Part:BBa_K1074006

Pgrac+RBS+SamyQ+TD1+GFP

This is the central gene circuit in our project to allow the high expression of various proteins (antigen, adjuvant, etc) which can transport through the intact skin.

Usage and Biology

In the circuit, promoter Pgrac is a strong regulated promoter induced by IPTG. SamyQ is the signal peptide directing the recombinant proteins out of the cytoplasm in Bacillus subtilis.TD1 can facilitate target proteins transporting through the intact skin.And the GFP,as a modular biobrick, can be substituted by various proteins via a modular PCR or standard cut/ligation method.

We have been trying to build a modular gene circuit just like PSB1C3, in which the GFP site fit the standard of four restriction sites. If so, any standard protein part can fused with TD1 and get the ability to transport through the intact skin just via standard cut/ligation method. Expression of secreted proteins also avoid the ordinarily complicated work of protein separation and purification.However, because the transdermal-enhancing activity of the TD1 was sequence specific, but standard restriction sites will produce unavoidable scars which will reduce the transdermal-enhancing activity of TD1. So it still remains a formidable task to build this modular gene circuit.

A modular gene circuit

In the experiment, we construct other four gene circuits based on this part. In these gene circuits, GFP is replaced by HBsAg, Ag85b, PAD4, LTB respectively. We transformed the recombinant Plasmids both in BL21 and Bacillus Subtilis WB800N and induced expression with IPTG . Results analyzed by SDS-page , mass spectrum and ELISA were proved positive. But proteins expressed in WB800N were far less than those in BL21. The antigenicity and transdermal function of N-terminal TD1 modified antigen HBsAg was also tested positive by ELISA.

figure 1 SDS-PAGE of N-terminal TD1 modified GFP

figure 2 SDS-PAGE of N-terminal TD1 modified LTB and HBsAg

figure 3 Fluorescence detection of N-terminal TD1 modified GFP expression with and without IPTG induction

figure 4 ELISA test transdermal function of N-terminal TD1 modified antigen HBsAg

References

SUN Zheng, WEN Long-ping.Transdermal delivery of fusion green fluorescent protein mediated by covalently associated TD1 peptide. OURNAL OF UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA.2009.Vol.39,No.4

Jung, J., et al., Improvement of surfactin production in Bacillus subtilis using synthetic wastewater by overexpression of specific extracellular signaling peptides, comX and phrC. Biotechnology and Bioengineering, 2012. 109(9): p. 2349-2356.

Ilk, N., et al., Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy. Microbial Cell Factories, 2011. 10. </div> Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 984