Part:BBa_K2985160
P43-liaH->Lipopeptide tolerance gene
Enhanced tolerant genes for lipopeptides, so that the liaH gene will not be expressed only when the surfactin reaches a certain concentration, but will begin to synthesize a specific tolerance protein during the growth process, thus protecting Bacterial cells.
The liaH gene cluster is mainly involved in the coding of the protein Lia protein, which can protect the integrity of the cell membrane. At the same time, liaH exists in the large gene cluster of lia, and the two-component system liaRS can effectively resist the damage of cells such as surfactin. It can enhance the tolerance of bacteria to lipopeptide antibiotics.
According to the research of Anna Barbara [1], we know that the protein encoded by liaH gene can protect the cell membrane of Bacillus subtilis. At the same time, Wu [2] and others also obtained higher yield of surfactin by strengthening liaH gene, which also provides reference for our experiment.
Figure 1: The addition of P43 promoter causes the overexpression of liaH gene
We obtained the sequence we needed through gene synthesis. We amplified the corresponding length sequence by PCR. The length of the sequence was tested by 0.8% agarose gel electrophoresis.
Figure 2:0.8% agarose gel electrophoresis and PCR validation. The product has a signal band at 678bp, consistent with the target length.
To verify the effect of the gene we expressed, we decided to detect the resistance of bacterial cells to surfactin.
Because surfactin influence for bacterial cells is no quantitative indicators, we also simply by adding different concentrations of surfactin in the culture medium to test the effects of concrete, we chose in 50 mg/L - 200 mg/L test the condition of bacillus subtilis on the experiment, the results found that concentrations in 200 mg/L has the strongest inhibitory on bacillus subtilis, and also the most obvious. We also obtained the corresponding bacteriostatic effect diagram.
Figure 3:The growth of wild-type bacillus subtilis containing 200mg/L of surfactin is shown on the right and normal LB medium on the left
At the same time, we also cultured our recombinant bacteria on coated plates to observe the morphological differences of colonies.
Figure 4:The left is the normal LB medium, and the right contains 200mg/L of surfactin of the modified bacillus subtilis
We can clearly see that liaH gene has a great effect on the improvement of colony tolerance. In addition, we also carried out high-performance liquid phase analysis of the yield of surfactin, which also proved that the parts for Bacillus subtilis production of surfactin ability. Under the cooperation of yerp gene, the yield of surfacin increased by 100% compared with the initial yield.part:BBa_K2985002 However, due to the increase in the yield of surfactin, which can not show the transcription level of the overexpressed liaH gene, we have not done further research.
Reference:
[1]Helmann JD,Hachmann AB,Angert ER.Genetic analysis of factors affecting susceptibility of Bacillus subtilis to daptomycin.[J].Antimicrobial agents and chemotherapy.,2009,53(4):1598-1609.
[2]Qun Wu, Yan Zhi, Yan Xua. Systematically engineering the biosynthesis of a green biosurfactant surfactin by Bacillus subtilis 168. Metabolic Engineering ,2019,53 : 87-97
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 149
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 149
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 149
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 149
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 149
Illegal NgoMIV site found at 173 - 1000COMPATIBLE WITH RFC[1000]
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