Difference between revisions of "Part:BBa K2382001"
(→‎Characterization of the MSMEG_5998)
Line 48: Line 48:
 
===Protein Expression Over Time===
 
===Protein Expression Over Time===
  
−
====Material and Method====
 
−
<p align="justify">
 
 
We transformed the plasmids that contained MSMEG_5998(BBa_K2382001) and Thioredoxin-MSMEG_5998 fusion protein(BBa_K2382009) respectively into competent cell E.coli BL21. After cultured overnight, measure the ABS600 and diluting the LB medium to O.D.=0.1. Then incubate at 37℃, 150 rpm until the O.D. of the samples reach 0.4 to 0.6 . Add 80ul 100mM IPTG( final concentration : 0.4mM ) to 125 ml flask and return to 37°C. From then on, after measure the O.D. values, transfer 1 ml from the induced sample and centrifuge at maximum speed for 60 seconds at RT and remove supernatant at 0, 1, 2, 3, 4, 5, 6, 7, 8 hours and 0, 0.5, 1.0, 1.5, 2.0 ,2.5 , 3.0, 3.5, 4 hours.  
 
We transformed the plasmids that contained MSMEG_5998(BBa_K2382001) and Thioredoxin-MSMEG_5998 fusion protein(BBa_K2382009) respectively into competent cell E.coli BL21. After cultured overnight, measure the ABS600 and diluting the LB medium to O.D.=0.1. Then incubate at 37℃, 150 rpm until the O.D. of the samples reach 0.4 to 0.6 . Add 80ul 100mM IPTG( final concentration : 0.4mM ) to 125 ml flask and return to 37°C. From then on, after measure the O.D. values, transfer 1 ml from the induced sample and centrifuge at maximum speed for 60 seconds at RT and remove supernatant at 0, 1, 2, 3, 4, 5, 6, 7, 8 hours and 0, 0.5, 1.0, 1.5, 2.0 ,2.5 , 3.0, 3.5, 4 hours.  
 
Then we use Western Blot mehtod to amalyze the quantaty of MSMEG_5998 at each time spot.
 
Then we use Western Blot mehtod to amalyze the quantaty of MSMEG_5998 at each time spot.
 
</p>
 
</p>
  
 +
[[File:growth curve 1.png|700px|thumb|left|Figure 1 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.]]
  
−
====Result====
+
[[File:growth curve BL21 2.png|700px|thumb|left|Figure 2 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.]]
  
 +
[[File:Synthetic MSMEG5998 western.png |700px|thumb|left|Figure 3 : Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.
 +
]]
  
−
[[File:growth curve 1.png|700px|thumb|center|Figure 1 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.]]
+
<br style="clear: both" />
−
 
+
−
[[File:growth curve BL21 2.png|700px|thumb|center|Figure 2 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.]]
+
−
 
+
−
[[File:Synthetic MSMEG5998 western.png |700px|thumb|center|Figure 3 : Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.
+
−
]]
+
  
 
===References===
 
===References===

Revision as of 11:07, 31 October 2017

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

This is an enzyme that could degrade aflatoxin with the aid of coenzyme F420. It belongs to the F420H2-dependent reductases family from Mycobacterium Smegmatis.


Characterization of the MSMEG_5998

The Test of Solubility

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: 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.


Protein Expression Over Time

We transformed the plasmids that contained MSMEG_5998(BBa_K2382001) and Thioredoxin-MSMEG_5998 fusion protein(BBa_K2382009) respectively into competent cell E.coli BL21. After cultured overnight, measure the ABS600 and diluting the LB medium to O.D.=0.1. Then incubate at 37℃, 150 rpm until the O.D. of the samples reach 0.4 to 0.6 . Add 80ul 100mM IPTG( final concentration : 0.4mM ) to 125 ml flask and return to 37°C. From then on, after measure the O.D. values, transfer 1 ml from the induced sample and centrifuge at maximum speed for 60 seconds at RT and remove supernatant at 0, 1, 2, 3, 4, 5, 6, 7, 8 hours and 0, 0.5, 1.0, 1.5, 2.0 ,2.5 , 3.0, 3.5, 4 hours. Then we use Western Blot mehtod to amalyze the quantaty of MSMEG_5998 at each time spot.

Figure 1 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.
Figure 2 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.
Figure 3 : Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.


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.