Part:BBa_K1841001
nisI
Research from Torsten et al. revealed that the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes, containing nisI, nisF, nisE, and nisG. Functional analyses provided evidence that NisI acts as a nisin-sequestering protein that expels nisin molecules from the cytoplasmic membrane into the environment.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2019-SZPT-China
1.Construction of pMG36e-nisl vector
Since the biological chassis is ultimately to be valued in the intestine, a food grade resistant expression vector is required. We replaced the erythromycin resistance gene of the original plasmid PMG36e with the nisin resistance gene nisl，which encode an antibacterial peptide for food preservation. Fig13 is the result of recombinant pMG36e-Nisl verification. The restriction and PCR result show that nisl with size 862bp was inserted in pMG36e successfully.
2.Verification of nisin resistance of MG1363(pMG36e-Nisl)
The MG1363 cells transferred with pMG36e-Nisl were spread on the plate with nisn and erythromycin respectively. The result is showed in Fig.14. we can see that the recombinant strain could grow on the nisin-resistant plate, could not grow on the Emr-resistant plate, indicating that the resistance gene replacement was successful.
The MG1363, MG1363 transferred with PMG36e and MG1363 transferred with PMG36e-nisI were inoculated in GM17 medium with 0,10,20,30,40,50,60,70,80,90,100 IU/ml nisin respectively. After 8 hours of culture, samples were taken and measured the OD600 to compare their growth (Fig15)
3.The study of nisin tolerance activity of MG1363(PMG36e-nisI)
We constructed a lactic acid bacteria food grade carrier with nisl resistance. In order to verify the function of the nisl part, we transferred the constructed vector into lactic acid bacteria to verify the tolerance of lactic acid bacteria with the part nisl.
3.1 The growth curve of MG1363, MG1363(PMG36e) and MG1363(PMG36e-nisI) in GM17medium
The MG1363, MG1363 transferred with PMG36e and MG1363 transferred with PMG36e-nisI were inoculated in GM17 medium respectively. Sample was taken every 2 hours to measure the OD600 to examine its growth. Fig.1 is the growth curve of these three strains.
3.2 The effect of nisin on the growth of MG1363, MG1363(PMG36e) and MG1363(PMG36e-nisI)
The MG1363, MG1363 transferred with PMG36e and MG1363 transferred with PMG36e-nisI were inoculated in GM17 medium with 0,10,20,30,40,50,60,70,80,90,100 IU/ml nisin respectively. After 12 hours of culture, samples were taken and measured the OD600 to compare their growth.
The result showed that the growth of MG1363 and MG1363 transferred with PMG36e was inhibited strongly in GM17 culture medium with nisin (Fig.2), while the MG1363 transferred with PMG36N still growth very well. This demonstrated that the gene nisl encoding lipoprotein nisl is expressed in MG1363 and confers the resistance to nisin in this strain. But as the concentration of nisin increases, the growth of this strain is also depressed.
3.3 The effect of different concentration of nisin on the growth of MG1363 transferred with PMG36N
The MG1363 transferred with PMG36e-nisI were inoculated in GM17 medium with 0,10,20,30,40,50,60,70,80,90,100 IU/ml nisin respectively. Sample was taken every 2 hours to measure the OD600 to examine its growth. Fig.3 is the growth curve of this strains.
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