Part:BBa_K2675081
Golden Gate adapter + sfGFP-LVAtag
This part is sfGFP-LVAtag (BBa_K2675006) 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) fused to LVA degradation tag (BBa_K2675006). sfGFP 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. LVA degradation tag (AANDENYALVA) is a ssrA tag that accelerates protein degradation in Escherichia coli at 37°C [3]. It is used in synthetic biology to rapidly degrade reporter genes and thus finely observe the synthetic gene networks dynamics.
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-LVAtag 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-LVAtag in BBa_K2675081.
In this BBa_K2675081, sfGFP-LVAtag is not preceded by an RBS nor by a promoter. Consequently, no sfGFP-LVAtag 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_K2675081 or BBa_K2675056. In BBa_K2675056 sfGFP-LVAtag 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-LVAtag by BBa_K2675056 and no sfGFP-LVAtag expression by this part BBa_K2675081.
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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