Composite

Part:BBa_K4886001

Designed by: Ruyi Shi   Group: iGEM23_Nanjing-BioX   (2023-07-03)
Revision as of 16:10, 11 October 2023 by Xuleyu (Talk | contribs) (→‎(2)Transfection of C. tyrobutyricum and its growth)


Pthl-F/Xpk(BD)

It is the part which is responsible for the expression of F/Xpk from Clostridium acetobutylicum ATCC824 with Pthl promotor. It consists of Pthl sequence (BBa_K3443002), 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 1986
    Illegal XbaI site found at 929
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1986
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1986
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1986
    Illegal XbaI site found at 929
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1986
    Illegal XbaI site found at 929
  • 1000
    COMPATIBLE WITH RFC[1000]

results

(1)Plasmid construction

We used Pthl-adhE2 from BBa_K4408008 as the template and X-pMTL-F and X-pMTL-R as primers to obtain a X-Pthl vector (5461bp) by amplification. We used the Clostridium acetobutylicum ATCC824 genome as a template to amplify F/Xpk(BD) fragment (2391bp). The vector and fragment were confirmed by gel electrophoresis (Figure 1 and 2). The F/Xpk(BD) fragment was ligated to the X-Pthl linear vector, using Gibson assembly. The plasmid was then transformed into E.coli JM109. After colony PCR (1000 bp) for the transformed bacterial colonies, positive colonies were inoculated, and plasmids were extracted. The recombinant plasmid pMTL-Pthl-F/Xpk(BD) obtained was confirmed by gene sequencing.

Figure 1 Gel electrophoresis of FXpk gene from Clostridium acetobutylicum ATCC824 (2391 bp)

Figure 2 Gel electrophoresis of X-pMTL-Pthl linear vector (5461bp)

(2)Transfection of C. tyrobutyricum and its growth

By using E. coli CA434 as a donor strain, pMTL-Pthl-F/Xpk(QS) plasmid and pMTL-Pthl-F/Xpk(BD) plasmid were transferred to C. tyrobutyricum, notated as Ct(Pthl F/Xpk-QS) and Ct(Pthl F/Xpk-BD), respectively. pMTL-Pthl-F/Xpk(QS) plasmid was constructed similar as pMTL-Pthl-F/Xpk(BD) using F/Xpk derived from Bifidobacterium adolescentis ATCC 15703 instead of F/Xpk(BD). Ct(Pthl F/Xpk-QS) , Ct(Pthl F/Xpk-BD) and native C. tyrobutyricum (the control) were fermented using glucose as carbon source. Fermentation experiment showed that the growth of Ct(Pthl F/Xpk-BD) was better than that of Ct(Pthl F/Xpk-QS), Figure 3.

Figure 3 Growth comparison of Ct(F/Xpk-QS) and Ct(F/Xpk-BD)

(3)Product yield of the transfected strain

Acetyl phosphate (AcP) is the final product of NOG pathway. AcP assay showed that the levels of AcP in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) were both higher than the control, which was in accordance with the growth of the strains. This indicated that NOG pathway was open in the engineered strains, Figure 4.

Figure 4 Levels of acetyl phosphate in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS)

HPLC experiment showed that after fermentation for 26h, the yields of butyric acid were 3.35 g/L and 3.31 g/L in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS), both higher than the yield in the control. The yields of acetic acid were 1.36 g/L and 1.28 g/L in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS), both lower than that in the control. Glucose consumption was much higher in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) compared with the control (Table 1). Ct(Pthl F/Xpk-BD) showed higher glucose consumption and butyric acid yield than Ct(Pthl F/Xpk-QS).

Butyric acid is a 4-carbon molecule, while acetic acid is a 2-carbon molecule. The increase in the butyric acid production and glucose consumption and decrease in the by-product acetic acid yield suggested that Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) both have higher efficiency of using glucose and less carbon loss in glycosis compared with the native strain. In addition, Ct(Pthl F/Xpk-BD) was better in reducing carbon loss than Ct(Pthl F/Xpk-QS).

Table 1 Metabolite levels in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) after 26h fermentation


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