Difference between revisions of "Part:BBa K2922012"

 
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<partinfo>BBa_K2922012 short</partinfo>
 
<partinfo>BBa_K2922012 short</partinfo>
  
This part contains the sequence for the protein kil regulated by constitutive promoter J23109 and the sequence for the protein beta-D-glucosidase 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 Beta-D-glucosidase regulated by T7 promoter. We used this part to achieve the secretion of Beta-D-glucosidase with the function of Kil secretion cassette.
 
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'''1. Kil Secretion Cassette'''
 
'''1. Kil Secretion Cassette'''
  
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.
+
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>
+
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>
  
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-''cex'' <partinfo>BBa_K118022</partinfo> of interest can be easily integrated.  
+
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. Bgl1A'''
 
'''2. Bgl1A'''
  
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.  
+
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.  
  
Beta-glucosidase is an 53 kDa enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in beta-D-glucosides and oligosaccharides, with release of glucose.<ref>M. Cox, D. Nelson, Lehninger Principles of Biochemistry.  (2000), vol. 5. New York: Worth Publishers. pp. 306–308.</ref>
+
Beta-glucosidase is a 53 kDa enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in Beta-D-glucosides and oligosaccharides, with release of glucose.<ref>M. Cox, D. Nelson, Lehninger Principles of Biochemistry.  (2000), vol. 5. New York: Worth Publishers. pp. 306–308.</ref>
  
Depending on the organism cellobiose may be cleaved extracellularly by β-glucosidases (cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.<ref>R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).</ref>
+
Depending on the organism cellobiose may be cleaved extracellularly by Beta-glucosidases (Cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.<ref>R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).</ref>
  
  
  
 
===Usage===
 
===Usage===
To construct this part, we moved J23109-kil (<partinfo>BBa_K2922009</partinfo>) and T7-RBS-bgl1A (linked by <partinfo>BBa_K525998</partinfo> and <partinfo>BBa_K2564000</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-''bgl1A'' (linked by <partinfo>BBa_K525998</partinfo> and <partinfo>BBa_K2564000</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'''
  
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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         <img src="https://2019.igem.org/wiki/images/3/33/T--XMU-China--J23109-kil-T7-RBS-cenA_SDS-PAGE.png" width="80%" style="float:center">
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         <img src="https://2019.igem.org/wiki/images/1/13/T--XMU-China--J23109-RBS-kil-T7-RBS-bgl1A_SDS-PAGE.png" width="80%" style="float:center">
 
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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-bgl1A: protein of BL21 (DE3) carrying J23109-kil-PT7-RBS-bgl1A (<partinfo>BBa_K2922012</partinfo>), target bands can be seen in both cells and the medium at about 53 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-bgl1A: protein of BL21 (DE3) carrying J23109-''kil''-T7-RBS-''bgl1A'' (<partinfo>BBa_K2922012</partinfo>), target bands can be seen in both cells and the medium at about 53 kDa; Lane control: protein of BL21 (DE3) carrying J23109-''kil''-T7-RBS (linked by <partinfo>BBa_K2922009</partinfo> and <partinfo>BBa_K525998</partinfo>).
  
  
 
'''2. HPLC'''
 
'''2. HPLC'''
  
We use HPLC to verify the activity of bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.  
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We use HPLC to verify the activity of Bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.  
  
 
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:'''Fig. 3''' SWC for J23019/J223112-kil-T7-RBS-bgl1A, made through the relationship between peak area and concentration.
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:'''Fig. 3''' SWC for J23109/J223112-''kil''-T7-RBS-''bgl1A'', made through the relationship between peak area and concentration.
  
  
Then mix the crude enzyme solution with cellobiose, 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 when and after interval time since reaction started. And then carry out HPLC on the sample.  
+
Then mix the crude enzyme solution with cellobiose, 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 when and after interval time since reaction started. And then carry out HPLC on the sample.  
  
 
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:'''Fig. 4''' The results of HPLC. (A): J23019-kil-T7-RBS-bgl1A supernatant; (B): J23019-kil-T7-RBS-bgl1A broken supernatant
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:'''Fig. 4''' Quantitative analysis for the enzymatic activity of Bgl1A (109-kil) was supported through HPLC. (A) The concentration of cellobiose and glucose of ''sup'' at different times. (B) The concentration of cellobiose and glucose of broken ''sup'' at different times.
 
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Result of the broken supernatant and supernatant of medium cultures with J23019-kil-T7-RBS-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained. Bgl1A could degrade D-cellobiose into D-glucose.
+
  
 +
Result of the broken supernatant and supernatant of medium cultures with J23109-''kil''-T7-RBS-''bgl1A'' part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained. Bgl1A could degrade D-cellobiose into D-glucose.
  
  
 
===Reference===
 
===Reference===
 
<references/>
 
<references/>
 
  
  

Latest revision as of 03:39, 22 October 2019


T7-RBS-bgl1A (Beta-D-glucosidase) functions in Kil secretion cassette with promoter J23109

This part contains the sequence for the protein Kil regulated by constitutive promoter J23109 and the sequence for the protein Beta-D-glucosidase regulated by T7 promoter. We used this part to achieve the secretion of Beta-D-glucosidase 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. Bgl1A

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.

Beta-glucosidase is a 53 kDa enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in Beta-D-glucosides and oligosaccharides, with release of glucose.[2]

Depending on the organism cellobiose may be cleaved extracellularly by Beta-glucosidases (Cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.[3]


Usage

To construct this part, we moved J23109-kil(BBa_K2922009) and T7-RBS-bgl1A (linked by BBa_K525998 and BBa_K2564000) 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 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 protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 109-kil-bgl1A: protein of BL21 (DE3) carrying J23109-kil-T7-RBS-bgl1A (BBa_K2922012), target bands can be seen in both cells and the medium at about 53 kDa; Lane control: protein of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).


2. HPLC

We use HPLC to verify the activity of Bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.


Fig. 3 SWC for J23109/J223112-kil-T7-RBS-bgl1A, made through the relationship between peak area and concentration.


Then mix the crude enzyme solution with cellobiose, 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 when and after interval time since reaction started. And then carry out HPLC on the sample.


Fig. 4 Quantitative analysis for the enzymatic activity of Bgl1A (109-kil) was supported through HPLC. (A) The concentration of cellobiose and glucose of sup at different times. (B) The concentration of cellobiose and glucose of broken sup at different times.

Result of the broken supernatant and supernatant of medium cultures with J23109-kil-T7-RBS-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained. Bgl1A could degrade D-cellobiose into D-glucose.


Reference

  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. M. Cox, D. Nelson, Lehninger Principles of Biochemistry. (2000), vol. 5. New York: Worth Publishers. pp. 306–308.
  3. R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).


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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1507
  • 1000
    COMPATIBLE WITH RFC[1000]