Team:ITB Indonesia/Part Collection

Introduction

To developed biosensor of Vibrio parahaemolyticus we used double plasmid expression system. Our plasmid was construct from various gene parts. Our plasmid was constructed using type IIS restriction enzymes (based on Golden Gate technology) and standard BioBricks assembly. Assembly methods using type IIS restriction enzymes have a great advantage such us increasing the efficiency of the assembly and reducing the time consumed. In addition, the Golden Gate technology allows to assemble more than two pieces and the backbone in a single reaction. These characteristics allows us to construct plasmid that consist of more than one structural gene.

As we have explained in our design page, we are use the combination of the Golden Gate and standard BioBricks assembly. To give added value to this collection, we have made an some characterization of these parts. Thus, we have included different types of basic parts so that, by combining them, we can create composite parts (transcriptional units) with different purpose.

Basic Parts

All our basic parts are inserted in the plasmid pSB1A3 as actuator module and pdCas9 as inverter module. For information on how these parts have been designed, see our design page.

Promoters

Our system contain three constitutive promoters of different strengths to express the structural gene. And pQrr4 short promoter as Lux cascade inducible promoter.

Table 1. Our promoter collections.

Part	Original BioBrick	Description
BBa_K3252021	p66	Constitutive promoter
BBa_K3252022	GabDP2	Constitutive promoter
BBa_K3252023	BBa_J23115	Constitutive promoter
BBa_K3252024	pQrr4 short promoter	Lux cascade inducible promoter

RBS

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

Table 2. Our RBS collections.

Part	Original BioBrick	Description
BBa_K3252025	BBa_B0034	Medium RBS (relative strength 0.38)
BBa_K3252026	BBa_B0032	Weak RBS (relative strength 0.047)
BBa_K3252027	BBa_B0033	Very weak RBS (relative strength 0.0035)

CDS

We have chosen different coding sequences of cascade Lux protein (LuxO, LuxU, and LuxN), fluoroproteins (GFPmut3b) as reporter gene, and dCas9 protein as inverter protein. They are used to design actuator module and inverter module.

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 collections.

Part	Original BioBrick	Description
BBa_K3252028	LuxO	LuxO protein coding sequence
BBa_K3252029	LuxU	LuxU protein coding sequence
BBa_K3252030	LuxN	LuxN protein coding sequence
BBa_K3252031	BBa_E0040	Green fluorescent protein (GFPmut3b) coding sequence
BBa_K3252032	dCas9	dCas9 protein coding sequence
BBa_K3252033	gRNA115	gRNA-J23115 coding sequence

Composite Parts

In order to make the necessary measurements to characterize the basic parts and demonstrate that they are functional, we have order composite parts by combining a promoter, an RBS, a CDS, and the terminator from IDT custom gene. To make functional composite parts to detect the present of V. parahaemolyticus we assemble that basic composite parts into functional composite parts (actuator module and inverter module).

Transcriptional units: complete table

This DNA Parts Collection also includes non-translational units for dCas9-gRNA complex inverter system.

Table 6. Our composite part complete collection

Composite Parts	Promoter	RBS	CDS	Terminator	Description
BBa_K3252036	BBa_K3252021	BBa_K3252025	BBa_K3252028	BBa_K3252034	LuxO transcription unit
BBa_K3252037	BBa_K3252021	BBa_K3252026	BBa_K3252029	BBa_K3252034	LuxU transcription unit
BBa_K3252038	BBa_K3252021	BBa_K3252027	BBa_K3252030	BBa_K3252034	LuxN transcription unit
BBa_K3252047	BBa_K3252024	None	BBa_K3252033	BBa_K3252035	gRNA transcription unit
BBa_K3252040	BBa_K3252022	BBa_K3252025	BBa_K3252032	BBa_K3252034	dCas9 transcription unit
BBa_K3252041	BBa_K3252023	BBa_K3252025	BBa_K3252031	BBa_K3252034	GFP transcription unit

Full system integration composite parts

Our full system integration composite parts contain translational and non-translational units to build actuator module and inverter module.

Table 7. Our full system integration composite part complete collection

Full Composite Parts	Composite Parts 1	Composite Parts 2	Composite Parts 3	Composite Parts 4	Description
BBa_K3252042	BBa_K3252036	BBa_K3252037	BBa_K3252038	BBa_K3252047	Actuator module
BBa_K3252043	BBa_K3252040	BBa_K3252041	None	None	Inverter module
BBa_K3252044	BBa_K3252042	BBa_K3252043	None	None	Full integration

Plasmids

Our transcriptional units were built by IDT custom gene which is compatible with the Golden Gate and BioBrick grammar. Therefore, we have created the BBa_K3252024 and BBa_K3252025 plasmid which is using pSB1A3 and pdCas9 backbone. By this way, the BioBrick and Golden Gate compatibility for each composite part is easily achieved using this plasmid as the destination vector.

Transformant screening

The transformant screening process when using the pSB1A3 and pdCas9 as backbond plasmid was rely on antibiotic resistant that carrying by that backbond plasmid. Positive transformants relies on the transformant ability to growth in antibiotic growth medium. PCR colony method was used in order to avoid miss ligation plasmid (see on result page). Thus, we have inserted LuxO transcriptional unit (BBa_K3252017), LuxU transcriptional unit (BBa_K3252018), LuxN transcriptional unit (BBa_K3252019), and gRNA transcriptional unit (BBa_K3252020) into pSB1A3 to act as the actuator module using combination Golden Gate and BioBricks assembly. GFP transcription unit was inserted into pdCas9 plasmid that has been contain the dCas9 transcription unit using BioBricks assembly. By this way, cloning is achieved in a more reliable, efficient, and low-cost way.

Table 8. Our designed plasmids

Part	Description
BBa_K3252024	Actuator plasmid
BBa_K3252025	Inverter plasmid