Difference between revisions of "Part:BBa K4885004"

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<partinfo>BBa_K4885004 short</partinfo>
 
<partinfo>BBa_K4885004 short</partinfo>
  
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This part is responsible for the expression of adhE2 gene with Pthl promotor and Cat1 gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Cat1 gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence  (BBa_K103015),  adhE2 sequence (BBa_K1462060), terminator sequence (BBa_K3585002), Pcat1 sequence (BBa_K4119011), strong ribosomal binding site (RBS) sequence  (BBa_K103015),  Cat1 gene sequence (BBa_K4119013) and terminator sequence (BBa_K3585002). adhE2 gene encodes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Cat1 gene codes the butyryl-CoA/acetate CoA transferase.  
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This part is responsible for the expression of adhE2 gene with Pthl promotor and Cat1 gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Cat1 gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence  (BBa_K103015),  adhE2 sequence (BBa_K1462060), terminator sequence (BBa_K3585002), Pcat1 sequence (BBa_K4119011), strong ribosomal binding site (RBS) sequence  (BBa_K103015),  Cat1 gene sequence (BBa_K4119031) and terminator sequence (BBa_K3585002). adhE2 gene encodes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Cat1 gene codes the butyryl-CoA/acetate CoA transferase.  
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===Usage and biology===
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This is the part collection that used to engineer C. tyrobutyricum to overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885006 were used to overexpress rate-limiting
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enzymes (bcd and crt). BBa_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate. BBa_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.
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==Results==
 
==Results==
 
===(1)Plasmid construction and transformation===
 
===(1)Plasmid construction and transformation===
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We used the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 as the template, Vad-f and Vad-r as the primers, and amplified to obtain the Vpthl-adhE2 vector (7985 bp). Using the genomic DNA of C. tyrobutyricum as a template, with primers Cat-f and Cat-r, we amplified a fragment of the butyryl-CoA/acetate CoA transferase (cat1) gene (1378 bp). The cat1 fragment and Vpthl-adhE2 linearized vector were ligated by Gibson assembly method. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transfected, plasmid was extracted, and the recombinant plasmid, pMTL-Pthl-adhE2-Pcat1-Cat1, was verified by gene sequencing.
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We constructed pMTL-Pthl-adhE2-Pcat1-cat1 recombinant plasmid to inhibit the synthesis of a by-product, acetic acid, so that the synthesis of butanol and butyrate was enhanced indirectly.
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===(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-cat1 ===
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Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template and Vad-f and Vad-r as primers, the Vpthl-adhE2 vector (7985 bp) was amplified. Using C. tyrobutyricum genome as template and Cat-f and Cat-r as primers, cat1 gene fragment (1378 bp) was amplified.And Pcat-f and Pcat-r primers were used to amplify Pcat1 fragment. Gibson assembly method was used to link the cat1 gene fragment and Pcat1 fragmentto the Vpthl-adhE2 linearized vector. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, the recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 was obtained.
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Ct(adhE2::cat1) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-cat1 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. C. tyrobutyricum L319 transfected with pMTL-Pthl-adhE2 plasmid was notated as Ct(adhE2). Ct(adhE2) was used as the control.
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===(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Pcat1-cat1 ===
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Ct(adhE2::cat1) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. C. tyrobutyricum L319 transfected with pMTL-Pthl-adhE2 plasmid was notated as Ct(adhE2). Ct(adhE2) was used as the control.
 
SDS-PAGE confirmed the overexpression of cat1 protein (47 kDa) in Ct(adhE2::cat1) (Figure1).  
 
SDS-PAGE confirmed the overexpression of cat1 protein (47 kDa) in Ct(adhE2::cat1) (Figure1).  
 
HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing cat1 in C. tyrobutyricum improved the yield of butyrate by 130%, and decreased the yield of butanol by 26% (Figure 2), increasing the butyrate-to-butanol molar ratio from 0.3 to 1.1. The increased product ratio was more favorable for the esterification of butyrate and butanol into butyl butyrate.
 
HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing cat1 in C. tyrobutyricum improved the yield of butyrate by 130%, and decreased the yield of butanol by 26% (Figure 2), increasing the butyrate-to-butanol molar ratio from 0.3 to 1.1. The increased product ratio was more favorable for the esterification of butyrate and butanol into butyl butyrate.
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In addition, the yield of acetate as a byproduct decreased significantly from 4 g/L to 1.2 g/L by overexpressing cat1.
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In addition, the yield of acetate as a byproduct decreased significantly from 4 g/L to 1.2 g/L by overexpressing cat1.
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/004-fig1.png">
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  <div class="unterschrift"><b>Figure 1 Verification of cat1 protein overexpression in Ct(adhE2::cat1) by SDS-PAGE</b>
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/004-fig2.png">
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  <div class="unterschrift"><b>Figure 2 Fermentation performance of butyrate, butanol and acetate of Ct(adhE2::cat1)</b>
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Latest revision as of 13:29, 12 October 2023


Pthl-adhE2-Pcat1-Cat1

This part is responsible for the expression of adhE2 gene with Pthl promotor and Cat1 gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Cat1 gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence (BBa_K103015), adhE2 sequence (BBa_K1462060), terminator sequence (BBa_K3585002), Pcat1 sequence (BBa_K4119011), strong ribosomal binding site (RBS) sequence (BBa_K103015), Cat1 gene sequence (BBa_K4119031) and terminator sequence (BBa_K3585002). adhE2 gene encodes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Cat1 gene codes the butyryl-CoA/acetate CoA transferase.

Usage and biology

This is the part collection that used to engineer C. tyrobutyricum to overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885006 were used to overexpress rate-limiting enzymes (bcd and crt). BBa_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate. BBa_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.

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]

Results

(1)Plasmid construction and transformation

We constructed pMTL-Pthl-adhE2-Pcat1-cat1 recombinant plasmid to inhibit the synthesis of a by-product, acetic acid, so that the synthesis of butanol and butyrate was enhanced indirectly. Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template and Vad-f and Vad-r as primers, the Vpthl-adhE2 vector (7985 bp) was amplified. Using C. tyrobutyricum genome as template and Cat-f and Cat-r as primers, cat1 gene fragment (1378 bp) was amplified.And Pcat-f and Pcat-r primers were used to amplify Pcat1 fragment. Gibson assembly method was used to link the cat1 gene fragment and Pcat1 fragmentto the Vpthl-adhE2 linearized vector. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, the recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 was obtained.

(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Pcat1-cat1

Ct(adhE2::cat1) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. C. tyrobutyricum L319 transfected with pMTL-Pthl-adhE2 plasmid was notated as Ct(adhE2). Ct(adhE2) was used as the control. SDS-PAGE confirmed the overexpression of cat1 protein (47 kDa) in Ct(adhE2::cat1) (Figure1). HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing cat1 in C. tyrobutyricum improved the yield of butyrate by 130%, and decreased the yield of butanol by 26% (Figure 2), increasing the butyrate-to-butanol molar ratio from 0.3 to 1.1. The increased product ratio was more favorable for the esterification of butyrate and butanol into butyl butyrate. In addition, the yield of acetate as a byproduct decreased significantly from 4 g/L to 1.2 g/L by overexpressing cat1.

Figure 1 Verification of cat1 protein overexpression in Ct(adhE2::cat1) by SDS-PAGE

Figure 2 Fermentation performance of butyrate, butanol and acetate of Ct(adhE2::cat1)