Difference between revisions of "Part:BBa K1031441"

Revision as of 16:23, 26 September 2013

J23106-HcaR

For detailed information concerning HcaR, please visit 2013 Peking iGEM Biosensor HcaR

Characterization

In order to fine-tune the performance of HcaR biosensor, we constructed a library of Pc constitutive promoters, collocating with RBS (Ribosome Binding Site) at different intensity. BBa_K1031441 is composed of three elements, the constitutive Pc promoter J23106[1], coding sequence of HcaR and terminator B0015[2]. (Fig 1). This circuit expressing HcaR is collocated with RBS B0034.

Fig 1 Construction of biosensor circuit Ph/J23106-HcaR. The orange arrowheads represent promoters, RBS is shown as a green oval, and the red circle stands for B0015 terminator.

Sequence and Features

Sequence and Features

Data shown

Tuning

Induction ratio of HcaR biosensor library test utilizes different Pc promoters, collocating with RBS (Ribosome Binding Site) at different intensity to control the expression of HcaR. The dashed box refers to data for Ph/J23106-HcaR biosensor circuit (Fig 2) in collocation with RBS B0034. This figure shows that when using B0034, the biosensor circuit adopting J23106 obtains the optimal performance.

Fig 2 Induction ratio of HcaR biosensor library test. Horizontal axis stands for different HcaR biosensor with Pc promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Partsregistry. Four kinds of aromatics shown with different color intensities, were tested following Test Protocol 1 [http://2013.igem.org/Team:Peking/Team/Notebook/Protocols]. Vertical axis represents the ON-OFF induction ratio. The dashed box refers to performance for Ph/J23106-HcaR biosensor circuit.

ON/OFF test

The best performed biosensor circuit Ph/J23106-HcaR is then subjected to the On-Off test about 78 aromatics according to protocol 1[1]. Results showed that HcaR worked as a specific sensor to PPA (Fig 4).

Fig 3 Results of On-Off test about biosensor HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ration over 2.

Dose-response curve

PPA was used to test dose-response curve of Ph/J23106-HcaR at the concentration at 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM (Fig 3)

Fig 3 Dose response curve of biosensor circuit Ph/J23106-HcaR induced by PPA. X-axis stands for concentration gradient of inducers at 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM. Y-axis shows induction ratio reflected via fluorescence intensity.