Team:BS United China/Contribution
Contribution
In order to prove our contribution to IGEM teams, we modified the part: BBa_K855008 and BBa_R0061 from the Hong Kong University team 2012 and Antiquity 2004. We added many details for them.
See detail in part BBa_K3882002 PVDQ has been known by us and the University of Hong Kong in 2012 as the degrading enzyme of AHL (the most important quorum sensing molecule of Gram-negative bacteria). But in the part we designed this year, we connected the PVDQ protein and eGFP protein with three HA protein linkers, and added two pairs of 6 his tags. Our design is more intuitive and easier to extract PVDQ protein. And we also used the T7 promoter to increase protein expression.
Our ultimate goal in designing this part is to apply the PVDQ protein to daily life. We also designed a plasmid that can detect the concentration of AHL in food. See details in BBa_K3882001 After connecting the two parts we designed, we can detect the growth of bacteria in fresh food and secrete PVDQ protein in time to prevent the flora from releasing toxic substances.
The experiments conducted by our team in the laboratory in 2021 proved:
1. PVDQ protein can inhibit the growth of bacteria (P. aeruginosa).It has been speculated that the weakening of PVDQ inhibitory effect after 9 hours is due to protein denaturation, decomposition and inactivation under a constant temperature culture environment of 37 degrees Celsius.
2. PVDQ-eGFP was successfully extracted by our non-denaturing elution method and can be stored at -40 degrees Celsius without inactivation.
3. IPTG can activate the T7 promoter to initiate the expression of PVDQ-GFP gene. As shown in the figure 1, the E. coli are producing the PVDQ-GFP. The growth of BL21 E. coli inserted into our plasmid is not significantly different from that of normal E. coli.
Fig.1 The PVDQ-eGFP produced by BBa_K3882002 induced by IPTG was observed under a fluorescence microscope.
4. PVDQ-eGFP has not been decomposed after 30 minutes of treatment in a simulated gastric acid environment. As shown in the figure 2, the PVDQ-GFP can resistant to the low pH environment. Thus, the fusion design of PVDQ-GFP parts can have a long time function in the reaction buffer.
Fig.2 The degradation of PVDQ-GFP (group1 is control group, while group2 is the group reacting with pH=0.61 acetic acid, and group3 is the group reacting with pH=2.14 hydrochloric acid).
5. PVDQ-GFP can not only prevent the growth of P. aeruginosa on the LB plate (Figure 3) but also can protect the shrimp and fish from the contamination of P. aeruginosa (Figure 4).
Fig.3 Statistic analysis of PVDQ-GFP in inhibiting the growth of P. aeruginosa.
Fig.4 The protective effect on the food storage using PVDQ-GFP.
Also see details in part BBa_K3882002. In our design, we also used LuxR regulatory protein to identify AHL in the environment. Through a series of experiments, we proved that LuxR protein and LuxR operon can correctly activate downstream genes.The LB media which contains AHL released from the P. aeruginosa can active the E. bsuahlscout and show visible red in the bottles (Figure 5).
Fig.5 The mCherry is expressed when E. bsuahlscout sensing the AHL in the environment.
Base on the new activator binding site and LuxR operator, our E. bsuahlscout can be more sensitive to the low AHL in the environment (Figure 6).
Fig.6 The standard curve of sensing the AHL in E. bsuahlscout.
Introduction
As an important quorum sensing molecule for Gram-negative bacteria, AHL has been studied by many IGEM teams. In this year’s laboratory work, we synthesized two plasmids and added them to BL21 expressing E. coli. The first plasmid allows our bacteria to respond to the AHL concentration in the environment and produce red fluorescent protein. The second plasmid allows our bacteria to produce PVDQ protein when the environmental AHL concentration reaches a certain level, and inhibits the production of toxins by P. aeruginosa. Through experiments, we prove that the promoter that senses the environmental concentration of AHL is effective, and it can correctly start the genes after it of the plasmid and translate to produce proteins.
Part1: E. bsuahlscout
Fig.7 Construction of E. bsuahlscout (Created with BioRender.com)
In the laboratory, we use IPTG to induce E. bsuahlscout engineered bacteria to produce red fluorescent protein to prove that our plasmid can function normally. In this way, we successfully expressed the red fluorescent protein and saw them under the fluorescence microscope.
Fig.8 E. bsuahlscout sequence
LuxR is the receptor protein gene of AHL. When it binds to AHL, it produces a signal peptide to activate the transcription of the downstream red mCherry fluorescent protein.
Fig.9 0.2um filter bottle bought from Fisher Scientific
We plan to put E. bsuahlscout and E. bsuahlterminator into real daily life use. To this end, we designed the device as shown in the picture above.
Part2: E. bsuahlterminator
Fig.10 Construction of E. bsuahlterminator (Created with BioRender.com)
Same as our experiment on E. bsuahlscout, we also induced the BL21 type E. coli with E. bsuahlterminator by IPTG in the laboratory to prove its normal function. We successfully obtained green fluorescent protein with PVDQ. Subsequent fluorescence microscopy observations and protein gel electrophoresis SDS-PAGE have proved this point.
Fig.11 Sequence of E. bsuahlterminator
When the AHL concentration in the environment is sufficient to start E. bsuahlscout, the E. bsuahlscout, the part 2: E. bsuahlterminator will continue to transcribe its downstream PVDQ and green fluorescent eGFP protein. Moreover, we designed them to have His tags, which can facilitate our extraction of them in the laboratory.