Difference between revisions of "Part:BBa K2382015"

 
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A fusion protein composed of thioredoxin and FGD(F420-dependent glucose-6-phosphate dehydrogenase). Between the two small proteins are flexible linkers.
 
  
 
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===Usage and Biology===
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==Usage and Biology==
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<span class='h3bb'>Sequence and Features</span>
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===<span class='h3bb'>Sequence and Features</span>===
 
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<partinfo>BBa_K2382015 parameters</partinfo>
 
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===Short Description===
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A fusion protein composed of thioredoxin and FGD(F420-dependent glucose-6-phosphate dehydrogenase). Between the two small proteins are flexible linkers.
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===Expression results===
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=====IPTG induction=====
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FGD were synthesized by Allbio Life Co., Ltd and put into
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the standard backbone pSB1C3. First, we transformed the plasmids into E. coli BL21 (DE3)
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strain to express our proteins. Then IPTG was used to induce the expression system,
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since all plasmids in our project had T7 promoter. We sonicated E. coli and did 9500 rpm
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and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To
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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
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cell supernatant (the 13000 Su group).
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[[File:Synthetic FGD.png|350px|thumb|left|figure 1]]
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Figure 1: Cell lysates in the process of two times centrifuge were analyzed by SDS-PAGE and coomassie
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brilliant blue staining. 9500 T meant the initial sample gotten after sonication; 9500 P and 13000 T
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meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant
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the pellet and the supernatant gotten after 13000 rpm for 20 min.
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===References===
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(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.
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(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.
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(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

Revision as of 23:39, 26 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.

Expression results

IPTG induction