Part:BBa_K5015002
Arabinose promoter+bacterial RNA cleave enzyme encoding gene
An unavoidable issue when utilizing genetically engineered bacteria strains is contamination, where our strains might accidentally leak out to the outer environment, potentially polluting the soil and impacting the ecology in a negative way. Hence, we designed an apoptosis system under the arabinose promoter sequence with the goal of proper cell death at contact with arabinose.
Usage and Biology
We used the plasmid skeleton pSB1A3, which insert the arabinose promoter, as the basis for the mazF gene expression. We then transformed these constructs into th E. Coli DH5a strains with the heat shock method. the mazF gene codes for a series of protein toxin enzymes that severs mRNA and hence inhibit cellular protein expression and consequentially kill the cells.
Figure 1 The design of arabinose promoter and mazF.
Figure 2. (A) Gel electrophoresis of the arabinose promoter and mazF. (B) Map of recombinant plasmid pSB1A3-pBAD-mazF.
Characterization
Figure 3 The effect of MazF.
All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD). Statistical significance between induced and non-induced cultures was determined using a Student's t-test, with p < 0.05 considered statistically significant.
The mRFP (monomeric Red Fluorescent Protein) gene was utilized as a reporter to assess the functionality of the arabinose promoter. The intensity served as an indicator of the activity of the arabinose promoter，the result is shown in Figure 3A. When the OD600 of the engineered bacteria and the control strain is 0.6, 1 mM arabic acid is added. After induction for 4 hours, the fluorescence intensity is measured using an enzyme-linked immunosorbent assay microplate reader and divided by OD600 to obtain the relative fluorescence intensity in Figure 3B .To test the response of the arabic acid promoter to different concentrations of arabic acid, as shown in Figure 3C, when the OD600 of the engineered bacteria is 0.6, 0.1, 0.5, 1, 2, and 4 mM arabic acid is added. After induction for 4 hours, the fluorescence intensity is measured and divided by OD600 to obtain the relative fluorescence intensity. To induce the expression of MazF, varying concentrations of arabinose were added to the cultures. Bacterial growth was monitored at specific time intervals (i.e., 2, 4, 6 hours post-induction) to assess the inhibitory effects of MazF expression，The results are shown in Figure 3D. In conclusion, it can be concluded that MazF induction has a significant inhibitory effect on bacterial growth compared to the uninduced control group. After 6 hours of induction, bacterial growth was significantly inhibited, and after 14 hours, growth almost completely stopped.
Potential application directions
Our experiment shows one possibility of controlling biological pollution by artificial bacterial death. This method can be widely applied to many different areas involving bacteria in nature such as organic fertilizers or waterculture. By creating a certain condition, in our case, exposure to arabinose, that stimulates a protein toxin gene to be expressed, bacteria growth can be well-controlled without widespread use of antibiotics or other synthetic chemicals.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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