Part:BBa_K4147004

DrsB1 Construct with LacI Promoter

Antimicrobial protein from the arboreal tree frog Phyllomedusa bicolor, dermaseptin-b1 gene (AKA MsrA2) with added parts as a single sequence to promote expression, being optimized for E. coli. Other than the original sequence for MsrA2 (BBa_K2577001), we added a LacI promoter (BBa_R0010), an RBS (BBa_B0032) and a double terminator (BBa_B0015).

Usage and Biology

Dermaseptins (DRSs) are peptides produced by Hylid frogs, a group of ɑ-helical shaped polycationic and short peptides (21-34 residues) containing a highly preserved tryptophan residue on N-terminal 3rd position, with hydrophobic residues and the polar cationic residues clusters in opposite sides [1]. The majority of the dermaseptin family peptides exhibit a wide variety of antibacterial activities. A broad range of microorganisms, including mollicutes, bacteria, fungi, protozoa, yeast, and enveloped viruses are all susceptible to the dermaseptins. Despite having a very similar sequence, the dermaseptins vary in how well they can neutralize different substances [2]. Dermaseptins also exhibit efficacy against various kinds of human cancer, making them anticancer peptides in addition to their antimicrobial capabilities.[1].

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]

Characterization of DrsB1 Construct with LacI Promoter

Before moving on to the experimental process, we first performed an in silico analysis in SnapGene®️ to simulate our ligated expression plasmid. The theoretical result was a sequence of 2,553 bp, as shown in Figure 1.

Figure 1. SnapGene®️ map of BioBrick BBa_K4147004.

Our insert of DrsB1 Construct with LacI Promoter was later synthetized by Twist Bioscience with the Biobrick prefix and suffix as well as adapters flanking the composite part for easiest restriction digest. EcoRI and PstI enzymes were used to digest both construct and vector. Once digested we proceed to ligate our insert into a pSB1C3 plasmid with Anza™ T4 DNA Ligase Master Mix. The ligation product was then transformed into E. coli BL21(DE3) cells by heat shock.

The next step was to confirm the presence of the vector of interest in our chassis after transformation, so we performed colony PCR using Forward: 5'-GTTTCTTCGAATTCGCGGCCGCTTCTA and Reverse: 5'-GTTTCTTCCTTCCTGCAGCGGCCGCTACTAG primers specific for the BioBrick prefix and suffix respectively. The PCR action from SnapGene®️ was used to predict the resultant agarose gel.

Figure 2.Left. SnapGene®️ amplification through PCR of BBa_K4147004 on an 0.8% agarose gel. Right A) NEB Quick-Load Purple 1Kb Plus DNA Ladder. B) Amplification of DrsB1 Construct with LacI Promoter at 50 °C as temperature of alignment where the highlighted band corresponds to approximately 540 bp as expected. C) Amplification of DrsB1 Construct with LacI Promoter at 55 °C as temperature of alignment where the highlighted band corresponds to approximately 540 bp as expected.

REFERENCES

[1] Bartels J. et al. Dermaseptins, Multifunctional Antimicrobial Peptides: A Review of Their Pharmacology, Effectivity, Mechanism of Action, and Possible Future Directions. [2] Nicolas, P., & el Amri, C. (2009). The dermaseptin superfamily: A gene-based combinatorial library of antimicrobial peptides. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1788(8), 1537–1550.