Bac to Bac TOPO System

Creative BioMart provides an efficient method to optimize the recombinant donor vector preparation section. TOPO blunt-end clone technique, a strategy that enables the rapid direct insertion of blunt-end amplified PCR fragment, is used to replace the conventional TA subclone when inserting the interest gene into donor vector. TOPO clone technology can quickly complete the connection reaction in 2-5 minutes, while the traditional TA clone requires 2-3 days. In addition, the TOPO clone positive rate is close to 100%.

Figure 1. Bac-to-Bac TOPO expression approach

Components of Bac-to-Bac TOPO System

The Bac-to-Bac TOPO system still relies on Tn7 site-specific transposition between donor plasmid and Bacmid to generate recombinant baculovirus. It shares the same advantages (such as efficiency to generate recombinant baculovirus, accurate post-translational modification and protein folding) with Bac-to-Bac, while it costs shorter time on constuction and screening of recombinant donor vector,

pFastBac TOPO Vector, donor plasmid modified from pFastBac donor vector
Bacmid which contains Tn7 target site
E. coli DH10 which hosts Bacmid and contains a helper plasmid

Features of Bac-to-Bac TOPO Protein Expression Service

Enable the highly efficient blunt end subcloning reaction, thus allowing the use of proofreading polymerases in the PCR amplification.
Save 2 days from subcloning.
Save 2-4weeks from multiple rounds of plaque purification.
Easy and quick to identify and purify recombinant baculovirus.
Allow rapid and simultaneous isolation of multiple recombinant baculovirus

Service Details of Bac-to-Bac TOPO System

Here is the service process, which is very flexible. Customers can choose to start from or stop at any step they want. For more technical information or enquiry, please feel free to contact us.

Step		Service Description	Timeline	Deliverables
1	System Selection （Optional）	Select donor vector (pFastBacTM TOPO) Select host insect cell line (Sf9, Sf21, High Five)	——	——
2	Generate Recombinant pFastBac Vector	Sequence design and codon optimization Gene synthesis Subclone the interest sequence to the donor plasmid	1weeks	Enzymatic / Sequencing validation report.
3	Generate Recombinant Bacmid	Transforming DH10Bac E. coli Isolating and analyzing recombinant Bacmid DNA	2days	Recombinant DNA Analyze report
4	Recombinant Baculovirus Production	Transfect the insect cells with recombinant Bacmid Produce P1 Virus stock (low titer), P2 Virus stock (high titer) P1, P2 Western Blot and titer assay	2 weeks	Recombinant virus suspension WB report Titer test report
5	Small-Scale Expression & Purification	P2 virus transfect insect cell Protein purification SDS-PAGE, Western Blot	1.5 weeks	Target protein sample Quantitative test report
6	Optional Services	System optimization Secondary purification Tag removal Activity assay	1-2 weeks	Optimization solution Protein product Protein activity report
7	Large-Scale Expression & Purification (1-10L)	Culture amplification Western Blot Purification	1-3 weeks	Purified Protein QC report

Donor Vector and Insect Cell Line

In order to optimize the expression approach, TOPO donor vectors and insect with unique advantage are available at Creative BioMart.

Donor Vector

Donor Vector	Advantage	Use
pFastBac CT-TOPO	Offers strong polyhedrin promoter Has a C-terminal 6xHis tag for sample purification Has a TEV protease cleavage site for tag removal	Suitable for high-level protein expression Allow C-terminal intercellular polyhistidine fusion proteins
pFastBac NT-TOPO	Offers strong polyhedrin promoter Has an N-terminal 6xHis tag for sample purification Has a TEV protease cleavage site for tag removal	Suitable for secreted protein Allow N-terminal intercellular polyhistidine fusion proteins

Insect Cell Line for Bac-to-Bac TOPO

Insect Cell	Advantage	Use
Sf9 (Spodoptera frugiperda)	Well-characterized host cell, good for plaquing	All general types of recombinant baculovirus production and protein expression
Sf21 (Spodoptera frugiperda)	High yield of intracellular protein	Intracellular recombinant baculovirus production and protein expression

Creative BioMart has more than 10 years of experience in protein expression and is competent to satisfy various protein expression desire. We are expert in protein expression (intracellular or secretory), signal peptide selection, insect cell selection and culture. Choosing our comprehensive service, customers only need to provide the coding sequence of your target protein. We will provide customizes service from codon optimization to large-scale expression and purification.

Please note that all products/services provided are for research use only. Not intended for any clinical use.