Composite

Part:BBa_K4644011

Designed by: Han Cao   Group: iGEM23_BUCT   (2023-10-06)


pCS27-lac-crnA-creJ-racE

Creatinine degradation module This part catalyze the hydrolysis of Creatinine to sarcosine and urea.

Fig 1. Degradation of Creatinine

Usage

1.Construction of expression plasmid

As part of the module validation, we wanted to investigate the performance data of the creatinine degradation module. Therefore, we conducted separate fermentation and supplementation experiments for this module. Considering the requirements for the expression levels of these two enzymes, we selected the pCS27 plasmid with medium copy number and used the lac promoter to express the desired proteins under IPTG induction. To do this, we separately constructed two plasmids, pCS-lac-crnA and pCS-lac-creJ, with the primers as mentioned above. Additionally, we amplified the plasmid backbone of pCS27 and the homologous recombination fragment of the glutamate racemase (details can be found in the complete pathway fermentation experiment). We first connected the three fragments using overlap PCR and then performed homologous recombination with the plasmid backbone to obtain the required plasmid for this module. The plasmid map is shown in the figure.

Fig 2. PCS27 lac crnA creJ lac race

2.Fermentation addition experiment

We will amplify the constructed plasmid in E.coli DH5α through chemical transformation method. The transformed cells will be spread onto solid LB agar plates supplemented with kanamycin for antibiotic selection. Positive colonies will be selected (Figure 3) and subjected to colony PCR screening (Figure 4) using primers overlapping the target region. The colonies carrying the correct plasmid will be cultured in liquid LB medium supplemented with kanamycin at 37°C on a shaker overnight. Plasmid DNA will be subsequently extracted. To ensure the correctness of the plasmid sequence, it will be sent to a genetic company for sequencing, confirming its accuracy.

Fig 3. The result of Plasmid transformation.

Fig 4. The result of Colony PCR.

The carrier bacterium to be used, Nissle 1917 (EcN), was pre-cultured overnight at 37°C in LB medium without antibiotics. Plasmid transformation was carried out using electroporation, and the transformed plasmids were selected on solid LB medium with kanamycin resistance. Positive colonies were picked and inoculated into liquid LB medium with kanamycin resistance, followed by overnight incubation at 37°C. Subsequently, they were inoculated into 50 mL of M9 medium with kanamycin resistance, induced with IPTG, and supplemented with the substrate creatinine (1 mM). The cultures were then incubated on a shaker at 30°C for 48hours.

Fig 5. The result of Electroconversion.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 56
    Illegal PstI site found at 284
    Illegal PstI site found at 2078
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 56
    Illegal NheI site found at 1703
    Illegal NheI site found at 2987
    Illegal PstI site found at 284
    Illegal PstI site found at 2078
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 56
    Illegal BglII site found at 2679
    Illegal BamHI site found at 866
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 56
    Illegal PstI site found at 284
    Illegal PstI site found at 2078
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 56
    Illegal PstI site found at 284
    Illegal PstI site found at 2078
    Illegal NgoMIV site found at 503
    Illegal NgoMIV site found at 745
    Illegal NgoMIV site found at 1835
    Illegal NgoMIV site found at 2000
    Illegal NgoMIV site found at 2867
  • 1000
    COMPATIBLE WITH RFC[1000]


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