Part:BBa_K3221005

HisG-MF

HisG is a domain which can transform adenosine to pentostatin, protecting the cordycepin deaminate by adenosine deaminase. The primers we use to get this part is provided in the reference.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 127
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This part is the CDS of HisG-MF, which is half of Cns3 containing the HisG domain and is designed from the reference. It has been validated by 2019 SCU-China first for expression and function in S. cerevisiae.

Cns3 is an enzyme originated from Cordyceps militaris, it catalyzes the biosynthesis of Pentostatin (2’-deoxycoformycin, PTN), an ada chemotherapeutic drug used for the treatment of several forms of leukemia.It has been proved that PTN can protect the stability of cordycepin from deamination by inhibiting adenosine deaminase (ADA) activity in the natural pathway of COR in C.militaris.Therefore, in our designs, we make use of Cns3, producing PTN together with COR in order to guarantee a high yield of COR.Previous studies have also proved that the NK domain of Cns3, one of it's two function domains, is responsible for PTN biosynthesis, which is why we only use part of Cns3.

As is mentioned above, this part is an expression cassette of HisG-MF that is half of Cns3 containing the HisG domain. 2019 SCU-China validated its both in expression and function in Saccharomyces cerevisiae.To verify it’s function, we did RT-qPCR to detect the existence of Cns3-MF mRNA. (Figure. 1) As for function validation, we performed HPLC to detect PTN, the catalysate of Cns3-MF(Figure. 2). And we successfully demonstrated the cns3-mf transcription and function finally.

Figure. 1 RT-qPCR of Cns3-HisG-mf\Cns3-HisG-only.
No-load is the S. cerevisiae without transformed plasmids. The reference gene is PGK1. The RNA of cns3 is detected successfully.

Figure. 2 HPLC analysis of PTN
a. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG-MF sample. The strain BY4741 was culturing in YPDG media for 3 days.
b. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG ONLY sample. The strain BY4741 was culturing in YPDG media for 3 days.
c. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.