Part:BBa_K2325103

An expression device for fusion protein of Formamidase and Phosphite dehydrogenase

The BBa_K2325001 harbors a coding sequence of and formamidase(FOR) and phosphite dehydrogenase (PTDH) with a linker between them, which can simultaneously catalyzes the conversion of formamide to ammonia and phosphite to phosphate, respectively. The device also contains GST tag for increasing the solubility of exogenous protein.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 821
  Illegal NgoMIV site found at 1308
  Illegal NgoMIV site found at 1503
  Illegal NgoMIV site found at 1650
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 159
  

Gel analysis

M：1kb DNA Marker P;
1: plasmid from BL21(DE3) (pGEX-for-ptx);
2&3: plasmid from BL21(DE3) (pETDuet-for-ptx).

Usage and Biology

We digested and ligated the pGEX-2T vector and fusion protein gene and then transformed it into E.coli strain BL21(DE3). We also constructed an co-expressed vector pETDuet-for-ptx where these two genes are expressed separately.

Figure 1:Growth curves of E.coli (with two ways of constructed plasmids possessing the formamidase gene and phosphite dehydrogenase gene) in a basal MOPS medium (Na₂HPO₃•5H₂0 (1.32mM) , NH₃CO (200mM)).

The results showed that the growth of recombinant strains of both construction ways are encouraging, indicating the recombinant strains were able to utilize formamide and phosphite at the same time, while the negative control group (BL21(DE3)) was unable to grow normally due to the lack of nitrogen and phosphorus sources.