Part:BBa_K4410003

Kan^R

This part is Kan^R gene, which is a kanamycin resistance gene from pKD4 vector. The gene encodes aminoglycoside phosphotransferase from Tn5，which confers resistance to neomycin, kanamycin, and G418 (Geneticin®).Therefore, only the bacteria carrying KanR gene can grow on the culture dish containing kanamycin, so as to achieve the goal of rapid screening of engineering bacteria. In our project, we used KanR gene to replace EcN1917 argR gene to eliminate argR feedback repression.

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 409
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1073
  Illegal NgoMIV site found at 1356
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 922
  Illegal SapI.rc site found at 1132
  

Results

ArgR protein acts as an arginine repressor of the arginine synthetic genes in EcN 1917, we successfully knocked down the argR gene in EcN1917 to increase the production of arginine in the strain. argR gene was replaced by Kan^R gene.

We first linearized the H1-argR-H2 fragment with upper and lower homologous arms by PCR from EcN1917 WT .The pMD19T vector and H1-argR-H2 were connected to form Recombinant pMD19T by TA cloning. H1-pMD19T-H2 was linearized by PCR. The Kan^R gene was linearized from pKD4 vector by PCR.Kan^R gene and H1-pMD19T-H2 gene were combined into recombinant pMD19T by one-step cloning. The recombinant pMD19T was heat transformed into a DH5α colony. Donor DNA (H1-Kan^R-H2) was linearly extracted from successfully transformed DH5a by PCR. Donor DNA was precipitated by alcohol and then electrotransformed into EcN1917 WT. We used Kan^R antibiotics to verify that the ArgR gene was knocked out. Hence, EcN^△argR strain, EcN with argR gene knocked out, was constructed successfully (Figure 1).

Figure 1: Gel electrophoresis results of EcN1917-△argR--Kan^R ( 2021bp)

From lines 2-10 of the result of gel electrophoresis, it can turn out that with ArgR knocked out from EcN, Kan^R sequence is successfully inserted into the bacteria.