Part:BBa_K2382001

MSMEG_5998

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 AgeI site found at 428
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

To degrade aflatoxin in intestine as protection. We have chosen an enzyme of F420H2-dependent reductases family from Mycobacterium smegmatis. which can reduce and degrade aflatoxin with the aid of coenzyme F420.

Characterization of the MSMEG_5998

IPTG induction

MSMEG_5998 ( plasmid is from Australia) were transformed into E. coli BL21 (DE3) strain to express the protein. Then IPTG was used to induce the expression system, since the plasmid in our project had T7 promoter. We sonicated E. coli and did 9500 rpm and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To confirm the suitable concentration of cell supernatant, we do western blot. The results are demonstrated in figure 1. After centrifuging for two times, we could find a high percentage of proteins in the cell supernatant (the 13000 Su group).

figure 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: Cell lysates in the process of two times centrifuge were analyzed by western blot. 9500 T meant the initial sample gotten after sonication; 9500 P and 13000 T meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant the pellet and the supernatant gotten after 13000 rpm for 20 min.

References

(1)Taylor, M.C., et al., Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular microbiology, 2010. 78(3): p. 561-575.

(2)Lapalikar, G.V., et al., F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the Actinomycetales. PLoS One, 2012. 7(2): p. e30114.