Cconboy:Terminator Characterization/Constructs

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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.

Table 1: Terminator Characterization Constructs
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