Composite

Part:BBa_K4885012

Designed by: Leyu Xu   Group: iGEM23_Nanjing-SDG   (2023-10-09)
Revision as of 07:24, 10 October 2023 by Xuleyu (Talk | contribs) (→‎Experiments and results)


Ptkt-adhE2

This part is responsible for the expression of adhE2 gene with Ptkt promotor. adhE2 gene is derived from Clostridium acetobutylicum. Ptkt is a native promoter that drives the expression of transketolase (tkt) gene in C. tyrobutyricum. This part consists of Ptkt sequence (BBa_K4886005), adhE2 sequence (BBa_K1462060), and terminator sequence (BBa_K3585002). adhE2 gene encodes alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor.

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]

Experiments and results

1.Plasmid construction and transformation: pMTL-Ptkt-adhE2

Using pMTL-Pthl-adhE2 plasmid (BBa_K4408008) as the template and Vtkt-F and Vtkt-R as the primers, a linearized vector Vtkt was amplified (7825 bp). Using the C. tyrobutyricum genome as the template and TKT-F and TKT-R as the primers, a tkt fragment was amplified (300 bp). The linearized vector Vtkt and the tkt fragment were ligated by Gibson assembly. Colony PCR was performed on the transformed colonies (300 bp) with TKT-F and TKT-R as the primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Ptkt-adhE2 was obtained.

Figure 1 Genetic circuit of recombinant plasmid pMTL-Ptkt-adhE2

Table 1 Primer sequences

Figure 2 Colony PCR verification of pMTL-Ptkt-adhE2 recombinant plasmid

2. Construction of C. tyrobutyricum L319 with pMTL-Ptkt-adhE2

Ct(Ptkt-adhE2) strain was obtained by conjugation of recombinant plasmid pMTL-Ptkt-adhE2 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(Pthl-adhE2) which was a C. tyrobutyricum strain transfected with pMTL-Pthl-adhE2 was used as the control. HPLC showed that after fermentation for 215 hours (Figure 3), compared with Ct(Pthl-adhE2), Ct(Ptkt-adhE2) had increased the yield of butyrate by 66.5% and decreased the yield of butanol by 24.3%. Therefore, by using the weaker promoter Ptkt instead of Pthl, we raised the butyrate-to-butanol molar ratio from 0.29 to 0.63, much closer to the optimal ratio of 1:1. In addition, the production of acetate was reduced in Ct(Ptkt-adhE2) which was in accordance with the increased synthesis of butyrate.

Figure 3 Butyrate, butanol and acetate fermentation performance of Ct(Ptkt-adhE2)

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