Difference between revisions of "Part:BBa K4886006"
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==Results==
 
==Results==
 
===(1) Plasmid construction===
 
===(1) Plasmid construction===
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By using a recombinant plasmid pMTL-Pthl-F/Xpk(BD) as a template, and X-PN-F and X-PN-R as primers, we obtained a X-F/Xpk(BD) vector (7670 bp). Ptkt fragment (300bp) was amplified from the genome template of C. acetobutylicum using P-Ptkt-F and P-Ptkt-R as primers, by PCR. DNA electrophoresis confirmed the lengths of the PCR products (Figure 2). Ptkt fragment was ligated with X-F/Xpk(BD) vector into a pMTL-Ptkt-FXpk(BD) recombinant plasmid by Gibson assembly. The plasmid was transformed into a E. coli JM109 strain. After verification by colony PCR and DNA electrophoresis, positive colonies were transferred and expanded. Gene sequencing was used to verify that the plasmid extracted from the colonies was pMTL-Ptkt-FXpk(BD).
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By using a recombinant plasmid pMTL-Pthl-F/Xpk(BD) as a template, and X-PN-F and X-PN-R as primers, we obtained a X-F/Xpk(BD) vector (7670 bp). Ptkt fragment (300bp) was amplified from the genome template of C. tyrobutyricum using P-Ptkt-F and P-Ptkt-R as primers, by PCR. DNA electrophoresis confirmed the lengths of the PCR products (Figure 2). Ptkt fragment was ligated with X-F/Xpk(BD) vector into a pMTL-Ptkt-F/Xpk(BD) recombinant plasmid by Gibson assembly. The plasmid was transformed into a E. coli JM109 strain. After verification by colony PCR and DNA electrophoresis, positive colonies were transferred and expanded. Gene sequencing was used to verify that the plasmid extracted from the colonies was pMTL-Ptkt-F/Xpk(BD).
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Figure 2 Verification of Ptkt (300bp) by DNA gel electrophoresis  
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  <img class="bild" src="https://static.igem.wiki/teams/4886/wiki/006-figure-2-verification-of-ptkt-300bp-by-dna-gel-electrophoresis.png">
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  <div class="unterschrift"><b>Figure 2 Verification of Ptkt (300bp) by DNA gel electrophoresis</b>
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===(2)Transfection and function analysis ===
 
===(2)Transfection and function analysis ===
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By using E. coli CA434 as a donor strain, pMTL-Ptkt-FXpk(BD) plasmid was transferred to C. tyrobutyricum, noted as Ct(Ptkt F/Xpk(BD)).  C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(BD) was used as the control. C. tyrobutyricum transfected with pMTL-Pfba-F/Xpk(BD) was noted as Ct(Pfba F/Xpk(BD)). pMTL-Pthl-F/Xpk(BD) and pMTL-Pfba-F/Xpk(BD) plasmids were same as pMTL-Ptkt-FXpk(BD) except using a different promoter, Pthl or Pfba. Xylose was used as the carbon source for fermentation.
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By using E. coli CA434 as a donor strain, pMTL-Ptkt-F/Xpk(BD) plasmid was transferred to C. tyrobutyricum, notated as Ct(Ptkt F/Xpk-BD). Ct(Pfba F/Xpk-BD) (refer to BBa_K4886004 ), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD) (refer to BBa_K4886001 ) were fermented using xylose as carbon source.
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Fermentation experiment showed that the growth of Ct(Ptkt F/Xpk-BD) was better than that of Ct(Pthl F/Xpk-BD), and the growth of Ct(Pfba F/Xpk-BD) was worse than that of Ct(Pthl F/Xpk-BD) (Figure 3).
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Fermentation experiment showed that the growth of Ct(Ptkt F/Xpk(BD)) was worse than that of the control and that of Ct(Pfba F/Xpk(BD)) (Figure 3).
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HPLC experiment showed that after culturing on xylose for 60.5h, the yield of butyric acid was 5.89 g/L in Ct(Ptkt F/Xpk-BD), higher than the 5.19 g/L yield in Ct(Pthl F/Xpk-BD) and the 5.21 g/L in Ct(Pfba F/Xpk-BD). Ct(Ptkt F/Xpk-BD) showed higher xylose consumption than Ct(Pthl F/Xpk-BD) and Ct(Pfba F/Xpk-BD) (Figure 4).  
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The results implied that among Pthl, Ptkt and Pfba, Ptkt was the best promoter for F/Xpk(BD) gene to construct NOG pathway in C. tyrobutyricum to have satisfactory growth, butyric acid production and carbon conservation.
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HPLC experiment showed that after culturing on xylose for 48h, the yield of butyric acid was 2.02 g/L in Ct(Ptkt F/Xpk(BD)), lower than the 2.40 g/L yield in the control and the 2.58 g/L yield in Ct(Pfba F/Xpk(BD)) (Figure 4).  
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From this, it can be concluded that using Ptkt as the promoter for F/Xpk(BD) gene was not satisfactory for growth and butyric acid production in the NOG pathway of the engineered C. tyrobutyricum, compared with Pthl and Pfba.
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  <img class="bild" src="https://static.igem.wiki/teams/4886/wiki/006-fig3.png">
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  <div class="unterschrift"><b>Figure 3 Growth performance of Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD) on xylose</b>
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Figure 3 Growth performance of  Ct(Pfba F/Xpk(BD)) and Ct(Ptkt F/Xpk(BD)) （Control：Ct(F/Xpk-BD) with Pthl promoter）
 
  
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Figure 4 Butyric acid yield and glucose consumption of Ct(Pfba F/Xpk(BD)) and Ct(Ptkt F/Xpk(BD))（Control：Ct(F/Xpk-BD) with Pthl promoter）
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  <img class="bild" src="https://static.igem.wiki/teams/4886/wiki/006-fig4.png">
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  <div class="unterschrift"><b>Figure 4 Butyric acid yield and xylose consumption of Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD)</b>
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Revision as of 06:08, 30 September 2023


Ptkt-P/Xpk(BD)

It is the part which is responsible for the expression of F/Xpk from Clostridium acetobutylicum ATCC824 with Ptkt promotor. It consists of Ptkt sequence (BBa_K4886005), strong ribosomal binding site (RBS) sequence (BBa_K103015), F/Xpk sequence (BBa_K4119076) and terminator sequence (BBa_K3585002). F/Xpk is a gene that encodes phosphoketolase. Phosphoketolase is an enzyme with both the Fpk and Xpk activity. It catalyzes the conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), and the conversion of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1675
    Illegal XbaI site found at 618
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1675
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1675
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1675
    Illegal XbaI site found at 618
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1675
    Illegal XbaI site found at 618
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

(1) Plasmid construction

By using a recombinant plasmid pMTL-Pthl-F/Xpk(BD) as a template, and X-PN-F and X-PN-R as primers, we obtained a X-F/Xpk(BD) vector (7670 bp). Ptkt fragment (300bp) was amplified from the genome template of C. tyrobutyricum using P-Ptkt-F and P-Ptkt-R as primers, by PCR. DNA electrophoresis confirmed the lengths of the PCR products (Figure 2). Ptkt fragment was ligated with X-F/Xpk(BD) vector into a pMTL-Ptkt-F/Xpk(BD) recombinant plasmid by Gibson assembly. The plasmid was transformed into a E. coli JM109 strain. After verification by colony PCR and DNA electrophoresis, positive colonies were transferred and expanded. Gene sequencing was used to verify that the plasmid extracted from the colonies was pMTL-Ptkt-F/Xpk(BD).

Figure 2 Verification of Ptkt (300bp) by DNA gel electrophoresis

(2)Transfection and function analysis

By using E. coli CA434 as a donor strain, pMTL-Ptkt-F/Xpk(BD) plasmid was transferred to C. tyrobutyricum, notated as Ct(Ptkt F/Xpk-BD). Ct(Pfba F/Xpk-BD) (refer to BBa_K4886004 ), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD) (refer to BBa_K4886001 ) were fermented using xylose as carbon source. Fermentation experiment showed that the growth of Ct(Ptkt F/Xpk-BD) was better than that of Ct(Pthl F/Xpk-BD), and the growth of Ct(Pfba F/Xpk-BD) was worse than that of Ct(Pthl F/Xpk-BD) (Figure 3). HPLC experiment showed that after culturing on xylose for 60.5h, the yield of butyric acid was 5.89 g/L in Ct(Ptkt F/Xpk-BD), higher than the 5.19 g/L yield in Ct(Pthl F/Xpk-BD) and the 5.21 g/L in Ct(Pfba F/Xpk-BD). Ct(Ptkt F/Xpk-BD) showed higher xylose consumption than Ct(Pthl F/Xpk-BD) and Ct(Pfba F/Xpk-BD) (Figure 4). The results implied that among Pthl, Ptkt and Pfba, Ptkt was the best promoter for F/Xpk(BD) gene to construct NOG pathway in C. tyrobutyricum to have satisfactory growth, butyric acid production and carbon conservation.

Figure 3 Growth performance of Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD) on xylose


Figure 4 Butyric acid yield and xylose consumption of Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD)