Part:BBa_K2382006

T7 promoter + Thioredoxin-MSMEG_5998 fusion protein

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 456
  Illegal XhoI site found at 462
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 901
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

By ligating these two different parts as a fusion protein, it is supposed to raise the solubility of our protein, MSMEG_5998.

Characterization of the Thioredoxin-MSMEG_5998 fusion protein

Expression results ( IPTG induction )

Two of our composition parts (*) were synthesized by Allbio Life Co., Ltd and put into the standard backbone pSB1C3. First, we transformed all of our plasmids into E. coli BL21 (DE3) strain to express our proteins. Then IPTG was used to induce the expression system, since all plasmids 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 ran western blot. The results are demonstrated in the Fig 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 SDS-PAGE and coomassie brilliant blue staining. 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.