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Revision as of 10:57, 2 October 2018

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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 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.

Part Description
BBa_K2656200 BioBricks compatible plasmid

CONTACT US igem.upv.2018@gmail.com