Difference between revisions of "Part:BBa K2382002"

(→‎Characterization of the)
(→‎Expression results ( IPTG induction ))
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since the plasmid in our project had T7 promoter. We sonicated E. coli and did 9500 rpm
 
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
 
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 ..........(the 13000 Su group).
+
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 small amount of protein exist in the supernatant ( the 13000T group )(the 13000 Su group).
  
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[[File:Australian FGD.png|350px|thumb|left|figure 1]]
+
 
 +
[[File:Australian FGD.png|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.]]
 
<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 
<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 
<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 
<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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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===
 +
(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
  
 
===References===
 
===References===

Revision as of 15:12, 29 October 2017

F420-Dependent Glucose-6-phosphate Dehydrogenase


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 646
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 695
    Illegal XhoI site found at 961
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 355
    Illegal NgoMIV site found at 553
    Illegal AgeI site found at 319
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

The working condition of MSMEG_5998 includes the help from coenzyme F420. F420-dependent glucose-6- phosphate dehydrogenase (FGD) is the enzyme that reduces the F420 being used by MSMEG_5998 and make it available again.

Characterization of the F420-Dependent Glucose-6-phosphate Dehydrogenase

Expression results ( IPTG induction )

FGD ( 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 small amount of protein exist in the supernatant ( the 13000T group )(the 13000 Su group).


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.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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.

(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

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