Cconboy:Terminator Characterization/Constructs: Difference between revisions
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All constructs were built on pSB1A1 in DH5? using biobricks standard assembly. [http://biobricks.ai.mit.edu/Assembly/BB_Assembly.htm] Expression of a polycistronic message containing the coding regions of ECFP and EYFP, separated by a transcriptional terminator of interest, was produced from the wildtype lac promoter (R0010). The ECFP and EYFP (E0021 and E0031) used in this study were derived from Clonetech expression vectors [http://orders.clontech.com/clontech/techinfo/vectors_dis/pECFP.shtml], [http://orders.clontech.com/clontech/techinfo/vectors_dis/pEYFP.shtml], [https://dspace.mit.edu/bitstream/1721.1/21167/1/dist-1-00.pdf], and include the original Clontech RBSs. (Fig. 1). Characterization constructs were built for six terminators in the forward and reverse directions (B0010-B0015 and B0020-B0025, respectively). (Table 1). | |||
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 control constructs expressed ECFP only (I7000) or EYFP only (I7001) from the same promoter. | |||
Fig.1:Schematic of terminator characterization construct. | Fig.1:Schematic of terminator characterization construct. | ||
[[Image:TerminatorCharacterization.jpg|center|600px]] | [[Image:TerminatorCharacterization.jpg|center|600px]] | ||
{| border = "1" cellpadding="5" cellspacing="0" align="left" | {| border = "1" cellpadding="5" cellspacing="0" align="left" | ||
|+ '''Table 1: Terminator Characterization Constructs''' | |+ '''Table 1: Terminator Characterization Constructs''' | ||
Revision as of 11:21, 22 March 2006
All constructs were built on pSB1A1 in DH5? using biobricks standard assembly. [1] Expression of a polycistronic message containing the coding regions of ECFP and EYFP, separated by a transcriptional terminator of interest, was produced from the wildtype lac promoter (R0010). The ECFP and EYFP (E0021 and E0031) used in this study were derived from Clonetech expression vectors [2], [3], [4], and include the original Clontech RBSs. (Fig. 1). Characterization constructs were built for six terminators in the forward and reverse directions (B0010-B0015 and B0020-B0025, respectively). (Table 1).
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 control constructs expressed ECFP only (I7000) or EYFP only (I7001) from the same promoter.
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 | Variaction 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 |
