Difference between revisions of "Part:BBa K2949012"

(→‎1.1 Construction of CA2(L203K)-N-LCTPSR) expression plasmid)
(→‎2. Identification of the function for CO2 capture)
 
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<partinfo>BBa_K2949012 short</partinfo>
 
<partinfo>BBa_K2949012 short</partinfo>
  
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we constructed this new biobrick by connecting the N-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) with the six-residue sulfatase submotif (LCTPSR). Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups, which can be immobilized via forming covalent bond with amino functionalized carriers (Unisil 30-100 NH2) through the Schiff base reaction.  
+
We constructed this new biobrick by connecting the N-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) with the six-residue sulfatase submotif (LCTPSR). Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups, which can be immobilized via forming covalent bond with amino functionalized support (Unisil 30-100 NH2) through the Schiff base reaction.  
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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[[File:T--AHUT_China--NewPart_01.jpg|800px|thumb|center|Fig.1 Map of CA2(L203K)-N-LCTPSR recombinant vector]]
 
[[File:T--AHUT_China--NewPart_01.jpg|800px|thumb|center|Fig.1 Map of CA2(L203K)-N-LCTPSR recombinant vector]]
  
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The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.2)and sequencing (Fig.3).
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The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.2) and sequencing (Fig.3).
 
[[File:T--AHUT_China--NewPart_02.jpg|350px|thumb|center|Fig.2 Agarose Gel Electrophoresis of CA2(L203K)-N-LCTPSR recombinant plasmid and its identification by enzyme digestion.<br>
 
[[File:T--AHUT_China--NewPart_02.jpg|350px|thumb|center|Fig.2 Agarose Gel Electrophoresis of CA2(L203K)-N-LCTPSR recombinant plasmid and its identification by enzyme digestion.<br>
 
Lane M: DL15000 marker; Lane 1: CA2(L203K)-N-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-N-LCTPSR recombinant plasmid, the length was 834 bp (the arrow indicated).]]
 
Lane M: DL15000 marker; Lane 1: CA2(L203K)-N-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-N-LCTPSR recombinant plasmid, the length was 834 bp (the arrow indicated).]]
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We induced pilot expression of CA2(L203K)-N-LCTPSR in E. coli TB1 by using isopropyl-1-thio-β-Dgalactopyrasonide(IPTG).
 
We induced pilot expression of CA2(L203K)-N-LCTPSR in E. coli TB1 by using isopropyl-1-thio-β-Dgalactopyrasonide(IPTG).
 
Briefly, recombinant plasmid of the CA2(L203K)-N-LCTPSR was transformed into E. coli TB1, and positive clone was screened by kanamycin resistance. Then, the recombinant E. coli TB1 were propagated and CA2(L203K)-N-LCTPSR expression were induced with IPTG. Cells were lysed by sonication on ice, and the obtained crude extracts were centrifuged to separate supernatant and debris, and the fraction was subjected to SDS-PAGE.
 
Briefly, recombinant plasmid of the CA2(L203K)-N-LCTPSR was transformed into E. coli TB1, and positive clone was screened by kanamycin resistance. Then, the recombinant E. coli TB1 were propagated and CA2(L203K)-N-LCTPSR expression were induced with IPTG. Cells were lysed by sonication on ice, and the obtained crude extracts were centrifuged to separate supernatant and debris, and the fraction was subjected to SDS-PAGE.
 +
 
The results showed that CA2(L203K)-N-LCTPSR could be successfully expressed in TB1 strain (Fig.4).
 
The results showed that CA2(L203K)-N-LCTPSR could be successfully expressed in TB1 strain (Fig.4).
 
[[File:T--AHUT_China--NewPart_04.png|400px|thumb|center|Fig.4 SDS-PAGE analysis for CA2(L203K)-N-LCTPSR cloned in pET-30a(+) vector and expressed in TB1 strain.
 
[[File:T--AHUT_China--NewPart_04.png|400px|thumb|center|Fig.4 SDS-PAGE analysis for CA2(L203K)-N-LCTPSR cloned in pET-30a(+) vector and expressed in TB1 strain.
 
Lane 1: CA2(L203K)-N-LCTPSR protein expression without IPTG induction; Lane 2: CA2(L203K)-N-LCTPSR protein expression with IPTG induction.]]
 
Lane 1: CA2(L203K)-N-LCTPSR protein expression without IPTG induction; Lane 2: CA2(L203K)-N-LCTPSR protein expression with IPTG induction.]]
  
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After confirming that CA2(L203K)-N-LCTPSR could be expressed in our chassis E.coli TB1, we successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-N-LCTPSR into E.coli TB1. Then the protein of CA2(L203K)-N-LCTPSR was further purified with nickel column for the following enzyme immobilization.
+
After confirming that CA2(L203K)-N-LCTPSR could be expressed in our chassis E.coli TB1, we successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-N-LCTPSR expression vector into E.coli TB1. Then the protein of CA2(L203K)-N-LCTPSR was further purified with nickel column for the following enzyme immobilization.
  
 
The results showed that CA2(L203K)-N-LCTPSR was purified with high purity as indicated by a significant single protein band by SDS-PAGE (Fig.5).
 
The results showed that CA2(L203K)-N-LCTPSR was purified with high purity as indicated by a significant single protein band by SDS-PAGE (Fig.5).
 
[[File:T--AHUT_China--NewPart_05.jpg|400px|thumb|center|Fig.5 SDS-PAGE analysis of purified CA2(L203K)-N-LCTPSR protein]]
 
[[File:T--AHUT_China--NewPart_05.jpg|400px|thumb|center|Fig.5 SDS-PAGE analysis of purified CA2(L203K)-N-LCTPSR protein]]
  
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===2. Identification of the function for CO2 capture===
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===2. Identification of the function for CO<sub>2</sub> capture===
  
 
====2.1 The efficiency of CA2(L203K)-N-LCTPSR protein immobilization====
 
====2.1 The efficiency of CA2(L203K)-N-LCTPSR protein immobilization====
  
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In order to improve the efficiency of the repeated utilization of the CA2(L203K)-N-LCTPSR protein, we achieved enzyme immobilization.
+
In order to furture improve the efficiency of the repeated utilization of the CA2(L203K)-N-LCTPSR protein, we achieved enzyme immobilization.
 
Briefly, we cultured CA2(L203K)-N-LCTPSR in shaking table with the immobilization support (Unisil 30-100 NH2), then added 10% sodium cyanoborohydrate and continued to immobilize it in shaking table culture.  
 
Briefly, we cultured CA2(L203K)-N-LCTPSR in shaking table with the immobilization support (Unisil 30-100 NH2), then added 10% sodium cyanoborohydrate and continued to immobilize it in shaking table culture.  
  

Latest revision as of 03:24, 22 October 2019


Carbonic Anhydrase 2(L203K)-N-LCTASR

We constructed this new biobrick by connecting the N-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) with the six-residue sulfatase submotif (LCTPSR). Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups, which can be immobilized via forming covalent bond with amino functionalized support (Unisil 30-100 NH2) through the Schiff base reaction.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



1. Engineered E.coli TB1

1.1 Construction of CA2(L203K)-N-LCTPSR) expression plasmid

The coding sequence of CA2(L203K)-N-LCTPSR was synthesized and cloned into the expression vector pET-30a(+), as presented in Fig.1:

Fig.1 Map of CA2(L203K)-N-LCTPSR recombinant vector

The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.2) and sequencing (Fig.3).

Fig.2 Agarose Gel Electrophoresis of CA2(L203K)-N-LCTPSR recombinant plasmid and its identification by enzyme digestion.
Lane M: DL15000 marker; Lane 1: CA2(L203K)-N-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-N-LCTPSR recombinant plasmid, the length was 834 bp (the arrow indicated).
Fig.3 Sequencing results

1.2 Expression and purification of CA2(L203K)-N-LCTPSR protein in E. coli TB1

We induced pilot expression of CA2(L203K)-N-LCTPSR in E. coli TB1 by using isopropyl-1-thio-β-Dgalactopyrasonide(IPTG). Briefly, recombinant plasmid of the CA2(L203K)-N-LCTPSR was transformed into E. coli TB1, and positive clone was screened by kanamycin resistance. Then, the recombinant E. coli TB1 were propagated and CA2(L203K)-N-LCTPSR expression were induced with IPTG. Cells were lysed by sonication on ice, and the obtained crude extracts were centrifuged to separate supernatant and debris, and the fraction was subjected to SDS-PAGE.

The results showed that CA2(L203K)-N-LCTPSR could be successfully expressed in TB1 strain (Fig.4).

Fig.4 SDS-PAGE analysis for CA2(L203K)-N-LCTPSR cloned in pET-30a(+) vector and expressed in TB1 strain. Lane 1: CA2(L203K)-N-LCTPSR protein expression without IPTG induction; Lane 2: CA2(L203K)-N-LCTPSR protein expression with IPTG induction.

After confirming that CA2(L203K)-N-LCTPSR could be expressed in our chassis E.coli TB1, we successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-N-LCTPSR expression vector into E.coli TB1. Then the protein of CA2(L203K)-N-LCTPSR was further purified with nickel column for the following enzyme immobilization.

The results showed that CA2(L203K)-N-LCTPSR was purified with high purity as indicated by a significant single protein band by SDS-PAGE (Fig.5).

Fig.5 SDS-PAGE analysis of purified CA2(L203K)-N-LCTPSR protein

2. Identification of the function for CO2 capture

2.1 The efficiency of CA2(L203K)-N-LCTPSR protein immobilization

In order to furture improve the efficiency of the repeated utilization of the CA2(L203K)-N-LCTPSR protein, we achieved enzyme immobilization. Briefly, we cultured CA2(L203K)-N-LCTPSR in shaking table with the immobilization support (Unisil 30-100 NH2), then added 10% sodium cyanoborohydrate and continued to immobilize it in shaking table culture.

Our formula for calculating the enzymatic immobilized efficiency is as follows：

η: The efficiency of immobilized CA2(L203K)-N-LCTPSR protein;
W1: The concentration of total CA2(L203K)-N-LCTPSR protein;
W2: The concentration of free CA2(L203K)-N-LCTPSR protein.


According to the formula, we got the efficiency of immobilized CA2(L203K)-N-LCTPSR protein is 32.35%.

2.2 Enzyme activity asssay of CA2(L203K)-N-LCTPSR protein

The enzyme activity of immobilized CA2(L203K)-N-LCTPSR protein were tested experimentally by esterase activity assay at 37℃ and 50℃.

As indicated in Fig.6, the immobilized CA2(L203K)-N-LCTPSR protein were stable and retained their activity at high temperature.

Fig.6 Esterase activity analysis of immobilized CA2(L203K)-N-LCTPSR protein at 37℃ and 50℃