Difference between revisions of "Part:BBa K4593004"

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==Team:BNDS-China 2023==
 
==Team:BNDS-China 2023==
 
===Design of the plasmid===
 
===Design of the plasmid===
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This part is constructed to verify the competence of P2 promoter, that if it can be successfully induced by AIPs. A common strategy of characterization is applied in our experiment. To reveal that P2 promoter is working, a plasmid containing two pathways is constructed: One is a constructive pathway expressing AgrA and AgrC, activated by LacI promoter; the other is an inductive pathway expressing sfGFP, activated by P2 promoter. As hypothesized, if lactose exists in the environment, AgrA and AgrC are expressed and are able to receive signals from AIPs. Thus, if AIPs once present, fluorescence will be observed; on the contrary, no fluorescence will exist in absence of AIPs. If this is the case, P2 promoter will be capable in later experiments. The plasmid is designed in Snapgene according to aforesaid directions, the picture of design is as shown in Figure 1.
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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.
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Figure 1. Design of P2 characterization plasmid.
 
Figure 1. Design of P2 characterization plasmid.

Revision as of 04:31, 11 October 2023


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

In previous preparations, the plasmid is divided to two parts (P1 and P2)to reduce the mistakes made in PCR. The PCR result is as shown in Figure 2. After obtained the amplified DNA, Goldengate Assembly is used to construct the plasmid. The plasmid is then transferred into Top 10 competent cells to amplify, following the standard protocols: Plate on LB plane with K+, select single colonies, and shake overnight in liquid culture. The plasmids are sequenced by biology companied after extracted from cells, and the outcome is correct.

Figure 2. Result of amplification of plasmid parts.

The plasmids are them transformed into BL21 to express proteins. After shaking overnight, IPTG is used to induce expression. Unfortunately, in the absence of AIPs, sfGFP is expressed and observed (Figure 3), indicating P2 is not a good induced promoter but somehow works constructively. Therefore, the use of P2 promoter is not valid in our experiment, and redesign of the gene circuit is needed.

Figure 3. Fluorescence observed in E. coli with no AIPs.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 883
    Illegal NheI site found at 906
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1294
    Illegal BamHI site found at 2178
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 121