Part:BBa_K4853004

Transdermal delivery chaperone

This sequence encodes a chaperone that modifies proteins to allow them to permeate through the dermis.

Usage and Biology

To test the expression and functionality of our TD-1 sequence, we placed the gene in the same plasmid as nisA. Each construct contains our TD-1 sequence along with a combination of a WELQ site, linker sequence, nisA sequence, and/or truncated leader sequence. Through SDS-Page analysis, we confirmed the presence of TD-1 fused to NisA in our E. coli BL21(DE3) cells. As seen in Figure 1, a protein gel was completed using NisA and TD-1 with different components, including a V8 cleavage site, a truncated leader, and a linker sequence. Each construct had an extra band confirming the presence of the protein, indicated by the red asterisks in Figure 1.

Figure 1. Coomassie blue-stained 8-18% Tris-glycine SDS-Page analysis of TD-1-NisA constructs. Lane 1 contains whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TL-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. Lane 2 contains the whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TL-TD1-V8-nisA with no IPTG induction. Lane 3 contains the whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. Lane 4 contains the whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TD1-V8-nisA with no IPTG induction. Lane 5 contains whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TD1-linker-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. Lane 6 contains the whole cell lysate of E. coli BL21(DE3), pRSFDuet-1-TD1-linker-V8-nisA with no IPTG induction. Protein bands are indicated by the red asterisks in lanes 1, 3, and 5 at approximately molecular weights of 11, 12, and 12 kDa., respectively

Figure 2. Western blot of TD-1-NisA constructs. The fifth lane contains the whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-TL-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. The seventh lane contains whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. The ninth lane contains whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TD1-linker-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. The presence of the bands of interest is indicated by a red asterisk.

To confirm the presence of the TD-1, a Western blot was conducted using a whole cell lysate of E. coli BL21(DE3) containing pRSFDuet1- TL-TD1-V8-nisA, pRSFDuet-1- TD1-V8-nisA, and pRSFDuet-1- TD1-linker-V8-nisA. The positive control used was a Posi-Tag, which has several proteins with different epitopes, including His6. Our construct has a His6 tag. Protein bands were observed at a molecular weight of 11 kDa, 12 kDa, and 12 kDa, respectively, indicated by the red asterisks in Figure 1. While these are not the predicted molecular weight of 6.5 kDa, 7.3 kDa, and 7.9 kDa, bands at this molecular weight are consistent with previous data from Chen and Kuipers (2021).

References

Chen, J., & Kuipers, O. P. (2021). Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: Further Insights into Their Cooperative Action. mBio, 12(5), e02585-21. https://doi.org/10.1128/mBio.02585-21

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
  COMPATIBLE WITH RFC[1000]