Part:BBa_K4609072
pET30a-LuxR-Lux pR-EphiX174p08-mCherry
This part is used to control the population density of Escherichia coli. When the population density of Escherichia coli reaches a certain level, AHL, which carries out signal communication between the populations, will enter the Escherichia coli cells, thus triggering the combination of LuxR and AHL to form the LUXR-AHL protein complex, which will cause the activation of LuxpR promoter. Induces transcription of the downstream lysing gene Ephix174p08 and the red fluorescence gene from phage. Ephix174p08 protein can cause E. coli cleavage to control its population density. The normal expression of the system was verified by detecting the expression effect of the red fluorescent gene.
The system mainly refer to the BBa_K4115039 (https://parts.igem.org/Part:BBa_K4115039), on the basis of the original, we introduced the phage cracking and red fluorescent genes, to control the effect of e. coli population density.
1. Design In order to control the population density of Escherichia coli and ensure that the metabolism of engineered bacteria can effectively perform its specific functions without over-reproduction, we designed quorum sensing with reference to BBa_K4115039, and inserted the LuxR and mCherry gene sequences successively into the downstream promoter of the plasmid. The composition of the plasmid includes pET30a-LuxR-LuxpR-mCherry. When LuxI expression produces a high concentration of AHL, AHL binds to LuxR to form a protein complex, which causes the activation of the LuxpR promoter and the transcription of the red fluorescent gene, thereby causing the cleavage of part of Escherichia coli to control its population density.
2. Build:
We sought the help of qualified companies for plasmid construction, and the relevant companies provided us with bacterial solutions with plasmids.
After extracting the plasmid, we selected BL21 as the protein expression system and used heat shock to transform the recombinant plasmid. Then the positive colonies were selected from kanamycin containing plates for amplification. They are sent for sequencing to verify conversion efficiency and avoid potential gaps or mismatch risks. The sequencing results are as follows:
The results show that our design matches perfectly with the extracted plasmids, indicating that we have theoretically achieved the goal of the project.
3.Test
We used this group of plasmids to initially test the growth curve of quorum sensing plasmids, compared with the growth curve containing only mcherry plasmids (E. coli). That is, when the OD600 value of the two tubularis solutions was 0.05, the two tubularis solutions were placed in a shaking table. In the experimental group, 10-8 molAHL inducer was added to measure the OD value of the two tube bacterial solution every 2.5 hours, and the results showed as follows. The bacterial solution of the experimental group began to decline when OD value reached about 0.76, as shown in the figure below. As time goes on, we can see that the OD value drops slightly, then decreases to a certain critical value and then continues to rise back to the peak.
4.Study
Due to the small reduction in OD value, the desired effect was not achieved, and the quorum sensing plasmid originally designed was not enough to meet our requirements for population density control, we decided to improve the plasmid structure. After searching various literatures, we decided to change the concentration of AHL inducer, set up three sets of experiments, and insert new effective lytic genes to enhance the quorum sensing effect.
5.Redesign
EphiX174p08 is a protein commonly associated with the genome of phage PhiX174. PhiX174, a single-stranded DNA phage, is one of the model systems for studying DNA replication and phage life cycle. EphiX174p08 has a cleavage effect, which can enhance the population density control effect of quorum sensing plasmid, and insert EphiX174p08 and gene sequences between LuxpR and mCherry, the downstream promoter of the plasmid. The composition of the plasmid includes pET30a-LuxR-Lux pR-EphiX174p08-mCherry. With the activation of the LuxpR promoter, the downstream cracking gene Ephix174p08 from phage is transcribed, thereby causing the cracking of part of Escherichia coli to control its population density.
6. Build:
We sought the help of qualified companies for plasmid construction, and the relevant companies provided us with bacterial solutions with plasmids.
After the transformation, it showed that the new plasmid sequencing results were successful, and we conducted a new round of experiments.
7.Test
We set up three experimental groups and one control group. 10-8, 10-7 and 10-10mol of AHL were added to the bacterial solution containing plasmid Ephix174p08 as the experimental group. The growth curves of the control group were the plasmids containing only red fluorescent protein (E. coli). The growth curves of the quorum sensing plasmids were tested again, respectively. When OD600 values of the four tubetube bacteria were all 0.05, they were placed in a shaker and OD values were measured every 2.5 hours. Both the peak and decreasing range of E. coli concentration increased significantly, and when the same amount of inducer (10-8mol) was added, the bacterial solution in our experimental group began to decrease significantly when OD reached about 0.72. This result is encouraging, which proves that our improved quorum sensing system is successful and has obvious effects.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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