Coding

Part:BBa_K5015002

Designed by: Rui Ke   Group: iGEM23_Thinker-SC   (2023-10-09)
Revision as of 12:11, 12 October 2023 by ProfessorLi (Talk | contribs)

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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.

The arabinose promoter is a crucial promoter sequence in molecular biology research. It is widely used in microbiology, especially in microorganisms like Escherichia coli, to investigate and regulate gene expression. One of the key features of the arabinose promoter is its specificity. It can precisely respond to the presence or absence of arabinose, thereby regulating the transcription of genes associated with arabinose metabolism. This specificity allows researchers to finely control the activity of specific genes as needed.

MazF is a type of toxin-antitoxin (TA) system that has been widely studied and whose mechanism of action is well understood. The mazF gene is located downstream of the TA system mazEF and encodes a stable toxin protein. MazF is a ribosome-independent mRNA interferase (ribonuclease) that can cleave single-stranded mRNA at specific sequence sites and is conserved in most microbes and some archaea.

In Escherichia coli, MazF can recognize ACA sequences and hydrolyze the phosphodiester bond at the first A position of either the 5' or 3' end, causing the ribosome to release from the cleaved mRNA and preventing protein synthesis. Subsequently, improperly encoded peptides are released and degraded by intracellular proteases, ultimately leading to cell death.

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.

Reference

1. Khlebnikov A, et al. (2000) Sugar utilization patterns and growth of Escherichia coli K-12 mutants lacking known sugar transport systems. J Mol Microbiol Biotechnol. 2(2):195-203. 2. Lee SY, et al. (2015) Development of an AraBAD promoter possessing strong activity in Corynebacterium glutamicum. Appl Microbiol Biotechnol. 99(13):5539-5549. 3. Lim HG, et al. (2017) Development of a plasmid addiction system using the arabinose-inducible promoter P(BAD) and Escherichia coli chromosomal araB promoter. Biotechnol J. 12(7):1600532. 4. Ryu JH, et al. (2019) Regulation of mazEF toxin-antitoxin module expression in Escherichia coli by intracellular ATP levels. J Microbiol Biotechnol. 29(4):687-696.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


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