Difference between revisions of "Part:BBa K2922007"
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===Usage===
 
===Usage===
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In order to let yebF help secrete our cellulase out of the ''E. coli'' membrane, we fused the cellulase gene fragment with yebF gene fragment at the N-terminal by Overlap Extension Polymerase Chain Reaction (OE-PCR), and inserted a flexible GS Linker (GGGGS). PCR product was identified by agarose gel electrophoresis (Fig.1).
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In order to secrete our cellulases out of the ''E. coli'' membrane, we fused the cellulase gene fragments with ''yebF'' gene fragment at the N-terminal using a flexible GS linker (GGGGS) by overlap extension polymerase chain reaction (OE-PCR). PCR product was purified by agarose gel electrophoresis (Fig. 1).  
  
 
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:'''Fig.1''' Agarose Gel Electrophoresis of ''yebF-cenA'' OE-PCR product. Lane M: Marker.  
 
:'''Fig.1''' Agarose Gel Electrophoresis of ''yebF-cenA'' OE-PCR product. Lane M: Marker.  
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===Characterization===
 
===Characterization===
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These parts were insert into the expression vectors with T7 and RBS (<partinfo>BBa_K525998</partinfo>) by restriction sites ''Eco''RI and ''Pst''I. Then transformed the expression vectors into ''E. coli'' DH5α, and the correct construction of this recombinant plasmid was confirmed by chloramphenicol, colony PCR and plasmid sequencing.
+
These parts were insert into the expression vectors with T7 and RBS (<partinfo>BBa_K525998</partinfo>) by restriction sites ''Eco''RI and ''Pst''I. Then the ligation mixture was transformed into ''E. coli'' DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.
  
 
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'''1. SDS-PAGE'''
 
'''1. SDS-PAGE'''
  
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We transformed the constructed plasmid into ''E. coli'' BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced to express by IPTG. The supernatant of culture medium was obtained by centrifugation. And we gain the total protein by ultrasonic crushing. The lysate was then centrifuged and the supernatant was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2).
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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)
  
 
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:'''Fig.2''' SDS-PAGE analysis of  ''E. coli'' BL21 (DE3) by Sliver staining. YebF-cenA: protein of ''E. coli'' BL21 (DE3) carrying T7-RBS-yebF-cenA (<partinfo>BBa_K2922007</partinfo>), target bands can be seen in the medium at 57.5 kDa; Control: protein of ''E. coli'' BL21 (DE3) carrying T7 and RBS (<partinfo>BBa_K525998</partinfo>).
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:'''Fig.2''' SDS-PAGE analysis of  ''E. coli'' BL21 (DE3) by Sliver staining. YebF-cenA: protein of ''E. coli'' BL21 (DE3) carrying T7-RBS-yebF-cenA (<partinfo>BBa_K2922007</partinfo>), target bands can be seen in the medium at 60 kDa; Control: protein of ''E. coli'' BL21 (DE3) carrying T7 and RBS (<partinfo>BBa_K525998</partinfo>).
  
  
 
'''2. Congo Red Assay'''
 
'''2. Congo Red Assay'''
  
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We used Congo Red assay to verify the enzyme activity of YebF-CenA, and this method is from [http://2018.igem.org/Team:UESTC-China iGEM18-UESTC-China]. Luria agar plates with 0.2% CMC are addd with the crude enzyme. Then the agar is flooded with 1 mg/mL Congo Red solution for 1 h. Congo Red solution is poured off into a toxic waste bottle and 1 M NaCl is added and left for another 1 h. Then pour off NaCl solution. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation<ref>S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation.  (2012).</ref>. The results are shown in Fig. 3.
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Congo Red assay was utilized to qualitatively test the enzymatic activity of CenA in the form of crude enzyme, and this method was from iGEM18-UESTC-China, who had a nice [https://2019.igem.org/Team:UESTC-China/Collaborations collaboration] with us this year. As Congo Red only binds to long chain polysaccharides but not short chain, the short chain therefore are washed off during staining procedure resulting in halo formation <ref>S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation.  (2012).</ref>. The results are shown in Fig. 3.
  
 
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Zone added with YebF-CenA boken supernatant and cluture supernatant were shown clearance zones produced by hydrolysis of CMC. The empty control didn't show any zone of clearance. The results showed that both intracellular and extracellular YebF-CenA had enzyme activity.
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Zones with the broken ''sup'' and ''sup'' of YebF-CenA added showed due to the hydrolysis of CMC (carboxymethyl cellulose) whereas the blank control didn't show any clearance zones. The obvious difference showed that the broken ''sup'' and ''sup'' of YebF-CenA had enzymatic activity. This was to say that the enzyme, YebF-CenA, which was expressed successfully, had a certain level of enzymatic activity to hydrolyze cellulose. Besides, what the results showed was in accordance with the results of SDS-PAGE (Fig. 2) as well.
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Revision as of 08:53, 20 October 2019


Expression system of YebF-CenA coding YebF-Endoglucanase A with T7 promoter

This part contains the sequence for the protein endoglucanase A with protein YebF fused to its N-terminus by GS Linker. We use T7 promoter and RBS related to achieve the high level secretion of Endoglucanase A with the function of YebF protein.


Biology

BBa_K2922007 is a composite of cenA (BBa_K118023) with yebF ( BBa_K1659003)expressed in T7-RBS (BBa_K525998) system with the related protein reported to be naturally secreted into the extracellular medium by E.coli BL21:


1. Endoglucanase A

Cellulose is a polymer composed of beta-1,4-linked glucosyl residues. Cellulases (endoglucanases), cellobiosidases (exoglucanases), and beta- glucosidases are required by organisms (some fungi, bacteria) that can consume it. These enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass.

Bacterium 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.

2. YebF

YebF is a 13 kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory E. coli strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form. Export from periplasm into the extracellular space takes places via the Omp pathway, whereby the electropositive dynamic region of YebF electrostatically helps load YebF onto the OmpF/OmpC porins at their electronegative periplasmic face, and after which the disordered N-terminal region of YebF gets threaded through the OmpF lumen. YebF has been used successfully to mediate the secretion of recombinant proteins [1].


Usage

In order to secrete our cellulases out of the E. coli membrane, we fused the cellulase gene fragments with yebF gene fragment at the N-terminal using a flexible GS linker (GGGGS) by overlap extension polymerase chain reaction (OE-PCR). PCR product was purified by agarose gel electrophoresis (Fig. 1).

Fig.1 Agarose Gel Electrophoresis of yebF-cenA OE-PCR product. Lane M: Marker.


Characterization

These parts were insert into the expression vectors with T7 and RBS (BBa_K525998) by restriction sites EcoRI and PstI. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

1. SDS-PAGE

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.2 SDS-PAGE analysis of E. coli BL21 (DE3) by Sliver staining. YebF-cenA: protein of E. coli BL21 (DE3) carrying T7-RBS-yebF-cenA (BBa_K2922007), target bands can be seen in the medium at 60 kDa; Control: protein of E. coli BL21 (DE3) carrying T7 and RBS (BBa_K525998).


2. Congo Red Assay

Congo Red assay was utilized to qualitatively test the enzymatic activity of CenA in the form of crude enzyme, and this method was from iGEM18-UESTC-China, who had a nice collaboration with us this year. As Congo Red only binds to long chain polysaccharides but not short chain, the short chain therefore are washed off during staining procedure resulting in halo formation [2]. The results are shown in Fig. 3.

Fig.3 Activity determination of YebF-CenA using Congo Red assay. Left: T7-RBS-yebF-cenA medium supernatant and control (LB liquid medium). Right: T7-RBS-yebF-cenA broken supernatant and control (PBS).


Zones with the broken sup and sup of YebF-CenA added showed due to the hydrolysis of CMC (carboxymethyl cellulose) whereas the blank control didn't show any clearance zones. The obvious difference showed that the broken sup and sup of YebF-CenA had enzymatic activity. This was to say that the enzyme, YebF-CenA, which was expressed successfully, had a certain level of enzymatic activity to hydrolyze cellulose. Besides, what the results showed was in accordance with the results of SDS-PAGE (Fig. 2) as well.


References

  1. ↑ https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003#Biology
  2. ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).