Part:BBa_K3606814

mcbBCD

Usage and Biology:

This part is an antibiotic coding gene cluster with proteins for MccB17 maturation.

Design:

Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we wanted to know whether one promoter can start three consecutive genes. We successfully cloned McbBCD into the pGEX plasmid together downstream one promoter and transcribed it into BL21. After 5 hours of induction of McbG expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbG product was produced.

Results:

Figure 1. SDS-PAGE result. lane 1: mcbA lane 2: mcbA+IPTG lane 3: mcbE lane 4: mcbE+IPTG lane 5: mcbG lane 6: mcbG+IPTG lane 7: mcbBCD lane 8: mcbBCD+IPTG lane 9: pGEX+ IPTG

Comparing the 3th and 4th lanes, after IPTG induction,the difference is small. This may be due to the low expression of McbBCD products. We still need to experiment more to find the problem.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2062
  Illegal PstI site found at 2095
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2062
  Illegal PstI site found at 2095
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1969
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2062
  Illegal PstI site found at 2095
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2062
  Illegal PstI site found at 2095
  Illegal NgoMIV site found at 1724
  Illegal AgeI site found at 1896
  
• 1000
  COMPATIBLE WITH RFC[1000]