Part:BBa_K4407013

Plac-aceE，Expression of aceE in response to lactose

This part is responsible for expressing the aceE protein under the control of a Plac promotor and a terminator. It is composed of BBa_K4119006 (Plac), BBa_K4407002 (aceE), and BBa_K3585002 (terminator). aceE protein is a component of the PDH complex and catalyzes the conversion of pyruvate to acetyl-CoA and CO2. It can function well in aerobic environment. In our project, we used this device to provide a new metabolic pathway for C. tyrobutyricum in aerobic environment.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 368
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 368
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 368
  Illegal BglII site found at 428
  Illegal BglII site found at 2277
  Illegal BglII site found at 2685
  Illegal BamHI site found at 1237
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 368
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 368
  Illegal NgoMIV site found at 3307
  Illegal AgeI site found at 2018
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

1）Plasmid construction: pMTL-Plac-aceE

Using the recombinant plasmid pMTL-Pthl-aceE, we amplified and obtained a linearized pMTL-dps vector. Then, using the pMTL-perR-HR-tetR plasmid as template, we obtained the Plac promoter fragment. The Plac promotor fragment and linearized pMTL-aceE vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E. coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Plac-aceE by gel electrophoresis and gene sequencing (Figure 2).

Figure 1. Construction of the recombinant plasmid pMTL-Plac-aceE

Figure 2. Gel electrophoresis of colony PCR experiment for verification of correct transformation of plasmid pMTL-Plac-aceE into E .coli JM109

2) C. tyrobutyricum conjugated with pMTL-Plac-aceE

Using E. coli CA434 as the donor strain, the recombinant plasmid pMTL-Plac-aceE was conjugated into C. tyrobutyricum to construct the recombinant bacteria [Ct (Plac-aceE) strain], and the empty plasmid pMTL82151 was conjugated into C. tyrobutyricum to construct a control strain, Ct(control). The expression of aceE protein (99.7 kDa) in the Ct (Plac-aceE) strain was confirmed by SDS-PAGE (Figure 3).

Figure 3. SDS-PAGE confirmation of aceE gene expression in the recombinant C. tyrobutyricum (Ct) [(Ct (Plac-aceE): Ct conjugated with pMTL-Plac-aceE; Ct(control): Ct conjugated with empty plasmid pMTL82151]

To determine how aceE overexpression in C. tyrobutyricum affected the viability of the bacteria under aerobic and anaerobic conditions. We used OD600 to compare the growth of the Ct (Plac-aceE) and Ct(control) strains under these two conditions, respectively.

Under anaerobic condition, the maximum biomass (OD600) of the Ct (Plac-aceE) strain was lower than that of the control strain (Figure 4). It is speculated that under anaerobic conditions, the aerobic and anaerobic pathways from pyruvate to acetyl CoA in the recombinant strain may compete, which has a partial impact on the growth of the strain.

Under aerobic condition (100 rpm), the growth of the control strain was affected, and its delay period was longer than that of the Ct (Plac-aceE) strain (Figure 5). In addition, the growth rate of the Ct (Plac-aceE) strain was significantly higher than that of the control strain.

Figure 4. Growth analysis of the recombinant strain in anaerobic conditions [(Ct (Plac-aceE): Ct conjugated with pMTL-Plac-aceE; Ct(control): Ct conjugated with empty plasmid pMTL82151]

Figure 5. Growth analysis of the recombinant strain and control strain in aerobic conditions [(Ct (Plac-aceE): Ct conjugated with pMTL-Plac-aceE; Ct(control): Ct conjugated with empty plasmid pMTL82151]