Difference between revisions of "Team:Valencia UPV/Part Collection"

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 forces, 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.

RBS

Regarding the RBS, we have selected 5 from the Registry of Standard Biological Parts, with different strengths.

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.