Cconboy:Terminator Characterization/Constructs
A series of terminator characterization constructs was built using BioBricks standard assembly. [1] Each construct encodes ECFP and EYFP on a polycistronic message, separated by the transcriptional terminator of interest. The ECFP and EYFP (E0021 and E0031) used in this study were derived from Clonetech expression vectors and include the native ribosome binding site. [2], [3], [4] The BioBricked wild-type lac promoter (R0010) drives expression of the construct. All constructs were built on pSB1A1 in DH5a. (Fig. 1). Characterization constructs were built for six terminators and their reverse counterparts (B0010-B0015 and B0020-B0025). A control construct lacking an internal terminator (I7003) was used to determine a baseline YFP to CFP fluorescence ratio. Output from this construct was assumed to represent zero percent termination efficiency. Additional constructs expressing ECFP only (I7000) and EYFP only (I7001) from the same promoter served as high-expression controls. (Table 1).
Fig.1: Schematic of terminator characterization construct.
| Construct | Terminator | Description | |
|---|---|---|---|
| -- | -- | Negative Control: DH5a only | |
| BBa_I7000 | -- | Positive Control: CFP only | |
| BBa_I7001 | -- | Positive Control: YFP only | |
| BBa_I7003 | -- | Postitive Control: CFP.YFP | |
| BBa_I7010 | B0010 | T1 terminator | |
| BBa_I7011 | B0011 | luxICDABEG operon terminator from V.fischeri | |
| BBa_I7012 | B0012 | TE terminator from T7 | |
| BBa_I7013 | B0013 | Variation on TE terminator from T7 | |
| BBa_I7014 | B0014 | Double terminator: B0012.B0011 | |
| BBa_I7015 | B0015 | Double terminator: B0010.B0012 | |
| BBa_I7021 | B0021 | Reverse terminator of B0011 | |
| BBa_I7022 | B0022 | Reverse terminator of B0012 | |
| BBa_I7023 | B0023 | Reverse terminator of B0013 | |
| BBa_I7025 | B0025 | Reverse terminator of B0015 |
