Coding

Part:BBa_K4644001

Designed by: Han Cao   Group: iGEM23_BUCT   (2023-09-23)


creJ

Arthrobacter nicotianae creJ protein coding region.This part is useful for forcatalyzing the hydrolysis of creatine to sarcosine and urea.

Biology

In the enzymatic measurement of creatinine, creatinase is an important medical enzyme forcatalyzing the hydrolysis of creatine to sarcosine and urea.

Fig 1. Degradation of Creatinine

Usage

The second part of PCs27 lac crnA creJ lac raceExpression box

We constructed the first expression box using these three genes. We first amplified the cloned fragments of crnA and creJ by PCR, and obtained the first plasmid through homologous recombination with the PCs27 plasmid. As we learned that creJ has higher activity than crnA, we placed it in a lower position. Subsequently, we co recombinant this complete expression box with lac race into the PCS27 plasmid.

Fig 2. PCS27 lac crnA creJ lac race

Characterization

1. Agarose Gel Electrophoresis:

        Since Arthrobacter nicotianae strain 23710 is not currently preserved in our laboratory, we ordered the gene sequence from BGI Genomics. We amplified the sequence from the PMV plasmid with the fused creJ gene obtained from the company. This sequence will be used for homologous recombination with the pcs27 plasmid. The amplification results are shown in Figure 2.

Fig 3. creJ(left)

        After attaining the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 2.

Fig 4. The result of plasmid cut with enzyme KpnI(F) and BamHI(R). Plasmid: PCS27.

Reference

Song Liu, Jun Dai, Zhen Kang, Jianghua Li, Jian Chen & Guocheng Du (2015) Production of novel NaN3-resistant creatine amidinohydrolase in recomcombinant Escherchia coli, Bioengineered, 6:4, 248-250, DOI:10.1080/21655979.2015.1052919

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 358
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 358
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 959
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 358
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 358
    Illegal NgoMIV site found at 115
    Illegal NgoMIV site found at 280
    Illegal NgoMIV site found at 1147
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds
//chassis/prokaryote/ecoli
Parameters
biologyArthrobacter nicotianae strain 23710
protein creJ