Part:BBa_K5238013
PsppA promoter and endosome LysKB317 to achieve suicide of Lactobacillus plantarum
In this composite part, we have selected the PsppA promoter and the lysin LysKB317 to achieve the self-destruction of Lactobacillus plantarum.
The PsppA promoter, derived from the sakacin P gene cluster, plays a role in regulating gene expression in Lactobacillus plantarum, particularly in the production of bacteriocins, which are typically regulated through a secretion-based peptide quorum-sensing mechanism. In the expression vector we constructed, the PsppA promoter works in concert with other regulatory elements to precisely control the expression of target genes, such as LysKB317. This mechanism is of significant importance for the development of novel microbial control strategies.
In relevant literature, researchers have found that swapping different promoters, such as the PsppA and PorfX promoters, affects the production of GusA and PepN. In experimental studies, it was discovered that the PsppA promoter can enhance the production of GusA in two host strains. Therefore, we chose the PsppA promoter from Lactobacillus plantarum to participate in the expression of target genes in the vector, thereby promoting the expression of the downstream target gene LysKB317 and achieving the function of a self-destruction switch. The core of our self-destruction switch is the lysin gene LysKB317, which originates from the phage genome. LysKB317 is an enzyme that degrades bacterial cell walls, leading to cell lysis. In our design, the expression of LysKB317 is strictly regulated by the inducible promoter PsppA, ensuring the safety of the engineered Lactobacillus plantarum.
In this component, for the first time, we students have utilized the PsppA promoter to regulate the expression of the self-destruction gene LysKB317. In our experiments, we designed a control group (without inducer), an experimental group (with inducer), and a blank group (vector without the self-destruction switch to exclude the possible impact of the inducer itself on bacteria). We verified the effectiveness of the self-destruction switch by plotting growth curves.
In the experiment, initially, we constructed a control strain (L168-pSIP403-PsppA-empty, abbreviated as EP) and a strain expressing a suicide switch (L168-pSIP403-PsppA-LysKB317, abbreviated as LysKB317). Subsequently, we established control groups (EP, EP+sakacin P) and experimental groups (LysKB317, LysKB317+ sakacin P), with the inducer concentration of sakacin P set at 25 ng/ml. We activated the corresponding bacteria, cultured them overnight, and prepared the culture medium under the respective conditions in a 96-well plate. Finally, we inoculated the activated EP and LysKB317 into the corresponding wells at a ratio of 1:50. After completion, we placed the 96-well plate into a microplate reader and adjusted the automatic timing measurement parameters of the reader: 37°C, 10 s of shaking before measurement, medium shaking intensity, wavelength at 600 nm, and a full-plate detection every hour for a continuous monitoring period of 24 hours. Below are the growth curves we detected. As shown in the figure, EP (blue circles) represents the empty vector control, EP+ (purple squares) represents the empty vector control with inducer, LysKB317 (black triangles) represents the strain with the suicide switch plasmid but without inducer, and LysKB317+ (red inverted triangles) represents the strain with the suicide switch plasmid and with inducer. We continuously monitored the growth of Lactobacillus plantarum over a 24-hour period, reflecting the growth of bacteria in different wells of the 96-well plate through the OD values detected by the microplate reader. The x-axis represents time in hours, with each small division representing an automatic OD value detection by the microplate reader every hour. The y-axis represents the detected OD values. (n=4)
First, we compared the EP (blue circles), which represents the empty vector control, with the EP+ (purple squares), which represents the control with inducer, to eliminate the influence of the inducer on bacterial growth. As observed from the graph, the purple line is slightly lower than the blue line, indicating that the addition of the inducer indeed has a subtle effect on bacterial growth.
Next, we focused on comparing LysKB317 (black triangles), which represents the strain with the suicide switch plasmid but without inducer, with LysKB317+ (red inverted triangles), which represents the strain with the suicide switch plasmid and with inducer. As observed from the graph, the red line is indeed lower than the black line, and the inhibitory effect on the growth of Lactobacillus plantarum L168 by the group with the suicide switch and inducer is greater than the subtle influence caused by the inducer alone. Therefore, this experiment demonstrates that the PsppA+LysKB317 module can suppress the growth of Lactobacillus plantarum L168. Additionally, from the graph, we can observe that after the addition of the inducer, around the 10th hour, the growth curve of Lactobacillus plantarum L168 begins to gradually slow down, indicating a decrease in growth rate. After the 12th to 13th hour, the red line with the suicide switch and inducer starts to flatten, indicating that Lactobacillus plantarum L168 has entered the stationary phase of growth.. This indicates that the PsppA+LysKB317 suicide switch can inhibit the growth of Lactobacillus plantarum within 12 to 13 hours. Assuming that our Lactobacillus plantarum L168 is administered as a medication to patients with sakacin P inducer, it can control the growth of Lactobacillus plantarum in the gut within about 12 hours, thereby controlling the amount of small molecules expressed and released by Lactobacillus plantarum. This effectively prevents the accumulation of excessive amounts of small molecules in the body, achieving controllable dosage. The sakacin P-induced promoter PsppA is being used for the first time in iGEM, and LysKB317 is a lysin targeting the cell wall of Lactobacillus plantarum. Theoretically, this design should effectively regulate the growth of Lactobacillus plantarum; this ensures the safety of Lactobacillus plantarum when applied for therapeutic purposes and prevents biological leakage.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 752
- 1000COMPATIBLE WITH RFC[1000]
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