Part Collection
Introduction
Assembly methods using type IIS restriction enzymes (methods based on the Golden Gate technology) have a great advantage over those using Type II: The reaction is performed in a single step, without the need for gel band purification which decreases the efficiency of the assembly and is time consuming. In addition, with the Golden Gate technology it is possible to make assemblies with more than two pieces and the backbone in a single reaction. For these reasons, the Golden Gate technology is very well adapted to Printeria because its automation is less complicated than in other assembly methods.
It is because of these advantages that the Golden Gate technology is increasingly used. However, finding collections of parts in the same standard optimized for E. coli and well characterized is very complicated. That's why we decided to create our collection of basic parts: promoters, RBS, CDS and a terminator. This collection is based on some of the most used parts in E. coli from the Registry of Standard Biological Parts. To create this collection we used the Golden Braid 3.0 standard (link to that page). In addition, to give value to this collection, we have made a characterization of these parts.
In our collection we have represented different types of basic parts so that, combining them, we can create composite parts (transcriptional units) of different types: constitutive, inducible, repressible, depending on another construction, to implement a more complex circuit, ...
Basic Parts
All our basic parts are in the plasmid BBa_P10500 and are compatible with BioBricks as they do not contain any illegal sites for RFC10. They have not sites for the type IIS restriction enzymes BsaI and BsmBI. For information on how these parts have been designed, see our design page (link).
Promoters
With respect to promoters, we found three constitutive promoters of different strengths, a strong promoter for T7 phage DNA polymerase and a promoter for the sigma 32 subunit of E. coli DNA polymerase. In addition, in our collection there are three promoters regulated by a transcription factor: the inducible pBad, positively regulated by AraC in the presence of L-arabinose, and the inducible and repressible lux promoters, respectively positively and negatively regulated by LuxR in the presence of Acyl-homoserine-lactone.
| Part | Original BioBrick | Description |
|---|---|---|
| BBa_K2656004 | BBa_J23106 | Constitutive promoter |
| BBa_K2656005 | BBa_J23102 | Constitutive promoter |
| BBa_K2656007 | BBa_J23101 | Constitutive promoter |
| BBa_K2656000 | BBa_I719005 | Strong promoter for T7 phage DNA polymerase |
| BBa_K2656001 | BBa_K338001 | Promoter for the sigma 32 subunit of E. coli DNA polymerase |
| BBa_K2656006 | BBa_K2442101 | pBad minimal promoter: positively regulated by AraC in the presence of L-arabinose |
| BBa_K2656002 | BBa_R0061 | Lux repressible promoter: negatively regulated by LuxR in the presence of Acyl-homoserine-lactone. |
| BBa_K2656003 | BBa_R0062 | Lux inducible promoter: positively regulated by LuxR in the presence of Acyl-homoserine-lactone. |
RBS
Regarding the RBS, we have selected 5 from the Registry of Standard Biological Parts, with different strengths.
| Part | Original BioBrick | Description |
|---|---|---|
| BBa_K2656008 | BBa_J61100 | Weak RBS |
| BBa_K2656009 | BBa_B0030 | Strong RBS |
| BBa_K2656010 | BBa_B0032 | Weak RBS |
| BBa_K2656011 | BBa_B0034 | Strong RBS |
| BBa_K2656012 | BBa_J61101 | Weak RBS |