F
    GFP-R     		CAACACGTGCTGTAATTTGAAGC
    CACTTGTACAGCTCGTCCATG
    9 Erythromycin TlpA-pTlpA-GFP 853bp ZT-pTlpA-GFP-F
    GFP-R     		CATAAGGGAGAGCGTCGAGATC
    CACTTGTACAGCTCGTCCATG
    10 Erythromycin TlpA-pTlpA-GFP 1456bp ZT-TlpA-F
    tlpA-R     		CGGCGTAGAGGATCGAGATCT
    CTGGCCACCGGTCTGTTTATTG


    Sensing Module

    Colony PCR

    fig.1
    fig.2
    fig.3

    Single colonies of recombinant bacteria after electrotransformation, selected via antibiotic screening, were picked from MRS agar plates containing erythromycin resistance for colony PCR. The picked colonies were retained for subsequent fluorescent protein expression verification. The video shows the sensing module recombinant Lacticaseibacillus rhamnosus selected through antibiotic screening, where the colonies appear smooth and off-white.

    The image shows the positive PCR results of the plasmid primers for the engineered bacterial sensing module, with bands at the correct positions. The experiment preliminarily confirms that the designed plasmid of the sensing module has been successfully transferred into Lacticaseibacillus rhamnosus.

    Response of the Sensing Module Under Viral Gradient Stimulation

    fig.4

    The inactivated influenza A virus (H1N1-PR8 strain) measured in chicken red blood cells had a HAI= 1024(Figure 4).

    fig.5

    Group 1: Engineered bacteria without inactivated influenza A virus.

    Group 2: Engineered bacteria with 10^1-fold diluted inactivated influenza A virus.

    Group 3: Engineered bacteria with 10^2-fold diluted inactivated influenza A virus.

    Group 4: Engineered bacteria with 10^3-fold diluted inactivated influenza A virus.

    Group 5: Engineered bacteria with 10^4-fold diluted inactivated influenza A virus.

    Group 6: Engineered bacteria with 10^5-fold diluted inactivated influenza A virus.

    Group 7: Engineered bacteria with 10^6-fold diluted inactivated influenza A virus.

    The virus was diluted gradiently to stimulate the recombinant bacteria, and the expression of fluorescent proteins was monitored using a microplate reader at specific wavelengths(Figure 5). Experimental data show that as the viral dilution gradient increased, the fluorescent protein expression of the recombinant bacteria gradually increased until the dilution factor reached 10⁷, where it leveled off compared to the control group. This suggests that the growth and viability of the recombinant bacteria were inhibited at high viral titers, while at low to medium viral loads, they sensed the presence of the virus and responded.

    Response Module

    Co-transformation of the Sensing and Response Module Plasmids


    fig.1
    fig.2

    Preliminary experiments were conducted using E. coli containing the respective module plasmids. The E. coli cells containing the functional sensing module plasmid were prepared into competent cells, which were then transformed with the response module plasmid and incubated. The successful electroporation parameters are shown above.


    Colony PCR


    fig.4

    Colonies returned two diagnostic bands corresponding to both the sensing plasmid and the newly introduced response plasmid. PCR validation showed correctly sized bands in the second batch of engineered bacteria.

    These results indicate that both the sensing module and response module plasmids were successfully transformed into the engineered bacteria.

    Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis


    fig.3

    SDS-PAGE of bacterial lysates indicated scFv protein expression, though mixed with faint non-specific signals.

    Hemagglutination Inhibition Assays

    fig.1
    fig.2
    fig.3

    Figure 1 8852mc group hemoconcentration level: +; 1G01 group hemoconcentration level: ++

    Figure 2 Virus group hemoconcentration level: ++++

    Figure 3 WT group hemoconcentration level: +++

    Supernatant from induced dual-plasmid strains tested by HA inhibition assay shows the reduction in hemagglutination, indicating successful secretion and neutralizing activity.

    Elisa


    fig.4

    Based on the ELISA indirect assay, we observed that the engineered bacterial group exhibited lower levels of unbound anti-His tag mouse mAbs compared to the wild-type bacterial group. Furthermore, the upward trend in antibody levels was more pronounced during serial dilutions. This indicated the presence of scFv antibodies within the total protein extracted from the engineered bacteria.

    Functional Plasmid Expression in Engineered Bacteria

    Fluorescence Verification

    fig.1 Sensing Module
    fig.2 Response Module
    fig.3 Sensing and Response Module
    fig.4 Safety Module
    fig.5 Safety Module
    fig.6 Safety Module

    Figure 1 shows the expression of the eGFP green fluorescent plasmid in colonies of the sensing module Lactobacillus casei rhamnosus.

    Figure 2 shows the expression of the mCHERRY red fluorescent plasmid in colonies of the response module Lactobacillus casei rhamnosus.

    Figure 3 shows the co-expression of the red and green fluorescent plasmids in colonies after co-transformation with both the sensing and response module plasmids.

    Figures 4-6 show the expression of the eGFP green fluorescent plasmid in colonies of the safety module Lactobacillus casei rhamnosus.

    All colonies retained were PCR-positive with bands at the correct positions. The three groups on each plate represent three different single colonies from the same antibiotic plate and the three adjacent colony spots originate from the same single colony picked via antibiotic screening, representing three biological replicates.

    All plates were MRS agar plates with erythromycin resistance. Observations indicate that the fluorescent plasmids designed for all the modules were successfully expressed.

    Safety Module

    Colony PCR


    fig.1 colony PCR for TlpA-pTlpA-GFP
    fig.2 Colony PCR for RNAthermo & eGFP

    fig.3 colony PCR for CI-pR
    fig.4 colony PCR for YF1-FixJ-FixK2-GFP
    fig.5 colony PCR for MazF

    PCR products matched the theoretical sizes predicted from primer design, confirming successful amplification of the target fragments


    Temperature-induced eGFP expression


    fig.1 TlpA system validation
    fig.2 RNA thermometer(ROSE) validation experiment

    fig.3 Secondary-structure prediction of RNA thermometers

    1. TlpA system: TlpA switch ON >37 °C in 3h as expected (Fig 1).

    2. RNA thermometer: dip 25–30 °C, peak 37 °C, opposite to ROSE rule; modeling shows identical long-stem loops 25–45 °C, melt only at 50 °C (Fig 2-3). Now, the modeling team has abandoned single-shot static predictions and is now collaborating with the experimental group to build a four-dimensional dataset of temperature–time–fluorescence–mRNA half-life


    Blue light kill switch assay

    fig.4 pdawn elements validation
    fig.5 CI-PR Induction Assay
    fig.6 MazF toxin validation

    1. Fluorescence detection: As anticipated, under dark conditions, the FixK2 promoter was activated, producing eGFP and yielding a markedly stronger fluorescence signal. Following 2 hours of blue light exposure, a pronounced suppression of fluorescence was observed, confirming that blue light successfully inhibited FixJ-YF1, thereby suppressing the expression of the eGFP fluorescent protein (Fig.4). Under 75 ng/mL IP-673 induction, the CI-PR system functions normally: CI represses eGFP expression downstream of PR.

    2. Serial-dilution spread-plate assay: Induction with 7 ng/mL IP-673 triggered MazF expression and confirmed its toxic effect for at least 9h (fig.6).