Part:BBa_K4817015:Design

AidH homoserine lactonase

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

We linked the coding sequences of AidH and its mutants AidH A147G/AidH A147V to LacO/LacI (BBa_K1624002, BBa_K3257045) and pT7 (BBa_K4609008). IPTG was used to induce protein expression, simulating quorum sensing-induced protein expression to verify the function of the AidH and AidH A147G/AidH A147V. LacO/LacI are commonly found in the pET series plasmids. IPTG (isopropyl β-D-1-thiogalactopyranoside) is a molecular analogue of allolactose and has the same function as allolactose. Both can act as inducers and bind to the repressor in the Lac operon, thereby preventing LacI from binding to LacO upstream of pT7 and ultimately initiating the expression of AidH.

AidH crude extract, AidH A147G crude extract, AidH A147V crude extract were collected and used to treat E.coli DH5α(with p15A-lux-sfGFP). Without AidH, the LuxR secreted by J23100 in plasmid p15A (BBa_C0062) interacts with AHLs and initiates LuxP, expressing the green fluorescent protein sfGFP to emit fluorescence. When AidH exists, AidH degrades AHLs, LuxP cannot turn on the expression of sfGFP, and the green fluorescence weakens.

Figure.1 AidH characterization gene circuit

Figure.2 AidH function verification circuit

Source

Ochrobactrum

References

[1] Mei GY, Yan XX, Turak A, Luo ZQ, Zhang LQ. AidH, an alpha/beta-hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase. Appl Environ Microbiol. 2010;76(15):4933-4942.
[2] Zhang Y, Wei W, Wen H, Cheng Z, Mi Z, Zhang J, Liu X, Fan X. Targeting Multidrug-Recalcitrant Pseudomonas aeruginosa Biofilms: Combined-Enzyme Treatment Enhances Antibiotic Efficacy. Antimicrob Agents Chemother. 2023 Jan 24;67(1):e0135822.
[3] Coffey, B.M., Anderson, G.G. (2014). Biofilm Formation in the 96-Well Microtiter Plate. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY.