Difference between revisions of "Part:BBa K2382001"
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| + | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K2382001 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2382001 SequenceAndFeatures</partinfo> | ||
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<partinfo>BBa_K2382001 parameters</partinfo> | <partinfo>BBa_K2382001 parameters</partinfo> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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This is an enzyme that could degrade aflatoxin with the aid of coenzyme F420. It belongs to the | 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. | F420H2-dependent reductases family from Mycobacterium Smegmatis. | ||
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| − | === | + | </p> |
| + | </html> | ||
| + | __TOC__ | ||
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| + | ==Characterization of the MSMEG_5998== | ||
| + | ===The Test of Solubility=== | ||
| + | <div style="text-align:justify;"> | ||
MSMEG_5998 ( plasmid is from Australia) were transformed into E. coli BL21 (DE3) | 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, | strain to express the protein. Then IPTG was used to induce the expression system, | ||
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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 | 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). | cell supernatant (the 13000 Su group). | ||
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[[File:Astralian MSMEG5998.png|350px|thumb|left|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 | [[File:Astralian MSMEG5998.png|350px|thumb|left|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 | 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.]] | ||
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| + | ===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. | ||
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| − | + | ====Result==== | |
Revision as of 11:00, 31 October 2017
MSMEG_5998
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 428
- 1000COMPATIBLE 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.
Contents
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).
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. Then we use Western Blot mehtod to amalyze the quantaty of MSMEG_5998 at each time spot.
Result
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.