Team:Jiangnan China/Experiments

Part 1. Selection of acid tolerance component ( msmK )

    The key point of this part is selecting the gene related to anti-acid characteristic. Because the mechanism of acid resistance is complicated, and genes are connected to each other, it is hard to confirm the most significant gene. Gnomic mutagenesis and high throughout screening are carried out to obtain anti-acid mutant strains with significant higher survival rate under specific acid environment (pH 4.0 ,5h). Then analyze the diff-genes data between mutant strain and parent strain on pH 4.0 and pH 7.0 respectively. A dimensionality reduction model is established and five possible acid-resistant genes are obtained. Finally, with experiment verification and pathway analysis, msmK gene is shown to be the key anti-acid gene.

    1. Genomic Mutagenesis and Highthroughput Screening

Fig 1 Screening procedures.

We take model strain Lactococcus lactis NZ9000 as parent strain to select

    Chemical mutagenesis
    The dose of DES for chemical mutagenesis was 0.5% (v·v-1), the treatment was 30 min, and the mortality rate was 85.3%. Under the condition of pH 5.0, two acid-resistant strains were screened from 20,000 strains, respectively. L. lactis WH101 and L. lactis WH102.

    UV mutagenesis
    The UV mutagenesis conditions were UV lamp power of 15 W, irradiation time of 50 s, irradiation distance of 30 cm, and lethality rate of 92.1%. Under the condition of pH 5.0, the acid-resistant strain L. lactis WH103 was screened from 15000 strains.

    High throughput screening
    With high throughput screening, we got 3 mutants that can survive at pH 5.0 for 4 hours, and we named them as L. lactis WH101、L. lactis WH102、L. lactis WH103.

    2. Compare the growth performance of acid-tolerant strains
    According to the 2% inoculation rate, the original strain L. lactis NZ9000, three mutant strains L. lactis WH101, WH102, WH103 were inoculated into 10 ml of liquid medium under the conditions of pH 4.5, 5.0, and 7.0, respectively. The OD value of the strains were measured every 0.5 hours, and the growth curve of the strains are shown as Fig 2.

Fig 2 (A) The growth curve of the four strains at pH 7.0; (B) The growth curve of the four strains at pH 5.0; (C) The growth curve of the four strains at pH 4.5; (D)The survival rate of the four strains at pH 4.0.

    Results analysis
1) The growth of acid-resistant strains under normal pH 7.0 conditions was not significantly affected.
2) The growth performance of acid-resistant strains at pH 5.0 and pH 4.5 was significantly improved compared to the original strain.
3) At pH 5.0, the biomass of the acid-tolerant strains WH101, WH102, WH103 was 7.0, 4.8 and 5.6 times that of the original strain, respectively.
4) At pH 4.5, they were 5.5, 3.6, and 4.3 times the original strain, respectively.
5) Under the condition of pH 4.0, the survival rate of L. lactis WH101, L. lactis WH102 and L. lactis WH103 was significantly higher than that of the original strain. And the survival rate increased gradually with the prolongation of stress time and acid resistance after 5 hours of stress. The strains were 16000, 351.4 and 264.3 times the original strain, respectively.

    Finally, through genomic mutation and high-throughput screening, we obtained a strain L. lactis WH101 with good acid resistance from 35,000 strains. Next, we will compare the NZ9000 and WH101 by transcriptomics analysis and mathematical model to obtain key acid resistant components.

    Transcriptomics analysis and modeling


Part 2. Add anti-cold gene ( cspD2 )

    According to previous research, we know that cspD2 can express cold shock protein in lactic acid bacteria. Therefore, we obtained the cspD2 fragment by in vitro synthesis, and constructed the cspD2 overexpression strain. Verify the cold resistance performance of the recombined bacteria with nisin as inducer.

Part 3. Demonstration

    Because we use nisA as promoter to express our parts, 0.5 ng/mL of Nisin is required to induce cell expression in all the following steps.

    Demonstration of msmK gene
    Copy msmK gene from Lactococcus lactis NZ9000 gene group with PCR. Construct msmK overexpression strain L.lactis NZ3900/pNZ8149-msmK with electrotransformation. Take strains L.lactis NZ3900/pNZ8149-msmK and L.lactis NZ3900/pNZ8149 from glycerin tube kept under -80℃, dilute 1/25 in 2 x 10 ml fresh medium (30 °C). Grow until the OD600=0.4. Count their number of colonies per ml at pH 4.0 (More details can be seen in protocol).

    We can get the following results:
Fig 3 Number of colonies at acid stress (pH 4.0).

Fig 4 Survival rate at acid stress (pH 4.0).

    The result shows that msmK is an effective anti-acid gene, which can make the recombinant strain has 213-fold higher survival rate than parent one.

    Demonstration of composite part msmK-cspD2
    It has been reported that cspD2 can effectively exert anti-freezing effect in the recipient bacteria. CspD2 was ligated to the pNZ8149/msmK plasmid by one-step cloning (seamless ligation), and the recombinant plasmid was introduced into the constructed L. lactis NZ3900/pNZ8149-msmk-cspD2 strain using electroporation.
    The gfp gene was inserted as a marker gene, and cell viability was characterized by fluorescence intensity. The strain was tested for acid resistance and freezing resistance using a flow cytometer. The process of acid stress and cold stress is similar with the above demonstration process.

    Acid stress
    Deal with the samples under pH 4.0 with nisin as an inducer.

Fig 5 Number of colonies at acid stress (pH 4.0).

Fig 6 Survival rate at acid stress (pH4.0).

    The result shows that composite part can make the recombinant strain has 243-fold higher survival rate than parent one under acid stress.
Fig 7 Electron microscopy of L.lactis NZ3900/pNZ8149-msmk-cspD2-gfp and L.lactis NZ3900/pNZ8149 before and after acid stress.

    Before the acid stress, the cell structure of the control strain and the recombinant strain remained intact. After 3 h of pH 4.0 stress, the cell membrane thickness became thinner and the surface became rough, and the cell membrane of some control strains ruptured. In comparison, the cell structure of the recombinant strain remains more intact, thereby effectively reducing the damage caused by acid stress on the cells.

    Cold stress
    L.lactis NZ3900/pNZ8149-msmk-cspD2-gfp and L.lactis NZ3900/pNZ8149 strains were inoculated with 4% inoculation. When cultured at 30 °C for 2.5 h (OD=0.35), add 0, 0.5 ng/mL of Nisin, and then culture for 8 ∼ 10 h (OD=0.8), centrifuge at 4000 r/min. Resuspend them in the same volume of fresh M17 medium and count the number of colonies. Four freeze-thaw stimulations were performed on all samples by 1 mL of the sample after counting, and placed in a refrigerator at −20 °C to cool rapidly, frozen for 24 h, then slowly frozen at 4 min and 30 °C. Count separately and calculate the survival rate.

Fig 8 The Comparison curve of survival rate under cold stress.

    After 4 consecutive repeated freeze-thaw tests, the recombinant strain was 47.5 times more viable than the control strain, indicating the antifreeze survival rate of the strain with increased overexpression of cspD2.

Fig 9 Electron microscopy before and after repeated freezing and thawing.

    Before freezing and thawing, the cell structure of the control strain and the recombinant strain remained intact. After repeated freezing and thawing for 4 times, the cell membrane of the cell became thin and rough, and some intracellular substances overflowed. In comparison, the cell structure of the recombinant strain remains more intact, and the damage of the cell membrane is alleviated, thereby effectively reducing the damage caused by freezing stress on the cells.

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