Part:BBa_K4694003

Sp-His-QQ-7

Usage and Biology

The enzyme QQ7 was proposed to be a quorum quenching enzyme which therefore inhibits biofilm formation of pathogens such as Candida albicans and Staphylococcus epidermidis [1]. The DNA sequence was identified during a metagenomic screen of bacteria that quench biofilm formation.

This sequence is taken from GenBank JX870909. Forbidden restriction sites were removed, a L. plantarum signal peptide (Lp_3050, [2]) and a 6xHis_tag flanked by GS linkers was added to the N-terminal, prefix and suffix sequences compatible with Type IIS cloning were added, and the sequence was synthesised by IDT.

For expression in L. plantarum, this CDS was inserted into plasmid pX1845 via Type IIS cloning. The plasmid has an E. coli origin of replication (pUC18) and antibiotic resistance gene (𝛽-lactamase) to allow for cloning in E. coli DH5𝛼, and an origin of replication and antibiotic resistance gene to allow for propagation in L. plantarum. Three constitutive promoters were tested: synthetic promoter P_48 [3], natural promoter from L. plantarum WCFS1 P_ldhL1 (GenBank NC_004567) and natural promoter from L. lactis P_32 [4]. The latter two promoters had integrated RBS sequences but P_48 was combined with the synthetic RBS SDOPT8 [5]. All constructs contained a terminator from L. lactis MG1363 pepN, called Lacto_term (GenBank AM406671).

For expression in E. coli, this CDS was inserted into plasmid pX1900 via Type IIS cloning. The plasmid has an E. coli origin of replication (pBR322) and antibiotic resistance gene (𝛽-lactamase) to allow for cloning and propagation within E. coli. The strong constitutive promoter BBa_J23100 combined with the strong RBS BBa_B0034 were tested as well as the IPTG inducible T7 promoter (original sequence from pET21a) were tested. All constructs contained the double terminator BBa_B0015.

Characterisation

In order to characterise this part and determine whether the enzyme would be able to inhibit biofilm formation in our modified L. plantarum we performed a series of experiments. Please refer to the Experiments page on our Wiki for the protocols.

Enzyme activity assays

References

[1] Weiland-Brauer, N., Malek, I. & Schmitz, R. A. 2019. Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis. PLoS One, 14, e0211366.

[2] Ben-David, Y., Morais, S., Stern, J., Mizrahi, I. & Bayer, E. A. 2019. Cell-surface display of designer cellulosomes by Lactobacillus plantarum. Methods Enzymol., 617, 241-263.

[3] Rud, I., Jensen, P. R., Naterstad, K. & Axelsson, L. 2006. A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiol., 152, 1011-1019.

[4]Liu, W. B., Lin, Z. W., Zhou, Y. & Ye, B. C. 2021. Overexpression of Capsular Polysaccharide Biosynthesis Protein in Lactobacillus plantarum P1 to Enhance Capsular Polysaccharide Production for Di-n-butyl Phthalate Adsorption. J. Microbiol. Biotechnol., 31, 1545-1551.

[5] Tauer, C., Heinl, S., Egger, E., Heiss, S. & Grabherr, R. 2014. Tuning constitutive recombinant gene expression in Lactobacillus plantarum. Microbiol. Cell. Fact., 13, 150.

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]