Part:BBa_K2675080
Golden Gate adapter + sfGFP
This part is sfGFP (BBa_K2675005) preceded by a Golden Gate adapter (BBa_K2675070) and followed by the strong terminator L3S2P56 (BBa_K2675030).
This part allows rapid engineering of reporter constructs using Golden Gate Assembly [1, 2].
Usage and Biology
The Golden Gate adapter contains two BbsI recognition sites in opposite direction (Figure 1).
The reporter gene is the superfolder GFP (sfGFP) (BBa_K2675005) which is a derivative of the Green Fluorescent Protein from Aequorea victoria with improved features in terms of intrinsic brightness, tolerance of circular permutation, resistance to chemical denaturants and folding kinetics at 37°C [3]. The protein contains 13 mutations compared to wild-type (Uniprot P42212) : S2R, S30R, Y39N, F64L, S65T, S72A, F99S, N105T, Y145F, M153T, V163A, I171V & A206V.
This part allows rapid engineering of reporter constructs using Golden Gate Assembly [1, 2] : a custom made Promoter + RBS sequence can be inserted upstream of sfGFP in one step (figure 1).
For this, the Promoter + RBS sequence must be flanked by BbsI restriction sites with appropriate cutting sites for this type IIS restriction enzyme (see figure 1 for details). These sites may be introduced either by PCR with the right set of primers or during the full fragment DNA synthesis.
Figure 1. Principle of the insertion of Promoters + RBS sequences upstream of sfGFP in BBa_K2675080.
In this BBa_K2675080, sfGFP is not preceded by an RBS nor by a promoter. Consequently, no sfGFP expression was observed (figure 2). This feature is very useful as an insertion marker for screening the right colonies during the cloning process.
Figure 2. Pictures of E. coli cells harbouring an empty pSB1C3 backbone, this part BBa_K2675080 or BBa_K2675066. In BBa_K2675066 sfGFP was equipped by a custom made RBS (BBa_K2675017) and placed under the control of the constitutive promoter (BBa_J23110). We can clearly see on the pictures a strong sfGFP by BBa_K2675066 and no sfGFP expression by this part BBa_K2675080.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 45
- 1000COMPATIBLE WITH RFC[1000]
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