Part Collection
Introduction
Assembly methods using type IIS restriction enzymes (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, therefore increasing the efficiency of the assembly and reducing the time consumed. In addition, the Golden Gate technology allows to assemble more than two pieces and the backbone in a single reaction. These characteristics permit the automation of the Golden Gate assembly method, what suits Printeria perfectly.
Despite these advantages of the Golden Gate technology, finding collections of parts in the same standard optimized for E. coli, and well characterized, is very complicated. This is the reason why we decided to create our collection of basic parts. 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 GoldenBraid 3.0 standard . In addition, to give added value to this collection, we have made an exhaustive characterization of these parts.
In our collection, we have included different types of basic parts so that, combining them, we can create composite parts (transcriptional units) with different behaviours: constitutive, inducible, 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.
Promoters
With respect to promoters, we include three constitutive promoters of different forces, 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, positively and negatively regulated by LuxR in the presence of Acyl-homoserine-lactone, respectively.
| 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 five from the Registry of Standard Biological Parts, with different strengths.
| Part | Original BioBrick | Description |
|---|---|---|
| BBa_K2656008 | BBa_J61100 | Very weak RBS |
| BBa_K2656009 | BBa_B0030 | Strong RBS |
| BBa_K2656010 | BBa_B0032 | Weak RBS |
| BBa_K2656011 | BBa_B0034 | Medium RBS |
| BBa_K2656012 | BBa_J61101 | Very 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_K2656013 | BBa_I746916 | Superfolder Green fluorescent Protein (sfGFP) coding sequence |
| BBa_K2656014 | BBa_E1010 | monomeric Red fluorescent protein (mRFP1) coding sequence |
| BBa_K2656021 | BBa_K592101 | 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 GoldenBraid Alpha 1 vector with the Golden Gate assembly protocol .
Plasmids
Our transcriptional units were built using a GoldenBraid 3.0 alpha 1 vector which is not compatible with the BioBrick grammar. Therefore, we have created the BBa_K2656200 plasmid, adapted from the standard pSB1C3 backbone, to convert our Golden Braid transcriptional units to BioBricks.
BBa_K2656200 is a modified version of BBa_P10500, so it includes BsmBI restriction sites flanking a RFP selection marker. When digesting this plasmid with BsmBI the result is complementary sticky ends to those formed in a Golden Braid alpha 1 plasmid when digesting with the same enzyme.
By this way, the BioBrick compatibility for each composite part in our collection is easily achieved using this plasmid as the destination vector in a Golden Gate one-pot reaction with BsmBI.
| Part | Description |
|---|---|
| BBa_K2656200 | BioBricks compatible plasmid |