Difference between revisions of "Part:BBa K118023:Experience"

Latest revision as of 10:50, 13 October 2022

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out. Cellulomonas fimi uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose. This part was codon optimized for the expression in Caulobacter crescentus and synthesized by IDT.

We improved the characterization of this part by optimizing the expression and attempted to express the enzyme on the surface layer of C.crescentus. We characterized the activity of the CenA insert in the C.crescentus s-layer. In initial activity analysis we saw no increased activity compared to wild type RsaA protein indicating that either the protein insert as we designed it does not fold correctly in the surface layer, further cloning attempts and design alterations must be made to try to improve the results.

It likely did not work as the folding of the protein may have been disrupted by the insertion into the surface layer; we would have to try different designs of the protein insertion by using different insertion sites or cloning in a altered, truncated, version of the protein.

Applications of BBa_K118023

Improvement by CAU_China

Group: [http://2019.igem.org/Team:CAU-China iGEM Team CAU-China 2019]
Author: Siwen Liang and Xueshan Yan

Overview

We improved this part by fusing with INP-N (see BBa_K3279008), the INP-N fused endoglucanase (INPN-CenA) can anchor in the cell membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE(Figure 2), and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay. We fused the INP with both CenA and Cex and conducted similar procedures.

Fig. 2 SDS-PAGE assay for fusion protein

Microscopy Observation

6His tag was added to the original protein CenA sequence as well as the fusion protein INPN-CenA as the antigen to be targeted by the primary antibody. Logically, since the CenA-6His is originally expressed in the interior of the cell, we would not detect the fluorescence in the sample of CenA and Cex, while the fluorescence is detectable for INPN-CenA-6His due to the cell membrane anchoring effect. We observed the E.coli cells expressing the original proteins (CenA and Cex)and the fusion proteins (INPN-CenA and INPN-Cex)under the fluorescence microscopy`s 20X objective (Figure 3) and confocal fluorescence microscopy`s 100X objective (Figure 4).

Fig. 3 E.coli cell immunofluorescence staining observation via fluorescence_microscopy 20x objective

Fig. 4 E.coli cell immunofluorescence staining observation via confocal fluorescence microscopy, 100x objective (images are local zoomed)

Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the unfused protein field are dimmer than those of the fusion protein field on average. To examine it more clearly, we observed the slices with the confocal fluorescence microscopy. The field of fusion protein samples showed that some foci are located on the borders of the cells, while this phenomenon was not observed in the field of the unfused protein samples. But due to the minuscule size of E.coli cells, our equipment falls short when trying to determine whether the fluorescent dot on a single cell is located on the outer membrane surface or not.

Fusion enzyme activity assay

The effect of fusion on enzyme activity 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. We measured the cellulose degradation abilities of the supernatant of disrupted cell contents as well as the undisrupted cell suspensions. According to the standard curve of glucose concentration, we determined the activities of unfused enzymes CenA and Cex, and fusion enzymes INPN-CenA and INPN-Cex.(Figure 5)

Fig. 5 Enzyme activities assay (pH4.8 50℃)

From the data, we summarized that the cellulases` activities were not affected remarkably with the presence of INP-N. Also, the difference of enzyme activities between the ultrasonic-disrupted samples and undisrupted samples provided another evidence of the anchoring effect of INP-N. Since the fusion protein is anchored in the outer membrane surface, which would appear in the sediments after centrifugation，the samples of suspension with fused cellulases showed the relatively low level of the activity, compared with samples of unfused ones.

Improvement UMA_MALAGA 2022

Group: UMA_MALAGA
Author: Molina Calvo, Alonso

Description

CenA encodes for the endoglucanase gene of Cellulomonas fimi (BBa_K118023). This enzyme is responsible of the degradation of cellulose working coordinated with the genes cex and bglX. In addition, this part includes the composition used by the team, which includes a strong rbs (BBa_B0030), a double terminator (BBa_B0015) as well as a promoter inducible by glucose concentration (BBa_K118011). Furthermore, we improve this composite adding a gen encoding a motor protein named YebF (BBa_K1610300) that secrete the enzyme out of the E. coli membrane. The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.

Enzyme digestion

Congo red assay

A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 μL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.

User Reviews

UNIQ79045d5f4ded689e-partinfo-00000005-QINU

BBa_K118023 Allan Crossman (Edinburgh 2011)

Edinburgh originally made this part in 2008. We came back to it in 2011 and found a double "mutation" from the Registry sequence. Upon checking with [http://www.ncbi.nlm.nih.gov/nuccore/NC_015514.1?from=3561504&to=3562853&report=genbank&strand=true a published genome sequence, NC_015514.1], we found that our "mutations" were in fact nothing of the sort, and must have been present all along. So the true sequence of K118023 is almost certainly that published under accession [http://www.ncbi.nlm.nih.gov/nuccore/NC_015514.1?from=3561504&to=3562853&report=genbank&strand=true NC_015514.1] (aside from the stop codons).

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BBa_K118023 Xueshan Yan (CAU_China 2019)

We fused this part with INP-N(see BBa_K3279008)and displayed the part on the surface of the E.coli cells. The immunofluorescence staining and DNSA method for activity assay were carried out to determine the presence of the fusion protein and anchoring effect on the enzyme activity. We found that the activity of the enzyme was not affected remarkably after fusion

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UNIQ79045d5f4ded689e-partinfo-00000008-QINU

@@ Line 22: / Line 22: @@
 ==== Microscopy Observation ====
-His tag was added to the original protein CenA sequence as well as the fusion protein INP-CenA as the antigen to be targeted by the primary antibody. Logically, since the CenA-6His is originally expressed in the interior of the cell, we would not detect the fluorescence in the sample of CenA and Cex, while the fluorescence is detectable for INP-CenA-6His due to the cell membrane anchoring effect. We observed the E.coli cells expressing the original proteins (CenA and Cex)and the fusion proteins (INP-CenA and INP-Cex)under the fluorescence microscopy`s 20X objective (Figure 3) and confocal fluorescence microscopy`s 100X objective (Figure 4).
+His tag was added to the original protein CenA sequence as well as the fusion protein INPN-CenA as the antigen to be targeted by the primary antibody. Logically, since the CenA-6His is originally expressed in the interior of the cell, we would not detect the fluorescence in the sample of CenA and Cex, while the fluorescence is detectable for INPN-CenA-6His due to the cell membrane anchoring effect. We observed the E.coli cells expressing the original proteins (CenA and Cex)and the fusion proteins (INPN-CenA and INPN-Cex)under the fluorescence microscopy`s 20X objective (Figure 3) and confocal fluorescence microscopy`s 100X objective (Figure 4).
 [[File:CAU-China_INP_fusion_protein_20x_fluorescence_microscopy.png|800px|thumb|center|'''Fig. 3'''  E.coli cell immunofluorescence staining observation via fluorescence_microscopy 20x objective]]
 [[File:CAU-China INP fusion protein 100x confocal fluorescence microscopy.png|750px|thumb|center|'''Fig. 4'''  E.coli cell immunofluorescence staining observation via confocal fluorescence microscopy, 100x objective (images are local zoomed)]]
@@ Line 28: / Line 28: @@
 ====Fusion enzyme activity assay ====
-The effect of fusion on enzyme activity 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. We measured the cellulose degradation abilities of the supernatant of disrupted cell contents as well as the undisrupted cell suspensions. According to the standard curve of glucose concentration, we determined the activities of unfused enzymes CenA and Cex, and fusion enzymes INP-CenA and INP-Cex.(Figure 5)
+The effect of fusion on enzyme activity 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. We measured the cellulose degradation abilities of the supernatant of disrupted cell contents as well as the undisrupted cell suspensions. According to the standard curve of glucose concentration, we determined the activities of unfused enzymes CenA and Cex, and fusion enzymes INPN-CenA and INPN-Cex.(Figure 5)
 [[File:CAU-China INP fused cellulases activity assays.png|500px|thumb|center|'''Fig. 5''' Enzyme activities assay (pH4.8 50℃)]]
 From the data, we summarized that the cellulases` activities were not affected remarkably with the presence of INP-N. Also, the difference of enzyme activities between the ultrasonic-disrupted samples and undisrupted samples provided another evidence of the anchoring effect of INP-N. Since the fusion protein is anchored in the outer membrane surface, which would appear in the sediments after centrifugation，the samples of suspension with fused cellulases showed the relatively low level of the activity, compared with samples of unfused ones.
+==Improvement UMA_MALAGA 2022==
+*'''Group:''' [https://2022.igem.wiki/uma-malaga/index.html UMA_MALAGA]
+*'''Author:''' Molina Calvo, Alonso
+==Description==
+''CenA'' encodes for the endoglucanase gene of ''Cellulomonas fimi'' (<partinfo>BBa_K118023</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with the genes ''cex'' and ''bglX''. In addition, this part includes the composition used by the team, which includes a strong rbs (<partinfo>BBa_B0030</partinfo>), a double terminator (<partinfo>BBa_B0015</partinfo>) as well as a promoter inducible by glucose concentration (<partinfo>BBA_K118011</partinfo>). '''Furthermore, we improve this composite adding a gen encoding a motor protein named ''YebF''''' (<partinfo>BBa_K1610300</partinfo>) that secrete the enzyme out of the ''E. coli'' membrane.
+The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.
+==Characterization==
+The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm).
+Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit.
+The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.
+===Enzyme digestion===
+[[File:cen.png|350px|center|]]
+===Congo red assay===
+A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 μL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.
+[[File:congo_red.png|350px|center|]]
 ===User Reviews===