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

Thus, we have included different types of basic parts so that, by combining them, we can create composite parts (transcriptional units) with different behaviors: 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 have 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.

Table 3. Our CDS Collection.

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.

Table 4. Our Transcriptional Terminator.

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 .

Printone

With Printeria, one of our main goals is to promote the Microbial Art among society. Thus, we decided to provide the bioartists with a complete ART DNA toolkit for its use with Printeria. As they use living cells as primary material for their artworks, we wanted to produce an extense palette: Printone.

Printone is composed of a colection of transcriptional units assembled with each of our fluorescent and coloured basic parts. By changing the promoters and RBS strengths, a wide range of different tonalities were obtained. Moreover, the LVA tag allowed us to reduce the brightness and so include degraded tones.

Image 1. Our Printeria colors: Some of the Printone transcriptional units, with variable expression levels of mRFP, YFP and amilCP proteins

Table 5. Our BioArt DNA toolkit palette: Printone.

Transcriptional unit	Intensity	CDS
BBa_K2656105	Medium: Low promoter, strong RBS	GFPmut3b
BBa_K2656106	Medium: low promoter, strong RBS	GFPmut3b
BBa_K2656107	Strong: Strong promoter, strong RBS	GFPmut3b
BBa_K2656117	Low: Low promoter, medium RBS	GFPmut3b
BBa_K2656108	Degradation tag: less colour intensity	GFPmut3b+LVA tag
BBa_K2656101	Medium: Low promoter, strong RBS	sfGFP
BBa_K2656100	Low: weak promoter, very weak RBS	sfGFP
BBa_K2656102	Low: weak promoter, weak RBS	sfGFP
BBa_K2656103	Medium: weak promoter, medium RBS	sfGFP
BBa_K2656104	Very low: weak promoter, very weak RBS	sfGFP
BBa_K2656109	Medium: Low promoter, strong RBS	mRFP1
BBa_K2656118	Low: Low promoter, weak RBS	mRFP1
BBa_K2656119	Medium: Low promoter, medium RBS	mRFP1
BBa_K2656110	Degradation tag: less colour intensity	mRFP1+LVA tag
BBa_K2656113	Medium: Low promoter, strong RBS	amilCP
BBa_K2656120	Medium: Low promoter, medium RBS	amilCP
BBa_K2656112	Medium: Low promoter, strong RBS	YFP
BBa_K2656111	Degradation tag: less colour intensity	YFP+LVA tag

Transcriptional units: complete table

This DNA Part Collection also includes non- constitutive transcriptional units and TU assemblies related with genetic circuits regulation, such as the HSP-GFPmut3b transcriptional unit, pLuxR-GFPmut3b transcriptional unit, LuxI constitutive transcriptional unit or AraC constitutive transcriptional unit.

Table 6. Our composite part complete collection

Composite Part	Promoter	RBS	CDS	Terminator	Description
BBa_K2656100	BBa_K2656004	BBa_K2656008	BBa_K2656013	BBa_K2656026	sfGFP transcriptional unit 1
BBa_K2656101	BBa_K2656004	BBa_K2656009	BBa_K2656013	BBa_K2656026	sfGFP transcriptional unit 2
BBa_K2656102	BBa_K2656004	BBa_K2656010	BBa_K2656013	BBa_K2656026	sfGFP transcriptional unit 3
BBa_K2656103	BBa_K2656004	BBa_K2656011	BBa_K2656013	BBa_K2656026	sfGFP transcriptional unit 4
BBa_K2656104	BBa_K2656004	BBa_K2656012	BBa_K2656013	BBa_K2656026	sfGFP transcriptional unit 5
BBa_K2656105	BBa_K2656004	BBa_K2656009	BBa_K2656022	BBa_K2656026	GFP transcriptional unit 1
BBa_K2656106	BBa_K2656005	BBa_K2656009	BBa_K2656022	BBa_K2656026	GFP transcriptional unit 2
BBa_K2656107	BBa_K2656007	BBa_K2656009	BBa_K2656022	BBa_K2656026	GFP transcriptional unit 3
BBa_K2656108	BBa_K2656004	BBa_K2656009	Bba_K2656023	BBa_K2656026	GFP_LVA transcriptional unit
BBa_K2656109	BBa_K2656004	BBa_K2656009	Bba_K2656014	BBa_K2656026	mRFP1 transcriptional unit 1
BBa_K2656110	BBa_K2656004	BBa_K2656009	Bba_K2656024	BBa_K2656026	mRFP1_LVA transcriptional unit 1
BBa_K2656111	BBa_K2656004	BBa_K2656009	BBa_K2656020	BBa_K2656026	YFP_LVA transcriptional unit 1
BBa_K2656112	BBa_K2656004	BBa_K2656009	BBa_K2656021	BBa_K2656026	YFP transcriptional unit 1
BBa_K2656113	BBa_K2656004	BBa_K2656009	BBa_K2656018	BBa_K2656026	amilCP transcriptional unit 1
BBa_K2656114	BBa_K2656004	BBa_K2656009	BBa_K2656016	BBa_K2656026	Constitutive LuxR transcriptional unit
BBa_K2656115	BBa_K2656001	BBa_K2656009	BBa_K2656022	BBa_K2656026	HSP-GFPmut3b transcriptional unit
BBa_K2656116	BBa_K2656002	BBa_K2656009	BBa_K2656022	BBa_K2656026	pLuxR-GFPmut3b transcriptional unit
BBa_K2656117	BBa_K2656004	BBa_K2656011	BBa_K2656022	BBa_K2656026	GFPmut3b transcriptional unit 4
BBa_K2656118	BBa_K2656004	BBa_K2656010	BBa_K2656014	BBa_K2656026	mRFP transcriptional unit 2
BBa_K2656119	BBa_K2656004	BBa_K2656011	BBa_K2656014	BBa_K2656026	mRFP transcriptional unit 3
BBa_K2656120	BBa_K2656004	BBa_K2656011	BBa_K2656018	BBa_K2656026	amilCP transcriptional unit 2
BBa_K2656121	BBa_K2656004	BBa_K2656011	BBa_K2656017	BBa_K2656026	AraC constitutive transcriptional unit
BBa_K2656122	BBa_K2656003	BBa_K2656011	BBa_K2656022	BBa_K2656026	pLux-GFPmut3b transcriptional unit
BBa_K2656123	BBa_K2656000	BBa_K2656010	BBa_K2656022	BBa_K2656026	pT7-GFPmut3b transcriptional unit
BBa_K2656124	BBa_K2656000	BBa_K2656011	BBa_K2656022	BBa_K2656026	pT7-GFPmut3b transcriptional unit 2
BBa_K2656125	BBa_K2656004	BBa_K2656009	BBa_K2656019	BBa_K2656026	LuxI constitutive transcriptional unit 1
BBa_K2656126	BBa_K2656004	BBa_K2656009	BBa_K2656025	BBa_K2656026	BMSTI transcriptional unit

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 GoldenBraid transcriptional units to BioBricks.

BBa_K2656200 is a modified version of BBa_P10500, so it includes BsmBI restriction sites flanking the selection marker. When digesting this plasmid with BsmBI, the result is complementary sticky ends to those formed in a GoldenBraid alpha 1 plasmid when digesting with the same enzyme. By this way, the BioBrick compatibility for each composite part is easily achieved using this plasmid as the destination vector in a Golden Gate BsmBI one-pot reaction.

Screening marker

Using the BBa_P1050 plasmid, selection of positive transformants relies on the white-blue screening method, which is based on the α-complementation phenomena. To do so, the host strain must carry the ω-peptide, while the vector contains the α-peptide. If both subunits are expressed (when there is not insertion in the vector), functional beta-galactosidase is reconstituted. Thus, blue-white screening is carry out by the addition of X-gal (D-lactose analog) and IPTG into the agar plates. In this way, if there is no insertion, colonies will be blue as a consequence of the X-gal hidrolysis. Although the principle is simple, this method requires the use of lacZ mutant strains and it is always necessary to add both reagents into the medium.

In order to avoid these limitations, we have decided to rely on a different visual method. Thus, we have inserted an mRFP1 transcriptional unit (BBa_K2656201) to act as the screening gene marker. By this way, cloning is achieved in a more reliable, efficient, low-cost way, so avoiding the necessity of adding chemical supplements and the possible non-function of these substances once on the solid medium. With this method, non-positive transformants will always express an intense red fluorescent protein, while non-recombinant transformants will not be able to express this protein.

Table 7. Our designed plasmids

Part	Description
BBa_K2656200	BioBricks compatible plasmid