Part:BBa_K4593023

Characterization device for P2 promoter in B. subtilis

Usage and Biology

The QS System is a mechanism employed by specific bacteria to sense their population density and subsequently regulate gene expression accordingly. In the case of S. aureus, it produces a signaling molecule known as autoinducing peptide (AIP) during its growth. AIPs are recognized by a membrane receptor called AgrC. AgrC, in turn, phosphorylates AgrA, leading to the activation of the downstream promoter P2. (Marchand & Collins, 2013)

This part is constructed for the characterization of Promoter P2 in B.subtilis, in which sfGFP downstream of the P2 promoter will be expressed under the presence of AIPs.

Team:BNDS-China 2023

Design

Improving upon the previous plasmid construction within E. coli, our team has chosen to construct the plasmid in Bacillus subtilis, a gram-positive bacterium. We changed the Ori of the plasmid to suit plasmid replication in B. subtilis. Additionally, we use xylose inducible promoter pXylA to control the expression of AgrA and AgrC to suit the gene expression in B. subtilis and to reduce the expression burden, which is consistent with the design of the original backbone.

Figure 1.The plasmid map of p2 characterization in B. Subtillus

Build

The characterization plasmid is built upon pXylA-agrCA-I, obtained from Addgene. (Addgene: PXylA-AgrCA-I Sequences, n.d.) First, PCR is employed to amplify our plasmid parts as shown in Figure 17. The fragment lengths are 3531bp, 4691bp, and 189bp, respectively. Gel extraction was successful for the first two groups, with concentrations of 30 ng/ul and 137 ng/ul, respectively. However, the gel extraction for the "62-1" group failed.

Figure 2. The AGE result of the PCR product for consrtucting the characterization plasmid. The PCR result for three different groups of templates and primers. (Bands from left to right are: DNA marker, BN23_0055+58 (lane 1), BN23_0056+61 (lane 2-6), two groups of BN23_0059+62 (lane 7-8)(failed), DNA marker)

Figure 3. The AGE result of the PCR product for consrtucting the characterization plasmid. The DNA concentration of the band from the annealing temperature of 62 °C and 63 °C was measured at 36ng/µl, and the band from the annealing temperature of 67°C was measured at 137ng/36ng/µl.

While the gel extraction did not meet our expectations, we used DNA purification to extract PCR products. Also, the primer’s annealing temperature has a great difference. Hence, the primers need to be fixed.

Figure 4. The AGE result of the PCR product for constructing the characterization plasmid. The bands did not indicate the correctness of the component.

Learn

The plasmid assembly for characterizing the p2 promoter was unsuccessful, and we don’t have time for further debug. It is suspected that this failure may be attributed to our lack of familiarity with the pXyl vector, which is not native to E. coli. However, during the process, we have gained valuable experience in plasmid construction.

Reference

Marchand, Nicholas, and Cynthia H. Collins. “Peptide-Based Communication System Enables Escherichia Coli to Bacillus Megaterium Interspecies Signaling: Peptide-Based Interspecies Communication System.” Biotechnology and Bioengineering 110, no. 11 (November 2013): 3003-12. https://doi.org/10.1002/bit.24975.