Difference between revisions of "Part:BBa K118022"

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		+	=='''Improved by NFLS 2020'''==
		+
		+	<p>Part improvement by NFLS 2020</p>
		+
		+	<p>Team NFLS improved the efficiency of this part by designing a new part called JTi1 consists of five repeats of 5′-GGGTTTCCCC-3′as NF-κB binding site and a minimal CMV promoter. The binding sites repeats is derived from gene vascular cell adhesion molecule 1, VCAM1. This new reconstructed promoter is more than twice as efficient as this original part.</p>
		+	<p>We added a Secreted Alkaline Phosphatase (SEAP) which commonly used as a reporter gene behind the promoter. The enzyme activity of SEAP reflects the efficacy of the promoter. </p>
		+	<p>The improved part is: [https://parts.igem.org/Part:BBa_K3513006''' BBa_K3513006''']</p>
		+
		+	[[File:T--NFLS--p1.jpg|600px|thumb|center|'''''']]
		+	<p>The standard curve: </p>
		+
		+	[[File:T--NFLS--p2.jpg|600px|thumb|center|'''''']]
		+
		+
		+
		+
		+	<p></p>
		+
		+
		+	<!-- -->
		+	<span class='h3bb'>Sequence and Features</span>
		+	<partinfo>BBa_K2976009 SequenceAndFeatures</partinfo>
		+
		+
		+	<!-- Uncomment this to enable Functional Parameter display
		+	===Functional Parameters===
		+	<partinfo>BBa_K2976009 parameters</partinfo>
		+	<!-- -->

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Revision as of 10:29, 9 December 2020

cex coding sequence encoding Cellulomonas fimi exoglucanase

The cellulolytic bacterium Cellulomonas fimi uses an exoglucanase (from cex, accession M15824) along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.

Sequence and Features

Contribution

• Group: [http://2018.igem.org/Team:UESTC-China iGEM Team UESTC-China 2018]
• Author: Liang Zhao, Yetao Zou
• Summary: Enzyme digestion and enzyme activity assay

Characterization from iGEM18-UESTC-China

Molecular weight

This gene codes for a protein of 485 amino acids with a molecular mass of 51.2 kDa.

Enzyme digestion

We did a codon optimization of this part before using it. And we verified it by enzyme digestion.

Filter paper assay

We constructed a plasmid containing cenA gene BBa_K118023, cex gene BBa_K118022. We transformed this plasmid into BL21(DE3). We used an intracellular fraction (crude enzyme solution) obtained by ultrasonication to carry out an experiment for measuring total enzyme activities by the method of filter paper assay.The result is shown on Fig. 2[1].

References

[1]Luciano Silveira MH, Rau M, Pinto da Silva Bon E & Andreaus J. 2012. A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper. Enzyme and Microbial Technology, 51: 280-285.

Contribution

• Group: iGEM Team CAU-China 2019
• Author: Liang siwen, Yan xueshan
• Summary: Enzyme activity assay (CMC-Na as substrate)

Characterization from iGEM19_CAU_China

We linked the cex gene BBa_K118022 into the pET30a(+) backbone, which contains lacI sequence so that the heterogeneous proteins can be induced by IPTG. The plasmids were transferred into BL21(DE3) strain and we induced the recombinant overnight under the condition of 16℃ 0.08 mM IPTG. The expression of the fusion protein was determined by SDS-PAGE (Figure 3).

Enzyme Activity Assay

The activity of Cex was detected by measuring the cellulose degradation ability using CMC-Na as the substrate. We employed the procedures used by UESTC-China yet under the condition of citric acid-sodium citrate buffer with pH 4.8 and 50 ℃ for reaction temperature. The cells were disrupted by ultrasonication and the suspension of the centrifugal cell contents was used as the crude enzyme. We measured the cellulose degradation abilities of the crude enzyme.(Figure 4)

Reference

[1] Z., Liu, X., Yi, L., Sun, et al.(2007) Analysis of the Current Situation of Biomass Waste Utilization in China. Environmental Science and Management, 32 (2): 104 - 106.

[2] M., Li, C., Lin, M., Li, et al. (2016)Ice-nucleation Protein and Its Application in Bacterial surface Display Technology. Amino Acids and Biological Resources, 38 (2): 7-11.

[3] van Bloois, E., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology, 29(2), 79-86.

Contribution

• Group: [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
• Author: Zinuo Huang, Jisheng Xie
• Summary: Quantitative Experiment of Exoglucanase Activity

Characterization from iGEM19 XMU-China

Molecular weight

The molecular weight of cex is 47 kDa^[1]. In Cex enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from Cex by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge^[2].

SDS-PAGE

This part was inserted into the expression vectors with T7 and RBS (BBa_K525998). Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

The constructed plasmid was transformed into E. coli BL21 (DE3). Positive clones that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely sup, was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely broken sup, was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2)

Fig. 1 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. cex: protein of BL21 (DE3) carrying T7-RBS-cex (linked by BBa_K525998 and BBa_K118022), target bands can be seen in cells at about 47 kDa; Control: protein of BL21 (DE3) carrying T7 and RBS (BBa_K525998).

Quantitative MUC Experiment

Methylumbelliferyl cellobioside (MUC) in the presence of Exoglucanase is broken down into methylumbelliferone and cellobiose. Methylumbelliferone fluoresces under long wave length (λ=366 nm) ultra-violet light. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant / crushed cell supernatant as reaction system. Add 200 μL MUC working solution (5×) into 800 μL LB Broth / PBS Buffer as background group. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction. Take out one tube of reaction system into boiling water bath for 8 minutes to stop the reaction after interval time since reaction started. Dilute reaction samples for 100 times and pipet 200 μL diluent into Black opaque 96-well plate, measure fluorescence (Excitation 364 nm, Emission 460 nm) with TECAN^® infinite M200 PRO. Using fluorescence intesity to determine the activity of Exoglucanase in test samples^[3]. Fig. 3 shows the results from the qualitative MUC assay.

Fig. 2 Assay for Quantitative Determination of cex Activity using MUC. (A) Supernatant and control. (B) Broken supernatant and control.

Assay for supernatant showed that no fluorescence intensity can be detected, which meant no enzymatic activity. But broken supernatant of culture with Cex protein can be detected fluorescence intensity.

Reference

1. ↑ L. E. Sandercock, A. Meinke, N. R. Gilkes, D. G. Kilburn, R. A. J. Warren, Degradation of cellulases in cultures of Cellulomonas fimi. FEMS Microbiology Letters 143, 7-12 (1996).
2. ↑ N. Gilkes, R. Warren, R. C. Miller, Jr., D. Kilburn, Precise excision of cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. The Journal of biological chemistry 263, 10401-10407 (1988).
3. ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).

Improved by NFLS 2020

Part improvement by NFLS 2020

Team NFLS improved the efficiency of this part by designing a new part called JTi1 consists of five repeats of 5′-GGGTTTCCCC-3′as NF-κB binding site and a minimal CMV promoter. The binding sites repeats is derived from gene vascular cell adhesion molecule 1, VCAM1. This new reconstructed promoter is more than twice as efficient as this original part.

We added a Secreted Alkaline Phosphatase (SEAP) which commonly used as a reporter gene behind the promoter. The enzyme activity of SEAP reflects the efficacy of the promoter.

The improved part is: BBa_K3513006

The standard curve:

Sequence and Features