Part:BBa_K3882002
Elimination of AHL by E. bsuahlterminator
What it is? Why it is necessary? Microorganisms often persist in their natural niches by attaching to surfaces and forming complex, sessile microbial communities known as biofilms. P. aeruginosa is one of the most common food pathogens which is able to form biofilms either independently by itself or jointly with other microorganisms. When the concentration of quorum sensing (QS) molecule, AHL, secreted by pathogens in the environment is above the threshold, P. aeruginosa will be coordinated by QS signal to construct the biofilms. AHL is the key signaling molecular and can initiate the bio-toxin production. Thus, we need a quick methods to detect the accumulation of AHL in contaminated food.
What it does? Our E. bsuahlteminator bio-brick contains 2 key elements including pvdq and eGFP. PVDQ is a protein coded by pvdq gene. The pvdq gene is regulated by T7 promoter, Lac operator and T7 terminator. The PVDQ protein can catalyze the deacylation of acyl-homoserine lactone (AHL) which can prevent the activation of LUXR by eliminating the AHL in the environment. In order to increase the stability and show the production quality in a real time manner. We use a protein linker and 3x HA tag to link the PVDQ protein with eGFP. We also use 3xHis tag at the c-terminal of eGFP to increase the efficiency to purify the recombination proteins. Our results shows the great stability in solutions and almost no aggregation happens after elusion.
How to use it? E. bsuahlteminator bio-brick can transform to any competent bacterial. We use E. Coli BL21 as a recipe and safe bacterial to do the transformation. We use the LB media to culture the E. bsuahlteminator. When the OD value reach to 0.8, we add 200 uM IPTG into the LB media to activate the E. bsuahlteminator bio-brick inside at 30 degree centigrade for 4 h. After that, we use protein purification kit from Beyotime company, followed by detecting the concentration of PVDQGFP. The protein can be directly added into the LB media and spray on the surface of LB-agar plate as well as shrimp, meat. It has an inhibitory function on P. aeruginosa growth and can last for at least 5-8 hours.
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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