Part:BBa_K1031922

J23114-XylB-Terminator

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

Characterization

We achieved the concept of Adaptor by connecting XylC with XylS, and we want to further confirm whether other degradation enzymes could be added. So we construct XylB adaptor circuit adopting J23114 as constitutive promoter and B0015 as terminator (Fig 1)

Fig 1 Construction of adaptor circuit J23114-XylB. The orange arrowhead refers to constitutive Pc promoter. RBS(Ribosome Binding Site) 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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 364

Data shown

A set of culturing conditions were tried respectively to optimize the performance of K1031922. They were listed in Table 1, marked in red.

For part K1031922 adopting constitutive Pc promoter J23114, the second and third protocol both works. Induction ratio is shown in Fig 2when exposed to BAL and 2-MeBAL. The dashed boxes refers to the performance of XylS Biosensor equipped with XylC and XylB Adptors adopting J23114.

Fig 2 Performance of different XylB Adptor with different culture condition. Final concentrations of inducer were 300 uM. Abbreviations used in this figure: BAL: benzyl alcohol; 2-MeBAL: 2-methylbenzyl alcohol; Coculture: M9 minimal media containing the aromatics mentioned above was processed by E. coli transformed with Adaptor XylB and E. coli transformed with XylC simultaneously; Double Trans.: double transformation. Dashed boxes refers to performance of XylS Biosensor equipped with Adaptor XylC and K1031922.

Dose-response Curves were plotted to further describe response performance when exposed to four inducers respectively at different concentrations. Each strain is marked in different colors. The curves in red and purple represents Biosensor equipped with K1031922 in different culture conditions respectively.

Fig 3 Characterization and comparison of biosensor XylS with or without Adaptor XylB and XylC. Final concentrations of inducers were 0, 0.3, 1, 3, 10, 30, 100, 300 uM in the characterization assay. (a) Dose-response curve for benzyl alcohol (BAL); (b) Dose-response curve for 2-methylbenzyl alcohol (2-MeBAL); (c) Dose-response curve for benzoate (BzO); (d) Dose-response curve for benzoate (2-MeBzO).