Difference between revisions of "Part:BBa K3279008"

Revision as of 06:47, 20 October 2019

cenA gene fused with INP-N sequence

This part is an improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can be displayed on the surface of outer membrane surface of E.coli cells.

Usage and Biology

We created this part by fusing CenA(BBa_K118023) with INP-N (BBa_K3279006), the INP-N fused endoglucanase (INP-CenA) can anchor in the cell membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE(Figure 1), and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay. (We fused the INP with both CenA and its twin part Cex(BBa_K118022) and conducted similar procedures.

Fig. 1 SDS-PAGE assay for fusion protein, lane 4 and 5

Microscopy Observation

6His 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 2) and confocal fluorescence microscopy`s 100X objective (Figure 3).

Fig. 2 E.coli cell immunofluorescence staining observation via fluorescence_microscopy 20x objective

Fig. 3 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 2018 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 4)

Fig. 4 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. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 333
Illegal BamHI site found at 733
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 75
Illegal NgoMIV site found at 408
Illegal AgeI site found at 1051
Illegal AgeI site found at 1414
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3279008 short</partinfo>
-This part is a improvement of the previous part cenA gene ( [https://parts.igem.org/Part:BBa_K118023 BBa_K118023]). With the fusion with INP-N, the endoglucanase coded by cenA gene can rivet on the surface and achieve E.coli surface display[1].
+This part is an improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can be displayed on the surface of outer membrane surface of E.coli cells.
 ===Usage and Biology===
-This part is a improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can rivete on the surface and achieve E.coli surface display.
-We linked this part into a pET30a(+) backbone, then transformed this plasmid into BL21. We induced this recombinant E.coli strain overnight under the condition of 16℃ 0.08 mM. See Fig.1.
-To confirm whether it worked or not, we first detected the presence of the target protein by immunofluorescence staining and compared the fluorescence pattern and compared it with a negative control where cellulases was not fused with INP. We attached the His-tag to the fusion protein, so that it could allow an anti-His-tag primary antibody to combine the fusion protein and then let a fluorescence secondary antibody recognize them. As the result, we could spot the fluorescence of GFP when INP-N was fused with cellulase. See Fig.2.
+We created this part by fusing CenA([https://parts.igem.org/Part:BBa_K118023 BBa_K118023]) with INP-N ([https://parts.igem.org/Part:BBa_K3279006 BBa_K3279006]), the INP-N fused endoglucanase (INP-CenA) can anchor in the cell membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE(Figure 1), and the anchoring effect is measured through immunofluorescence staining and enzyme activity assay. (We fused the INP with both CenA and its twin part Cex([https://parts.igem.org/Part:BBa_K118022 BBa_K118022]) and conducted similar procedures.
+[[File:CAU China INP fusion proteins SDS-PAGE.png|500px|thumb|center|'''Fig. 1''' SDS-PAGE assay for fusion protein, lane 4 and 5]]
-Then the effect of fusion on enzyme activity was detected by measuring the cellulose degradation ability using CMC-Na as substrate. See Fig.3.
+==== 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 2) and confocal fluorescence microscopy`s 100X objective (Figure 3).
+[[File:CAU-China_INP_fusion_protein_20x_fluorescence_microscopy.png|800px|thumb|center|'''Fig. 2'''  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. 3'''  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 2018 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 4)
+[[File:CAU-China INP fused cellulases activity assays.png|500px|thumb|center|'''Fig. 4''' 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.
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 <span class='h3bb'>Sequence and Features</span>