● msmK (BBa_K2606001)
It is a fragment cloned from Lactococcus lactis NZ9000 gene group, which is verified to be a new and effective anti-acid gene in Lactococcus lactis NZ3900.
Usage and function
The msmK overexpression strain has 213-fold higher survival rate than parent one at pH 4.0 after 3 hours.
Fig 1 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.
Fig 2 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 hours 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.
● cspD2 (BBa_K2606003)
The cspD2 gene is a reported that can express cold shock protein in Lactococcus lactis, and can help bacteria survive at low temperature.
Kegg number: LLNZ_06470.
Fig 3 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.
● gfp (BBa_K2606004)
It can express green fluorescent protein. We use it as a marker gene in the process to characterize the number of cells.
● msmK-cspD2 (BBa_K2606005)
It is the final composite biobrick of our project, we overexpress anti-acid gene msmK and anti-cold gene cspD2 via the NICE system.
Usage and biology
The gene 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.
Deal with the samples under pH 4.0 with nisin as an inducer.
Fig 4 Number of colonies at acid stress (pH 4.0).
Fig 5 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 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 msmK-cspD2.