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

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 or inducible, dependant of another genetic construction, single parts of genetic circuits such as oscillators, etc.

Basic Parts

All our basic parts are inserted in the plasmid BBa_P10500 and are compatible with BioBricks grammar, as they do not contain any illegal sites for RFC10. Moreover, they do not sites for the type IIS restriction enzymes BsaI and BsmBI, used in the Golden Braid assembly. 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 stregths, a strong promoter for T7 phage DNA polymerase, and a promoter for the sigma 32 subunit of E. coli DNA polymerase.

In addition, we have included 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.

RBS

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

CDS

As E. coli is our bacterial chassis, all these coding sequences have been codon optimized for this chassis organism.

We have chosen different coding sequences of pigmented proteins, both fluoroproteins (sfGFP, mRFP1, YFP and GFPmut3b) and chromoproteins (amilCP), as they are completely useful to design genetic constructions where there are necessary protein reporters of gene expression.

Moreover, another choice we have made is the addition of the LVA degradation tag to the mRFP1, YFP and GFPmut3b sequences, respectively. The LVA tag causes an increase in protease activity and, therefore, a faster degradation of the reporter protein in the cell. Thus, comparison between protein expression with and without the tag is a useful information to integrate in complex genetic oscillators design.

On the other hand, we have also selected the BSMT1 enzyme coding sequence, which will allow our bacteria to smell like mint in the presence of salicylic acid, to demonstrate odor gene expression.

Additionally, this collection also includes the transcriptional factors AraC and LuxR, that control the regulable promoters previously explained, as well as the CDS of LuxI, that acts producing the Acyl-Homoserine-Lactone to which LuxR responds. For this last CDS, we had to codon optimize its sequence for E. coli

Finally, we have picked a lysis gene from the enterobacteria phage phiX174, which allows us to create genetic circuits which functionality implies the death of part of the population.

Thus, all these coding sequences are a toolkit of useful basic pieces to supply the user with the necessary pieces to construct the basis of common bacterial genetic circuits.