Part:BBa_K4593004
Characterization device for P2 promoter in E.coli
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) For further details, please refer to the Design page. During this engineering cycle, our primary objective is to verify the proper functioning of the P2 promoter.
Team:BNDS-China 2023
Design of the plasmid
Initially, our objective is to confirm the inducibility of the P2 promoter by AIPs. In our plasmid design, sfGFP serves as a reporter to indicate whether AgrA activated the transcription of genes downstream P2 promoter in the presence of AIPs. Consequently, the presence of observable fluorescence would signify the effective activation of the P2 promoter, validating its utility in our project. The plasmid design is depicted below.
Figure 1. Design of P2 characterization plasmid.
Construction of characterization plasmid
First, PCR is employed to amplify our plasmid parts (P1 and P2), as shown in Figure 2. Goldengate Assembly is then applied to construct our plasmid. The plasmid is transformed into TOP 10 competent cells, and the cells are spread on an LB agar plate with K+. After individual clones are selected and allowed to shake overnight, the plasmid is extracted and sent for sequencing, with the outcome being confirmed as correct.
Figure 2. Result of amplification of plasmid parts.
The extracted plasmid is introduced into BL21 to enable the expression of sfGFP. After an overnight incubation period, IPTG is introduced to induce expression. In conditions where S. aureus is not present, we observed the expression of sfGFP. This observation suggests that P2 is not induced but rather expressed constitutively. As a result, it becomes evident that P2 is not functioning as originally intended in our experiment, and a redesign of the gene circuit is required.
Figure.3 The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.
To determine the cause of the failure, the dry lab component was employed, and the transcriptional rates of the P2 promoter in E. coli were assessed using the De novo DNA calculator (LaFleur et al., 2022). The result is shown below.
Figure 4. The predicted transcription rates of p2 specifically in E. coli.
The calculated result reveals that it is evident that the transcription rate at the beginning of the sequence is exceptionally high even without an activator, which is consistent with the wet lab result.
Two main reasons contribute to the failure of our design. First, the structure of RNA polymerase and the microenvironment for transcription is very different in E. coli and S. aureus, causing the transcription activity of P2 to be exceptionally high. Also, as E. coli is a gram-negative bacteria and S. aureus is a gram-positive bacteria, the membrane protein AgrC may not locate itself successfully on the cell membrane of E. coli, causing the signal transduction to fail. For those reasons, we decided to move the QS detection module to a gram-positive bacteria, Bacillus subtilis, which has a more shared feature with S. aureus.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 883
Illegal NheI site found at 906 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1294
Illegal BamHI site found at 2178 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 121
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