Part:BBa_K3882000
Sensing the AHL accumulation by E. bsuahlscout
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. bsuahlscout bio-brick contains 3 key elements including LuxR, QS promoter, and mCherry. LUXR is a protein coded by LuxR gene. The LuxR gene is regulated by T7 promoter, Lac operator and T7 terminator. The LUXR protein can combine the AHL to formate a LUXR-AHL complex which can bind to the QS promoter and recruit RNA polymerase to start the down-stream gene expression. QS promoter is a DNA element to react with LUXR-AHL complex. The mCherry gene is down-stream report gene of QS promoter and codes a red fluorescence protein.
How to use it? E. bsuahlscout 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. bsuahlscout. When the OD value reach to 0.2-0.4, we add 200 uM IPTG into the LB media to activate the E. bsuahlscout bio-brick inside at 30 degree centigrade for 4 h. After that, we make the AHL detecting kit and 1 cube is from LB-agar plate which contains 500 uM IPTG. The sample LB media which contains AHL will culture the activated E. bsuahlscout at 30 about 1-2 hours until the red color show up.
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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