Difference between revisions of "Part:BBa K3453103"

Revision as of 16:55, 3 October 2020

Golden Gate (BbsI) adapter + sfGFP-LVAtag + SBa_000587 terminator

This part is sfGFP-LVAtag (BBa_K2675006) preceded by a Golden Gate adapter with with BbsI cloning sites (BBa_K2675070) and followed by the strong terminator SBa_000587 for the T7 RNA polymerase (BBa_K3453000).

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 K3453103.

In this BBa K3453103, sfGFP-LVAtag is not preceded by an RBS nor by a promoter. Consequently, no sfGFP-LVAtag expression was observed. This feature is very useful as an insertion marker for screening the right colonies during the cloning process.

In this BBa K3453103, sfGFP-LVAtag is followed by the strong synthetic transcriptional terminator SBa_000587 [5]. This terminator is recognised by the T7 RNA polymerase and should only be used in conjunction with a promoter recognised by T7 RNA polymerase too.

References

[1] Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One (2008) 3, e3647.

[2] Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One (2009) 4, e5553.

[3] Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol (2006) 24, 79-88.

[4] Purcell O, Grierson CS, Bernardo Md, Savery NJ. Temperature dependence of ssrA-tag mediated protein degradation. J Biol Eng (2012) 6, 10.

[5] Temme K, Hill R, Segall-Shapiro TH, Moser F, Voigt CA. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res (2012) 40: 8773-8781.

===Sequence and Features===