Difference between revisions of "Part:BBa K4593004"

Revision as of 03:02, 10 October 2023

Characterization device for P2 promoter in E.coli

Usage and Biology

Quorum Sensing System (QS System) is a way that certain kinds of bacteria use to detect its population and accordingly regulate its gene expression. S. aureus produces a signal molecule, autoinducing peptide (AIPs), during its growth. AIPs are received by a membrane receptor called AgrC. AgrC then transfers a phosphate group to AgrA, which activates a downstream promoter P2 to express the Agr operon (including AgrBDCA). Simultaneously, the other promoter P3 is activated to express a virulence gene, releasing toxins as a result. When the population of S. aureus reaches a threshold level, the concentration of AIPs raises and AgrC is activated, switching on the whole pathway.

Team:BNDS-China 2023

Design of the plasmid

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.

Figure 1.

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.

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.

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

@@ Line 8: / Line 8: @@
 ==Team:BNDS-China 2023==
-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.
+===Design of the plasmid===
+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.
+Figure 1.
 ===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.
+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.