Device

Part:BBa_K2606005

Designed by: Weijia Liu   Group: iGEM18_Jiangnan_China   (2018-10-09)


anti_acid and anti_cold part


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 954
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

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.

Acid stress

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

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

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Fig 2 Survival rate at acid stress (pH4.0).

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Fig 3 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.


Source

This part is designed by Jiangnan_China team. It is a composite mode of all the previous basic parts.

References

1. Nezhad MH, Hussain MA, Britz ML. 2015. Stress responses in probiotic Lactobacillus casei. Crit Rev Food Sci Nutr 55:740-749.

2. Wu CD, Huang J, Zhou RQ. 2014. Progress in engineering acid stress resistance of lactic acid bacteria. Appl Microbiol Biotechnol 98:1055-1063.

3. Zhang J, Caiyin Q, Feng WJ, Zhao XL, Qiao B, Zhao GR, Qiao JJ. 2016. Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44. Sci Rep 6:12.

4. Martani F, Berterame NM, Branduardi P. 2017. Microbial stress: From molecules to systems (Sitges, November 2015). New Biotechnology 35:30-34.

5. Bosma EF, Forster J, Nielsen AT. 2017. Lactobacilli and pediococci as versatile cell factories - Evaluation of strain properties and genetic tools. Biotechnol Adv 35:419-442.

6. Teusink B, Bachmann H, Molenaar D. 2011. Systems biology of lactic acid bacteria: a critical review. Microbial Cell Factories 10:17.

7. Yinghua Zhang, Yuting Lei, Guicheng Huo. 2008. Expression of cold-shock-protein genes from Lactococcus lactis and analysis of the cryoprotection function. Acta Microbiologica Sinica 48(9)：1203~1207.

8. Ermolenko DN, Makhatadze GI. Bacterial cold-shock proteins. Cell Mol Life Sci, 2002, 59: 1902−1913

9. Zhou, J., Huang, L., Liu, L., & Chen, J. (2009). Enhancement of pyruvate productivity by inducible expression of a F0F1-ATPase inhibitor INH1 in Torulopsis glabrata CCTCC M202019. Journal Of Biotechnology, 144(2), 120-126.

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