Team:Valencia UPV/Part Collection

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    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 transcriptional units of different types: constitutive, inducible, repressible, depending on another construction …

    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, 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 Description
    BBa_K2656004 J23106 Constitutive promoter
    BBa_K2656005 J23102 Constitutive promoter
    BBa_K2656007 J23101 Constitutive promoter
    BBa_K2656000 Strong promoter for T7 phage DNA polymerase
    BBa_K2656001 Promoter for the sigma 32 subunit of E. coli DNA polymerase
    BBa_K2656006 pBad minimal promoter: positively regulated by AraC in the presence of L-arabinose
    BBa_K2656002 Lux repressible promoter: negatively regulated by LuxR in the presence of Acyl-homoserine-lactone.
    BBa_K2656003 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 Description
    BBa_K2656008 RBS J61100
    BBa_K2656009 RBS B0030
    BBa_K2656010 RBS B0032
    BBa_K2656011 RBS B0034
    BBa_K2656012 RBS J61101

    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. 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 Description
    BBa_K2656013 superfolder Green fluorescent protein (sfGFP) coding sequence
    BBa_K2656014 monomeric Red fluorescent protein (mRFP1) coding sequence
    BBa_K2656021 Yellow fluorescent protein (YFP) coding sequence
    BBa_K2656022 Yellow fluorescent protein (GFPmut3b) coding sequence
    BBa_K2656018 amilCP (Blue chromoprotein) coding sequence
    BBa_K2656024 mRFP1 with the LVA tag coding sequence
    BBa_K2656023 GFPmut3b with the LVA tag coding sequence
    BBa_K2656020 YFP with the LVA tag coding sequence
    BBa_K2656025 BSMT1 coding sequence
    BBa_K2656017 AraC transcription factor coding sequence
    BBa_K2656016 LuxR transcription factor coding sequence
    BBa_K2656019 LuxI (Acyl-Homoserine-Lactone synthase) coding sequence

    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 Description
    BBa_K2656026 B0015 transcriptional terminator