Difference between revisions of "Part:BBa K4694005"
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===Characterisation=== | ===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. | 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. | ||
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| + | Unfortunately we were unable to express this enzyme in <i>L. plantarum</I>. We did see expression in <i>E. coli</I>, see Figures below, but we were unable to demonstrate activity. | ||
<h2> | <h2> | ||
<span class="mw-headline" id="Western Blot analysis">Western Blot analysis</span> | <span class="mw-headline" id="Western Blot analysis">Western Blot analysis</span> | ||
</h2> | </h2> | ||
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| + | <img src="https://static.igem.wiki/teams/4694/wiki/results-docs/western-blot-pj23100.jpg" width="49%"> | ||
| + | <img src="https://static.igem.wiki/teams/4694/wiki/results-docs/western-blot-t7.jpg" width="49%"> | ||
| + | <p> | ||
| + | (Left) Western blot showing protein expression of enzymes downstream of the strong constitutive promoter J23100. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a positive control (mCherry) whilst 4 is an E. coli ‘wild type’ of DH5a (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA | ||
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| + | (Right) Western blot showing protein expression of our enzymes downstream with the IPTG inducible ‘T7’ promoter. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a postive control (mCherry), whilst 4 is E. coli ‘wild type’ of BL21 (DE3) (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA). It seems like there is a lot of non-specific binding or protein overloading which has led to smearing on this western blot, making it harder to read. | ||
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Latest revision as of 15:38, 12 October 2023
His-LuxS
Usage and Biology
LuxS catalyses the formation of (S)-4,5-dihydroxy-2,3-pentanedione (DPD). DPD is the precursor of autoinducer 2 (AI-2). AI-2 is a signal molecule which is involved in inhibiting biofilm formation of Gram-positive bacteria. [1, 2]
This sequence is taken from L. plantarum WCFS1 (GenBank NC_004567). Forbidden restriction sites were removed, 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.
Unfortunately we were unable to express this enzyme in L. plantarum. We did see expression in E. coli, see Figures below, but we were unable to demonstrate activity.
Western Blot analysis
(Left) Western blot showing protein expression of enzymes downstream of the strong constitutive promoter J23100. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a positive control (mCherry) whilst 4 is an E. coli ‘wild type’ of DH5a (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA
(Right) Western blot showing protein expression of our enzymes downstream with the IPTG inducible ‘T7’ promoter. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a postive control (mCherry), whilst 4 is E. coli ‘wild type’ of BL21 (DE3) (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA). It seems like there is a lot of non-specific binding or protein overloading which has led to smearing on this western blot, making it harder to read.
References
[1] Jiang, L., Luo, Y., Cao, X., Liu, W., Song, G. & Zhang, Z. 2021. LuxS quorum sensing system mediating Lactobacillus plantarum probiotic characteristics. Arch. Microbiol., 203, 4141-4148..
[2] Man, L. L. & Xiang, D. J. 2021. LuxS-mediated quorum sensing system in Lactobacillus plantarum NMD-17 from koumiss: induction of plantaricin MX in co-cultivation with certain lactic acid bacteria. Folia Microbiol., 66, 855-871.
[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.