Part:BBa_K1162306
Antimicrobial spider silk generator with C-terminus 10x His-Tag
This part was created as an antimicrobial spider silk protein generator, composed of a promoter+rbs BBa_K208010, eight repeats of a spider silk protein BBa_844004, a 10x His-Tag BBa_K844000, followed by the standard double transcriptional terminator BBa_B0015.
Design
The LL-37 part, BBa_K1162006, was codon optimized for E. coli using the Life Technologies GeneArt ® software program. By design, this part is Assembly Standard #23 (Silver Fusion) so that all proteins are expressed in frame, and the LL-37 has a Methionine(atg) residue at the start of its coding region. Furthermore, this LL-37 and 8x spider silk protein sequences do not have any stop codons, which allows for the composite BioBrick construction. A 10x His-Tag BBa_K844000 was fused on the C-terminus of the 8x spider silk protein for purification purposes.
Experience
Figure 1. The image above of protein purification fractions demonstrates that there is expression of LL-37 and WAM-1 in E. coli through GFP fluorescence (GFP is at the C-terminal end). Unfortunately, there were issues with binding to the nickel affinity column, which will be troubleshooted in future work.
Figure 2. This image is an SDS-PAGE gel of the protein products from a 10L fermentation run for the LL37-spider silk generating construct using E. coli as the host organism. In the elution fraction of the gel image below, we can see a protein band at approximately 55-60 kDa, the size of the LL37-spider silk protein. From this study we have demonstrated that antimicrobial spider silk can be produced in E. coli and scaled-up. Since we also have additional AMP-spider silk constructs (see [http://2013.igem.org/Team:Utah_State/Parts BioBricks] page for a complete list), the next step would be to manufacture more of these using a similar approach to the LL37-spider silk. In future work, optimization of LL37-spider silk production will also take place.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| n/a | LL-37 antimicrobial peptide fused with 8x spider silk protein, with 10x His-Tag |