Difference between revisions of "Part:BBa K4593004"

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===Usage and Biology===
 
===Usage and Biology===
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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.
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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).  
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This part is constructed for the characterization of Promoter P2 in E. coli, in which sfGFP downstream of the P2 promoter will be expressed under the presence of AIPs. However, it should be noted that this part does NOT work as expected!
  
 
==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 was to confirm the inducibility of the P2 promoter by AIPs. In our plasmid design, sfGFP serves as a reporter to indicate whether AgrA activates 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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<HTML>
  
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/es/figure-12.png" width="400" height="auto"/>
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<br>
 
Figure 1. Design of P2 characterization plasmid.
 
Figure 1. Design of P2 characterization plasmid.
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</p>
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</html>
  
 
===Construction of characterization plasmid===
 
===Construction of characterization plasmid===
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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.
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The PCR was employed to amplify our plasmid; the AgrC and AgrA were directly amplified from the plasmid pXylA-agrCA-I obtained from Addgene (n.d.). P2 promoter was synthesized according to the previous existing sequence (Part: BBa_K1960900) that iGEM 16_XMU-China designed. The sfGFP was obtained from 2002p derived from BNDS-China 2021. All the fragments were added to the pET28a (+) backbone through Goldengate Assembly.  
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The plasmid was transformed into TOP 10 competent cells, and the cells were spread on the LB agar plate with K+. After individual clones were selected and allowed to shake overnight, the plasmid was extracted and sent for sequencing, with the outcome being confirmed as correct.
  
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Figure 2. Result of amplification of plasmid parts.
 
  
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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.
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<HTML>
  
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Figure 3. Fluorescence observed in E. coli with no AIPs.
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/pcr/pcr-qs-pcr-22.png" height="400" height="auto"/><img src="https://static.igem.wiki/teams/4593/wiki/pcr/qs-1-con.jpg" height="400" height="auto"/>
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<br>
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Figure 2. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component’s length.  
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</p>
  
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</html>
  
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The RBS was directly synthesized according to the previous existing sequence (Part: BBa_B0034), and PCR was employed to amplify the plasmid parts(P1 and P2) to add RBS downstream of the promoter. The plasmid underwent the same procedure, transformed into TOP 10 competent cells, and was sent for sequencing, with the outcome being confirmed as correct.
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<HTML>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/pcr/qs-rbs-final.jpg" height="300" height="auto"/>
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<br>
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Figure 3. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component’s length. Lane 1: 2428bp; Lane 2: 4373bp.
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</p>
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</html>
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The extracted plasmid was introduced into BL21 to enable the expression of sfGFP. After an overnight incubation period, IPTG was introduced to induce expression. In conditions where S. aureus was not present, the expression of sfGFP was observed. This observation suggested that P2 was not induced but rather expressed constitutively. As a result, it became evident that P2 was not functioning as originally intended in our experiment, and a redesign of the gene circuit was required.
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<HTML>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/es/figure-14.png" height="300" height="auto"/>
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<br>
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Figure 4. The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.
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</p>
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</html>
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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.
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<HTML>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/es/figure-15.png" width="400" height="auto"/>
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<br>
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Figure 5. The predicted transcription rates of p2 specifically in E. coli.
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</p>
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</html>
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The calculated result revealed that the transcription rate at the beginning of the sequence was exceptionally high even without an activator, which was consistent with the wet lab result.
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Two main reasons contribute to the failure of our design. First, the structure of RNA polymerase and the microenvironment for transcription are 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 more shared features with S. aureus (see part BBa_K4593023 ).
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===Reference===
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Addgene: pXylA-agrCA-I Sequences. (n.d.). Www.addgene.org. Retrieved October 11, 2023, from https://www.addgene.org/53437/sequences/
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Marchand, N., & Collins, C. H. (2013). Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling. Biotechnology and bioengineering, 110(11), 3003–3012.
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https://doi.org/10.1002/bit.24975
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LaFleur, T. L., Hossain, A., & Salis, H. M. (2022). Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-32829-5
  
 
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<!-- Uncomment this to enable Functional Parameter
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===Functional Parameters===
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<partinfo>BBa_K4593004 parameters</partinfo>
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Latest revision as of 14:32, 12 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).

This part is constructed for the characterization of Promoter P2 in E. coli, in which sfGFP downstream of the P2 promoter will be expressed under the presence of AIPs. However, it should be noted that this part does NOT work as expected!

Team:BNDS-China 2023

Design of the plasmid

Initially, our objective was to confirm the inducibility of the P2 promoter by AIPs. In our plasmid design, sfGFP serves as a reporter to indicate whether AgrA activates 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

The PCR was employed to amplify our plasmid; the AgrC and AgrA were directly amplified from the plasmid pXylA-agrCA-I obtained from Addgene (n.d.). P2 promoter was synthesized according to the previous existing sequence (Part: BBa_K1960900) that iGEM 16_XMU-China designed. The sfGFP was obtained from 2002p derived from BNDS-China 2021. All the fragments were added to the pET28a (+) backbone through Goldengate Assembly. The plasmid was transformed into TOP 10 competent cells, and the cells were spread on the LB agar plate with K+. After individual clones were selected and allowed to shake overnight, the plasmid was extracted and sent for sequencing, with the outcome being confirmed as correct.



Figure 2. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component’s length.

The RBS was directly synthesized according to the previous existing sequence (Part: BBa_B0034), and PCR was employed to amplify the plasmid parts(P1 and P2) to add RBS downstream of the promoter. The plasmid underwent the same procedure, transformed into TOP 10 competent cells, and was sent for sequencing, with the outcome being confirmed as correct.


Figure 3. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component’s length. Lane 1: 2428bp; Lane 2: 4373bp.

The extracted plasmid was introduced into BL21 to enable the expression of sfGFP. After an overnight incubation period, IPTG was introduced to induce expression. In conditions where S. aureus was not present, the expression of sfGFP was observed. This observation suggested that P2 was not induced but rather expressed constitutively. As a result, it became evident that P2 was not functioning as originally intended in our experiment, and a redesign of the gene circuit was required.


Figure 4. 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 5. The predicted transcription rates of p2 specifically in E. coli.


The calculated result revealed that the transcription rate at the beginning of the sequence was exceptionally high even without an activator, which was 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 are 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 more shared features with S. aureus (see part BBa_K4593023 ).

Reference

Addgene: pXylA-agrCA-I Sequences. (n.d.). Www.addgene.org. Retrieved October 11, 2023, from https://www.addgene.org/53437/sequences/

Marchand, N., & Collins, C. H. (2013). Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling. Biotechnology and bioengineering, 110(11), 3003–3012. https://doi.org/10.1002/bit.24975

LaFleur, T. L., Hossain, A., & Salis, H. M. (2022). Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-32829-5

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 121