Part:BBa_K1031624

J23114-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_K1031624 is composed of three elements, the constitutive Pc promoter J23114[1], coding sequence of NahF and terminator B0015[2]. (Fig 1)

Fig 1 Construction of adapter circuit J23114-NahF. The orange arrowhead refers to constitutive Pc promoter. RBS is shown in green oval. The sequare 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). The dashed box refers to data for Psal/NahR biosensor equipped with J23114-NahF adaptor (Fig 2b).

Fig 2 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. 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 represents the ON/OFF induction ratio. The dashed box refers to performance for J23114-NahF/NahR biosensor.