Difference between revisions of "Part:BBa K4407012"

(→‎2) C. tyrobutyricum conjugated with pMTL-Plac-dps)
(→‎1) Plasmid construction)
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Using the recombinant plasmid pMTL-Pthl-dps (refer to BBa_K4407010), 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-dps 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-dps by gel electrophoresis and gene sequencing (Figure 2).
 
Using the recombinant plasmid pMTL-Pthl-dps (refer to BBa_K4407010), 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-dps 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-dps by gel electrophoresis and gene sequencing (Figure 2).
 
[[File:fig-12-1.png|200px|thumb|left|Figure1. Construction of the recombinant plasmid pMTL-Plac-dps]]
 
[[File:fig-12-1.png|200px|thumb|left|Figure1. Construction of the recombinant plasmid pMTL-Plac-dps]]
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[[File:fig-12-2.png|200px|thumb|left|Figure2. Gel electrophoresis of colony PCR experiment for verification of correct transformation of plasmid pMTL-Plac-dps into E .coli JM109]]
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[[File:fig-12-2.png|200px|thumb|center|Figure2. Gel electrophoresis of colony PCR experiment for verification of correct transformation of plasmid pMTL-Plac-dps into E .coli JM109]]
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===2)  C. tyrobutyricum conjugated with pMTL-Plac-dps===
 
===2)  C. tyrobutyricum conjugated with pMTL-Plac-dps===
 
Using E. coli CA434 as the donor strain, the recombinant plasmid pMTL-Plac-dps was conjugated into C. tyrobutyricum to construct the recombinant bacteria [Ct (Plac-dps) strain], and the empty plasmid pMTL82151 was conjugated into C. tyrobutyricum to construct a control strain, Ct(control). The expression of dps protein in the Ct (Plac-dps) strain was confirmed by SDS-PAGE (Figure 3).  
 
Using E. coli CA434 as the donor strain, the recombinant plasmid pMTL-Plac-dps was conjugated into C. tyrobutyricum to construct the recombinant bacteria [Ct (Plac-dps) strain], and the empty plasmid pMTL82151 was conjugated into C. tyrobutyricum to construct a control strain, Ct(control). The expression of dps protein in the Ct (Plac-dps) strain was confirmed by SDS-PAGE (Figure 3).  

Revision as of 08:46, 8 October 2022


Plac-dps，Expression of dps in response to lactose

This part is responsible for expressing dps protein in response to lactose under the control of a Plac promotor and a terminator. It is composed of BBa_K4119006 (Plac), BBa_K4407001 (dps), and BBa_K3585002 (terminator). dps protects the bacteria from oxidative damage. It protects DNA against oxidative damage by binding with Fe2+ to prevent it from reacting with H2O2 (Fenton’s reaction). In our project, we used this device to enhance the viability of 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 368
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 368
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

1) Plasmid construction

Using the recombinant plasmid pMTL-Pthl-dps (refer to BBa_K4407010), 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-dps 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-dps by gel electrophoresis and gene sequencing (Figure 2).

Figure1. Construction of the recombinant plasmid pMTL-Plac-dps
Figure2. Gel electrophoresis of colony PCR experiment for verification of correct transformation of plasmid pMTL-Plac-dps into E .coli JM109

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

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

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

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

Under anaerobic condition, the maximum biomass (OD600) of the Ct (Plac-dps) strain was similar to that of the Ct(control) strain (9.67 vs 9.18), with overlapping error bars (Figure 4). Therefore, dps expression in the Ct (Plac-dps) strain made no significant difference in the maximum growth rates.

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

Under aerobic condition (100 rpm), the growth of the Ct(control) strain was affected, with a longer lag phase (Figure 5). Moreover, although there was no significant difference between the maximum biomasses of the two strains, the growth rate of the Ct (Plac-dps) strain was greater than the control strain. Therefore, overexpressing dps protein could improve the growth of C. tyrobutyricum under aerobic conditions.

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