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 |
CDS
For CDS we have chosen different coding sequences of reporter proteins: fluoroproteins (sfGFP, mRFP1, YFP and GFPmut3b) and chromoproteins (amilCP). Another choice we have made is the addition of the LVA degradation tag to the mRFP1, YFP and GFPmut3b sequences. We have also selected the BSMT1 enzyme coding sequence that will allow our bacteria to smell of mint in the presence of salicylic acid. Additionally, we have added to the collection the CDS of the transcription factors AraC and LuxR that act on our regulable promoters, as well as the CDS of LuxI that acts producing the Acyl-Homoserine-Lactone to which LuxR responds. For this last CDS, we have codon optimized its sequence for E. Coli. Finally, we have picked a lysis gene from the enterobacteria phage phiX174 which allows us to create genetic circuits whose functionality implies the death of part of the population. All these coding sequences are codon optimized for E. coli.
| Part | Original BioBrick | Description |
|---|---|---|
| BBa_I746916 | BBa_K2656013 | Superfolder Green fluorescent Protein (sfGFP) coding sequence |
| BBa_K2656014 | BBa_E1010 | monomeric Red fluorescent protein (mRFP1) coding sequence |
| BBa_K592101 | BBa_I746916 | Yellow fluorescent protein (YFP) coding sequence |
| BBa_K2656022 | BBa_E0040 | Green fluorescent protein (GFPmut3b) coding sequence |
| BBa_K2656018 | BBa_K592009 | amilCP (Blue chromoprotein) coding sequence |
| BBa_K2656024 | BBa_K1399001 | mRFP1 with the LVA tag coding sequence |
| BBa_K2656020 | None | YFP with the LVA tag coding sequence |
| BBa_K2656023 | BBa_K1399004 | GFPmut3b with the LVA tag coding sequence |
| BBa_K2656025 | BBa_J45004 | BSMT1 coding sequence |
| BBa_K2656017 | BBa_K2442103 | AraC transcription factor coding sequence |
| BBa_K2656016 | BBa_C0062 | LuxR transcription factor coding sequence |
| BBa_K2656019 | BBa_C0161 | LuxI (Acyl-Homoserine-Lactone synthase) codon optimized coding sequence |
| BBa_K2656015 | None | Lysis gene from the enterobacteria phage phiX174 |
Transcriptional terminators
Lastly, as far as terminators are concerned, we have only chosen one, as we do not think that introducing several would have an influence on the functioning of Printeria. The terminator chosen is B0015 because it is the most used in E. Coli.
| Part | Original BioBrick | Description |
|---|---|---|
| BBa_K2656026 | BBa_B0015 | Double transcriptional terminator |
Composite Parts
In order to make the necessary measurements to characterize the basic parts and demonstrate that they are functional, we have built some composite parts by combining a promoter, an RBS, a CDS and the terminator in a Golden Braid Alpha 1 vector (link a Golden Braid 3.0) with the Golden Gate assembly protocol (link a protocolos).
Plasmids
Our transcriptional units assemblies are assembled using a Golden Braid 3.0 alpha1 plasmid which is not compatible with the BioBrick standard. Therefore, we have created the BBa_K2656200, adapted from the standard pSB1C3 backbone, to convert our Golden Braid transcriptional units to BioBricks.
BBa_K2656200 is a modified version of BBa_P10500. It includes BsmBI restriction sites flanking a RFP selection marker. When digesting this vector with BsmBI the result are complementary sticky ends with those formed in a transcriptional unit when digesting with BsmBI a Golden Braid alpha 1 vector. By this way, the BioBrick compatibility for each composite part is easily achieved using this plasmid as the destination vector in a Golden Gate reaction with the BsmBI endonuclease.