Difference between revisions of "Part:BBa K2382015"
(→‎Expression results ( IPTG induction ))
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and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To
 
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
 
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).
+
pellet (the 8500 p group).
  
 
[[File:Synthetic FGD.png|350px|thumb|left|figure 1]]
 
[[File:Synthetic FGD.png|350px|thumb|left|figure 1]]
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brilliant blue staining. 9500 T meant the initial sample gotten after sonication; 9500 P and 13000 T
 
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
 
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.  
+
the pellet and the supernatant gotten after 13000 rpm for 20 min.
  
 
===References===
 
===References===

Revision as of 14:56, 29 October 2017

Thioredoxin-FGD fusion protein


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1030
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1079
    Illegal BamHI site found at 367
    Illegal XhoI site found at 373
    Illegal XhoI site found at 1345
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 739
    Illegal NgoMIV site found at 937
    Illegal AgeI site found at 703
  • 1000
    COMPATIBLE WITH RFC[1000]


Short Description

A fusion protein composed of thioredoxin and FGD(F420-dependent glucose-6-phosphate dehydrogenase). Between the two small proteins are flexible linkers.

Characterization of the Thioredoxin-FGD fusion protein

Expression results ( IPTG induction )

FGD were synthesized by Allbio Life Co., Ltd and put into the standard backbone pSB1C3. First, we transformed the 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 pellet (the 8500 p 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.

(3)Bashiri G, Rehan AM, Greenwood DR, Dickson JMJ, Baker EN. Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis. PLoS ONE 5(12): e15803. doi:10.1371/journal.pone.0015803