Difference between revisions of "Part:BBa K2922015"
 
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<partinfo>BBa_K2922015 short</partinfo>
 
<partinfo>BBa_K2922015 short</partinfo>
  
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This part contains the sequence for the protein kil regulated by constitutive promoter J23109 and the sequence for the protein endoglucanase A regulated by T7 promoter. We used this part to achieve the secretion of endoglucanase with the function of kil secretion cassette.
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This part contains the sequence for the protein Kil regulated by constitutive promoter J23109 and the sequence for the protein endoglucanase A regulated by T7 promoter. We used this part to achieve the secretion of Endoglucanase with the function of Kil secretion cassette.
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'''1. Kil Secretion Cassette'''
 
'''1. Kil Secretion Cassette'''
  
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In wild-type ''E.coli'' exist a plasmid named colE1, the kil gene of the ColE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of ''kil'' gene expression is critical for utilization in a protein secretion system.
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In wild-type ''E.coli'' exists a plasmid named colE1, the ''kil'' gene of the colE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of ''kil'' gene expression is critical for utilization in a protein secretion system.
  
 
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The main problem when using constitutive promoters for kil gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the ''kil'' gene under the control of the weak constitutive promoter enabled viability to be maintained<ref>G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).</ref>.
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The main problem when using constitutive promoters for ''kil'' gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the ''kil'' gene under the control of the weak constitutive promoter enabled viability to be maintained<ref>G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The ''kil'' gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).</ref>.
  
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Here, we use <partinfo>BBa_J23109</partinfo>, <partinfo>BBa_J23112</partinfo> and <partinfo>BBa_J23114</partinfo> to demonstrate the effect of the kil gene controlled by the J21309 series promoters (<partinfo>BBa_J23109</partinfo>) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the kil gene and constructed secretion cassettes, where target genes <partinfo>BBa_K118022</partinfo> of interest can be easily integrated.  
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Here, we use <partinfo>BBa_J23109</partinfo>, <partinfo>BBa_J23112</partinfo> and <partinfo>BBa_J23114</partinfo> to demonstrate the effect of the ''kil'' gene controlled by the J21309 series promoters (<partinfo>BBa_J23109</partinfo>) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the ''kil'' gene and constructed secretion cassettes, where target genes of interest can be easily integrated.  
  
 
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'''2. Endoglucanase A'''
 
'''2. Endoglucanase A'''
  
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Cellulose is a polymer composed of beta-1,4-linked glucosyl residues.
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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.
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Cellulases (endoglucanases), cellobiosidases (exoglucanases), and beta-
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glucosidases are required by organisms (some fungi, bacteria) that can
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consume it. These enzymes are powerful tools for degradation of plant
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cell walls by pathogens and other organisms consuming plant biomass.
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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.
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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.
  
 
===Usage===
 
===Usage===
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To construct this part, we moved J23109-kil (<partinfo>BBa_K2922009</partinfo>) and T7-RBS-cenA (linked by <partinfo>BBa_K525998</partinfo> and <partinfo>BBa_K118023</partinfo>) into the expression vector pSB1C3 by standard assembly.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.
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To construct this part, we moved J23109-''kil'' (<partinfo>BBa_K2922009</partinfo>) and T7-RBS-''cenA'' (linked by <partinfo>BBa_K525998</partinfo> and <partinfo>BBa_K118023</partinfo>) into the expression vector pSB1C3 by standard assembly. 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 clonies 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 protein in ''E. coli'' BL21 (DE3) cells and the medium by Coomassie blue staining. 109-kil-cenA: protein of BL21 (DE3) carrying J23109-kil-PT7-RBS-cenA (<partinfo>BBa_K2922021</partinfo>), target bands can be seen in both cells and the medium at about 47 kDa; Control: protein of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by <partinfo>BBa_K2922009</partinfo> and <partinfo>BBa_K525998</partinfo>).
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:'''Fig.2''' SDS-PAGE analysis of protein in ''E. coli'' BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 109-kil-cenA: protein of BL21 (DE3) carrying J23109-''kil''-T7-RBS-''cenA'' (<partinfo>BBa_K2922021</partinfo>), target bands can be seen in both cells and the medium at about 47 kDa; Lane control: protein of BL21 (DE3) carrying J23109-RBS-''kil''-T7-RBS (linked by <partinfo>BBa_K2922009</partinfo> and <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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     </figure>
 
     </figure>
 
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:'''Fig.3''' Activity determination of CenA using Congo Red assay. CenA: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109/J23112/J23114-kil-T7-RBS-cenA (<partinfo>BBa_K2922015</partinfo>/<partinfo>BBa_K2922016</partinfo>/<partinfo>BBa_K2922017</partinfo>); Control: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by <partinfo>BBa_K2922009</partinfo> and <partinfo>BBa_K525998</partinfo>).
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:'''Fig.3''' Activity determination of CenA using Congo Red assay. CenA: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109/J23112/J23114-RBS-''kil''-T7-RBS-''cenA'' (<partinfo>BBa_K2922015</partinfo>/<partinfo>BBa_K2922016</partinfo>/<partinfo>BBa_K2922017</partinfo>); Control: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109-''kil''-T7-RBS (linked by <partinfo>BBa_K2922009</partinfo> and <partinfo>BBa_K525998</partinfo>).
  
  
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Zone added with 109-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 109-CenA had enzyme activity.
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Zones with the broken ''sup'' and ''sup'' of 109-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 109-CenA had enzymatic activity. This was to say that the enzyme,  Endoglucanase (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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===References===
 
===References===
 
<references/>
 
<references/>
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Latest revision as of 02:41, 22 October 2019


T7-RBS-cenA (Endoglucanase A) functions in Kil secretion cassette with constitutive promoter J23109

This part contains the sequence for the protein Kil regulated by constitutive promoter J23109 and the sequence for the protein endoglucanase A regulated by T7 promoter. We used this part to achieve the secretion of Endoglucanase with the function of Kil secretion cassette.


Biology

1. Kil Secretion Cassette

In wild-type E.coli exists a plasmid named colE1, the kil gene of the colE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of kil gene expression is critical for utilization in a protein secretion system.


The main problem when using constitutive promoters for kil gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the kil gene under the control of the weak constitutive promoter enabled viability to be maintained[1].

Here, we use BBa_J23109, BBa_J23112 and BBa_J23114 to demonstrate the effect of the kil gene controlled by the J21309 series promoters (BBa_J23109) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the kil gene and constructed secretion cassettes, where target genes of interest can be easily integrated.



2. 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.

Usage

To construct this part, we moved J23109-kil (BBa_K2922009) and T7-RBS-cenA (linked by BBa_K525998 and BBa_K118023) into the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.


Characterization

1. SDS-PAGE

The constructed plasmid was transformed into E. coli BL21 (DE3). Positive clonies 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 protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 109-kil-cenA: protein of BL21 (DE3) carrying J23109-kil-T7-RBS-cenA (BBa_K2922021), target bands can be seen in both cells and the medium at about 47 kDa; Lane control: protein of BL21 (DE3) carrying J23109-RBS-kil-T7-RBS (linked by BBa_K2922009 and 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 CenA using Congo Red assay. CenA: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109/J23112/J23114-RBS-kil-T7-RBS-cenA (BBa_K2922015/BBa_K2922016/BBa_K2922017); Control: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).


Zones with the broken sup and sup of 109-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 109-CenA had enzymatic activity. This was to say that the enzyme, Endoglucanase (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. ↑ G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).
  2. ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NotI site found at 328
    Illegal NotI site found at 1472
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1385
    Illegal BamHI site found at 521
    Illegal XhoI site found at 883
    Illegal XhoI site found at 1132
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 663
    Illegal NgoMIV site found at 1588
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 567