Part:BBa_K1031622

J23106-NahF-Terminator

For detailed information concerning NahF, please visit 2013 Peking iGEM Adaptors

Characterization

In order to fine-tune the performance of NahF adapter for NahR biosensor, we constructed a library of Pc constitutive promoters at different intensity. BBa_K1031621 is composed of three elements, the constitutive Pc promoter J23106, coding sequence of NahF and terminator B0015. (Fig 1)

Fig 1 Construction of adaptor circuit J23106-NahF. The orange arrowhead refers to constitutive Pc promoter. RBS is shown in green oval. The square in dark red stands for terminator B0015.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 906
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1144
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1042
Illegal BsaI.rc site found at 1196

Data shown

It is necessary to fine-tune the expression level of NahF in E.coli. We built a library of constitutive promoters for tuning the expression of NahF, and NahR biosensor was used to detect the possible salicylates transformed from salicylaldehydes (Fig 2a). We adopted SaD and 5-ClSaD as inducers. The dashed box refers to data for Psal/NahR biosensor equipped with J23106-NahF adaptor (Fig 2b). From data it is shown that NahF adopting J23106 obtains the optimal performance, so this adaptor circuit is subjected to further tests.

Construction and test results of NahR biosensor equipped with NahF adaptor a. Schematic diagrams for the plasmid circuits used as Adaptor: NahF and the Sensor: NahR. A constitutive promoter library for the expression of NahF was constructed to obtain the most appropriate expression level of NahF enzyme in E.coli. The number of the Standard Biological constitutive promoter Parts used in this study and its initiation strength is listed in the left portion of the figure. Promoters are presented in orange, RBS in light green, coding sequence in dark cyan, and terminators in dark red.intensity b. Horizontal axis stands for NahR biosensor equipped with NahF adapters adopting Pc promoters of different strength. The expression intensity of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Part registry. Two kinds of aromatics, namely SaD and 5-ClSaD, shown with different color intensities, were tested following Test Protocol 1. Vertical axis stands for ON/OFF induction ratio calculated from fluorescence intensity. Dashed box refers to data for J23106-NahF/NahR biosensor circuit.

Dose response curves for salicylaldehydes of NahR equipped with Adaptor NahF were also obtained (Fig.3b). Comparing it with the dose-response curve for salicylaldehydes of NahR following the same test protocol (Fig.3b,c), we demonstrated that Adaptor NahF also functioned to lower the detection limit (the concentration of inducer at which an output three times the basal single is generated) of salicylaldehyde (from 300 μM to 1 μM) (Fig.3b) and 5-chloro-salicylaldehyde (from 300 μM to 3 μM) (Fig. 3c). NahR's dose response curves for corresponding acids processed by NahF were obtained as well, showing that adding Adaptor NahF does not significantly influence the original characteristics of the biosensor. (Fig.3d).

Fig.3 Data plots that demonstrate the performance of Apator NahF.
(a) Induction ratio of biosensor NahR and NahR equipped with Adaptor NahF elicited by salicylaldehyde at the concentration of 1 mM and 5-chloro-salicylaldehyde at the concentration of 0.1 mM. Biosensor NahR with Adaptor NahF showed higher induction ratio than NahR. (b) Dose-response curves of Biosensor NahR and NahR equipped with NahF for salicylaldehyde. Use of Adaptor NahF significantly reduced the detection limit by more than 100 folds. (c) Dose-response curves of Biosensor NahR and NahR equipped with NahF for 5-chloro-salicylaldehyde. (d) Dose-response curves of Biosensor NahR and NahR equipped with NahF for salicylate and 5-chloro-salicylate. The almost overlapping curves for the two compounds showed that the Adaptor NahF did not interfere the performance of NahR biosensor.