Parts

Features

- Function: encodes BLIP-I/BLIP-II, Beta-Lactamase Inhibitory Protein, with high potency against multiple ambler class A Beta-Lactamases.
- Design Optimization: Codon-Optimized for E.coli to maximize protein yield.
- Binding Properties: Maintains tight binding with small interaction face.
- Assembly Compatibility: Biobrick RFC[10], [1000]
- Applications: Designed for Beta-Lactamase inhibition assays, antibiotic resistance research, and antimicrobial resistance combat strategies.
- Safety Note: Non-toxic, non-virulent; qualifies under iGEM White List criteria for safe parts.

As the problem mentioned in design, our BLIP sequence was obtained from the National Institutes of Health (NIH)[1], but we could not get the protein sequence provided and its gene sequence to match. Since other sources relied on the same flawed sequence, we chose instead to reverse translate the verified protein sequence. This approach ensures more reliable structural and functional annotation.

For BLIP-II we again found mismatches between the provided gene sequence and the reverse-translated protein sequence. For consistency and reliability, we chose to use the protein sequence for reverse translation, as we did with BLIP-I.

Both BLIP-I and BLIP-II were reverse translated using Benchling and SnapGene. During the incipient stages of codon optimization we used the screen sequence function from Integrated DNA Technology (IDT) and its output of complexity number as index. And then we compared the numbers from SnapGene and Benchling, using the one with the lower complexity score to start optimizing with. The majority of codon was optimized with Benchling, and the rest were hand adjusted according to the codon table. For BLIP-I we were able to drop the complexity score from 78.1 to 1.7. But with BLIP-II the least we could manage to get to was 6.8. During the process of ordering the gene sequence we encountered some obstacles in getting authentication from IDT, so we went with Twist BioScience and Genescript instead. Twist was only capable of producing BLIP-I, so for BLIP-II we had to rely on Genescript.

Figure 1. BLIP-I Insertion Biobrick Diagram
Figure 2. BLIP-II Insertion Biobrick Diagram

The composite part used in experiments had two main coding sites embedded in the plasmid. The BLIP and the fluorescent protein mCherry. Initially mCherry was designed for validation purposes. Later in the project, our focus for validation shifted from confirming successful conjugation to testing the efficacy of BLIP, making the mCherry reporter unnecessary at that stage. However, as the wet lab encountered several experimental challenges, our validation focus shifted once more—this time toward confirming the successful transformation of BLIP into E. coli S17-1. Under this new goal, the mCherry reporter was reintroduced as a means of validation.

References:
Kang, S. G., Park, H. U., Lee, H. S., Kim, H. T., and Lee, K. J. Streptomyces exfoliatus beta-lactamase inhibitory protein BLIP-I precursor (bliA) gene, complete cds. Accession no. AF201389.1. National Center for Biotechnology Information. Available at: https://www.ncbi.nlm.nih.gov/nuccore/AF201389?report=genbank (accessed 2025-10-02).