Team:Jiangnan China/Demonstrate

    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 1 Number of colonies at acid stress (pH 4.0).

Fig 2 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 3 Number of colonies at acid stress (pH 4.0).

Fig 4 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 5 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 6 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 7 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.