Part:BBa_K1058009

Bla sensor with egfp(blaI+blaR1+PblaZ+egfp)

When there is no bla(beta-lactam), blaI will repress the blaZ promoter. When bla is present, it will be combined with blaR1 and active the depression of blaI, the PblaZ will be opened, then active the expression of dowmstream egfp.

Function

Beta-Lactam antibiotics have become less effective for the treatment of staphylococcal infections as a result of the bacteria's resistance to Beta-Lactam increases sharply during the past few years. Researches have shown that the resistance is mediated by beta-lactamase (encoded by blaZ) that hydrolyzes penicillin whose transcription is regulated by related regulators (encoded by blaI). The purified repressor(BlaI) of beta-lactamase production has been shown to bind specifically to two regions of dyad symmetry, known as operators, which are located between the divergently transcribed beta-lactamase structural gene(blaZ) and the gene(blaR1) encoding the putative transmembrane sensor protein.

The bla operon has been found that is induced by beta-lactam.



Hypothesis identified bla as a beta-lactam-sensing operon of beta-lactamase expression, so we designed two devices working in E.coli (DH5α) to build the beta-lactam biosensor.



This device will work to detect the concentration of Beta-Lactam in dairy products. At the same time, we designed another two devices to decrease the detection limit.

Data

We prepared a series of bera-lactam solution of which the concentration was respectively 0 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 5 μg/mL, 100 μg/mL and 200 μg/mL, then we add the solution to the bacteria liquid with BBa_K1058009 of which the OD is around 0.2~0.3 with the ratio of 1:1000 respectively, and the concentration of beta-lactam in the environment of the engineering E.coli in 8 different tubes is respectively 0 ng/mL, 10 ng/mL, 20 ng/mL, 30 ng/mL, 5 ng/mL, 100 ng/mL and 200 ng/mL. The samples were taken to two 96-well plates once per hour or once per 30 minutes. The intensity of green fluorescence was tested with a fluorescence microplate reader. The results are as follows.





We constructed the beta-lactam sensor BBa_K1058009 successfully. The characterization data of the beta-lactam sensor showed a great linear relationship between the fluorescence intensity and the concentration of the beta-lactams to be detected in a certain range (0-200ng/mL for the beta-lactam sensor).

Acknowedgment

[1] Leticia I. Llarrull, Marta Toth, Matthew M. Champion, and Shahriar Mobashery, J Biol Chem. 2011 November 4; 286(44): 38148–38158.

[2] Ana Amoroso, Julien Boudet, Stéphanie Berzigotti, Valérie Duval, Nathalie Teller, Dominique Mengin-Lecreulx, André Luxen, Jean-Pierre Simorre, and Bernard Joris1, PLoS Pathog. 2012 March; 8(3): e1002571.

[3] Martin K. Safo, Qixun Zhao, Tzu-Ping Ko, Faik N. Musayev, Howard Robinson, Neel Scarsdale, Andrew H.-J. Wang and Gordon L. Archer, J. Bacteriol. 2005, 187(5):1833.

[4] Clarke, S. R., and K. G. H. Dyke. 2001. J. Antimicrob. Chemother. 47:377–389 Sequence and Features

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]