Difference between revisions of "Part:BBa K1162306"
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===Design=== | ===Design=== | ||
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+ | The LL-37 part, [https://parts.igem.org/Part:BBa_K1162006 BBa_K1162006], was codon optimized for <i>E. coli</i> using the Life Technologies GeneArt ® software program. By design, this part is [https://parts.igem.org/Assembly_standard_23 Assembly Standard #23 (Silver Fusion)] and contains a Methionine(atg) residue at the start of the LL-37 coding region. Furthermore, this LL-37 sequence does not have any stop codons, which allows for composite BioBrick construction. All coding regions are [https://parts.igem.org/Assembly_standard_23 Assembly Standard #23 (Silver Fusion)] so that all proteins are expressed in frame. | ||
===Experience=== | ===Experience=== |
Revision as of 03:23, 28 September 2013
Antimicrobial spider silk generator with C-terminus 10x His-Tag
LL-37 + 8x spider silk
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) and contains a Methionine(atg) residue at the start of the LL-37 coding region. Furthermore, this LL-37 sequence does not have any stop codons, which allows for composite BioBrick construction. All coding regions are Assembly Standard #23 (Silver Fusion) so that all proteins are expressed in frame.
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.
References
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]