Difference between revisions of "Part:BBa K4694006"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K4694006 short</partinfo> | <partinfo>BBa_K4694006 short</partinfo> | ||
| + | ===Usage and Biology=== | ||
| + | AHL-lactonase breaks down N-acyl homoserine lactones (AHLs) of Gram-negative bacteria into N-acyl-homoserine. | ||
| + | The amino acid sequence was taken <i>Bacillus thuringiensis serovar kurstaki</i> (29339) [1]. The DNA sequence was codon optimised using the IDT software for <i>L. lactis</i>, forbidden restriction sites removed, a <i>L. plantarum</i> 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. | ||
| β | + | For expression in <i>L. plantarum</i>, this CDS was inserted into plasmid pX1845 via Type IIS cloning. The plasmid has an <i>E. coli</i> origin of replication (pUC18) and antibiotic resistance gene (π½-lactamase) to allow for cloning in <i>E. coli</i> DH5πΌ, and an origin of replication and antibiotic resistance gene to allow for propagation in <i>L. plantarum</i>. Three constitutive promoters were tested: synthetic promoter P_48 [3], natural promoter from <i>L. plantarum</i> WCFS1 P_ldhL1 (GenBank NC_004567) and natural promoter from <i>L. lactis</i> 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<i> L. lactis</i> MG1363 pepN, called Lacto_term (GenBank AM406671). | |
| β | < | + | For expression in <i>E. coli</i>, this CDS was inserted into plasmid pX1900 via Type IIS cloning. The plasmid has an <i>E. coli</i> origin of replication (pBR322) and antibiotic resistance gene (π½-lactamase) to allow for cloning and propagation within <i>E. coli</i>. The strong constitutive promoter [https://parts.igem.org/Part:BBa_J23100 BBa_J23100] combined with the strong RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] were tested as well as the IPTG inducible T7 promoter (original sequence from pET21a) were tested. All constructs contained the double terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]. |
| β | === | + | |
| + | |||
| + | ===Characterisation=== | ||
| + | In order to characterise this part and determine whether the enzyme would be able to inhibit biofilm formation in our modified <i>L. plantarum</i> we performed a series of experiments. Please refer to the [https://2023.igem.wiki/exeter/experiments Experiments page] on our Wiki for the protocols. | ||
| + | |||
| + | <h2> | ||
| + | <span class="mw-headline" id="Western Blot analysis">Western Blot analysis</span> | ||
| + | </h2> | ||
| + | |||
| + | |||
| + | <h2> | ||
| + | <span class="mw-headline" id="Biofilm formation assay">Biofilm formation assay</span> | ||
| + | </h2> | ||
| + | |||
| + | |||
| + | <h2> | ||
| + | <span class="mw-headline" id="Enzyme activity assays">Enzyme activity assays</span> | ||
| + | </h2> | ||
| + | |||
| + | |||
| + | |||
| + | ===References=== | ||
| + | [1] iu, D., Momb, J., Thomas, P. W., Moulin, A., Petsko, G. A., Fast, W. & Ringe, D. 2008. Mechanism of the quorum-quenching lactonase (AiiA) from <i>Bacillus thuringiensis</i> 1. Product-bound structures. <i>Biochemistry</i>, 47, 7706-14. | ||
| + | |||
| + | [2] Ben-David, Y., Morais, S., Stern, J., Mizrahi, I. & Bayer, E. A. 2019. Cell-surface display of designer cellulosomes by <i>Lactobacillus plantarum</i>. <i>Methods Enzymol.</i>, 617, 241-263. | ||
| + | |||
| + | [3] Rud, I., Jensen, P. R., Naterstad, K. & Axelsson, L. 2006. A synthetic promoter library for constitutive gene expression in <i>Lactobacillus plantarum</i>. <i>Microbiol.</i>, 152, 1011-1019. | ||
| β | < | + | [4]Liu, W. B., Lin, Z. W., Zhou, Y. & Ye, B. C. 2021. Overexpression of Capsular Polysaccharide Biosynthesis Protein in <i>Lactobacillus plantarum</i> P1 to Enhance Capsular Polysaccharide Production for Di-n-butyl Phthalate Adsorption. <I>J. Microbiol. Biotechnol.</i>, 31, 1545-1551. |
| β | + | ||
| β | < | + | |
| + | [5] Tauer, C., Heinl, S., Egger, E., Heiss, S. & Grabherr, R. 2014. Tuning constitutive recombinant gene expression in <i>Lactobacillus plantarum</i>. <i>Microbiol. Cell. Fact.</i>, 13, 150. | ||
| β | <!-- | + | <!-- |
| β | === | + | <span class='h3bb'>Sequence and Features</span>--> |
| + | ===Sequence and Features=== | ||
<partinfo>BBa_K4694006 parameters</partinfo> | <partinfo>BBa_K4694006 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
Revision as of 12:03, 12 October 2023
His-AHL-Lactonase
Usage and Biology
AHL-lactonase breaks down N-acyl homoserine lactones (AHLs) of Gram-negative bacteria into N-acyl-homoserine.
The amino acid sequence was taken Bacillus thuringiensis serovar kurstaki (29339) [1]. The DNA sequence was codon optimised using the IDT software for L. lactis, forbidden restriction sites 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 (π½-lactamase) to allow for cloning in E. coli DH5πΌ, 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 (π½-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.
Western Blot analysis
Biofilm formation assay
Enzyme activity assays
References
[1] iu, D., Momb, J., Thomas, P. W., Moulin, A., Petsko, G. A., Fast, W. & Ringe, D. 2008. Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis 1. Product-bound structures. Biochemistry, 47, 7706-14.
[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.