Description

To create a phage vaccine system for immunization study, we’ve developed and collected parts for a strong antigen gene expression in the engineered T7 bacteriophage-infected E. coli. First, we strengthened the T7 promoter-regulated expression system by characterizing an existing part (T7P-RBS-RFP-T7T/pSB1C3, BBa_K3431048) and improved for a better expression by adding a T7 g10 leader sequence in front of RBS (g10.RBS) (T7P-g10.RBS-RFP-T7T/pSB1C3, BBa_K4150002). Then, we replaced RFP with His-tagged GFP gene (T7P-g10.RBS-His.GFP-T7T/pSB1C3, BBa_K4150005), which was utilized as a reporter in engineering phage and measuring the kinetics of bacterial cell growth and gene expression levels. Finally, we put a model antigen, ovalbumin, gene with a N-terminal His tag in the same context (T7P-g10.RBS-His.Ova-T7T/pSB1C3, BBa_K4150006), which were engineered onto the T7 bacteriophage genome. The quantification and qualification of ovalbumin produced in the engineered phage-infected E. coli were verified by ELISA assay of the lysates and SDS-PAGE analysis of purified protein, respectively. For other antigen of interest as vaccine candidate, the collection will benefit to create a phage vector-based vaccine.

Chart

Diagram	Part No. & Name	Function or Characterization	Type
Existing Part
	BBa_K3431048 T7P-RBS-RFP-T7T/pSB1C3	RFP expression in IPTG-induced transformed E. coli in LB agar plate or broth	Composite
Improved Part
	BBa_K4150002 T7P-g10.RBS-RFP-T7T/pSB1C3	Enhaned RFP expression in IPTG-induced transformed E. coli in LB agar plate or broth	Composite
New Parts
	BBa_K4150000 g10.RBS/pSB1C3	Canonical RBS with a T7 g10 leader sequence	Basic: RBS
	BBa_K4150001 T7P-g10.RBS/pSB1C3	T7 promoter connected with g10.RBS	Composite
	BBa_K4150003 His.GFP/pSB1C3	His-tagged GFP reporter	Basic: Reporter
	BBa_K4150004 His.Ova/pSB1C3	His-tagged model antigen of chicken ovalbumin	Basic: Coding
	BBa_K4150005 T7P-g10.RBS-His.GFP-T7T/pSB1C3	GFP can be kinetically read at ex/em = 483/513 in the engineered phage-infected E. coli	Composite
	BBa_K4150006 T7P-g10.RBS-His.Ova-T7T/pSB1C3	Ova can be produced in the engineered phage-infected E. coli, which is quantified by ELISA assay and is qualified by purification through Nickel column.	Composite