Difference between revisions of "Part:BBa K2382009"
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==Usage and Biology==
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===<span class='h3bb'>Sequence and Features</span>===
 
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<partinfo>BBa_K2382009 SequenceAndFeatures</partinfo>
 
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===Short Description===
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===Usage and Biology===
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A fusion protein composed of thioredoxin and MSMEG_5998. Between the two small proteins are flexible linkers.
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This is a fusion protein of Thioredoxin (BBa_K2382004) and MSMEG_5998 (BBa_K2382001).We also call this part the "Synthetic MSMEG5998" in order to make a distinction with the original MSMEG_5998 from Australia. By our experiments, we found that the Synthetic MSMEG5998's ability of aflatoxin degrading is better than MSMEG_5998 alone.
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__TOC__
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==Characterization of the Thioredoxin-MSMEG_5998 fusion protein==
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===Protein Expression Over Time===
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We transformed the plasmids that contained this part (BBa_K2382006) 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.
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Then we use Western Blot mehtod to amalyze the quantaty of Thioredoxin-MSMEG_5998 (Synthetic MSMEG_5998) at each time spot.
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'''Discussion'''
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[[File:growth curve 1.png|450px|thumb|left|Figure 4 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.]]
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According to the data shown above, the growth curve of E.coli BL21 with Synthetic MSMEG_5998 reached the ceiling when the O.D. value was approximately at 2 while the  amount of Synthetic MSMEG_5998 were still increasing.
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Though the amount of Synthetic MSMEG_5998 increased consistently with time, we could not jump to conclusions that it was proper to incubate E.coli as long as possible. Another consideration was the time it would take. Just as our expected, it growed fast at the first 2.5 hours. That’s why we also chose 2.5hr after induced by IPTG when we  extracted Synthetic MSMEG_5998 from total cell lysate in other experiments.
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===Expression results===
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[[File:growth curve BL21 2.png|450px|thumb|left|Figure 5 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.]]
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=====IPTG induction=====
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<p align="justify">
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MSMEG_5998 was 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 ranwestern 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 MSMEG5998.png|350px|thumb|left|figure 1]]
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Based on previous experience, if the E.coli was incubated over 4 hours, the protein that it expressed may be degraded or mis-folded, leading to malfunction. As a result, it was also an important issue for this modeling. However, because of the lack of F420, we did not have the chance to check the enzyme activity of each time spot.  It was still unknown whether the titer of the Synthetic MSMEG_5998 would change or not and awaited further research.
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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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[[File:Synthetic MSMEG5998 western.png |450px|thumb|left|Figure 6 : 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.]]
  
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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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Latest revision as of 16:10, 1 November 2017

Thioredoxin-MSMEG_5998 fusion protein


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 367
    Illegal XhoI site found at 373
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 812
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This is a fusion protein of Thioredoxin (BBa_K2382004) and MSMEG_5998 (BBa_K2382001).We also call this part the "Synthetic MSMEG5998" in order to make a distinction with the original MSMEG_5998 from Australia. By our experiments, we found that the Synthetic MSMEG5998's ability of aflatoxin degrading is better than MSMEG_5998 alone.

Characterization of the Thioredoxin-MSMEG_5998 fusion protein

Protein Expression Over Time

We transformed the plasmids that contained this part (BBa_K2382006) 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 Thioredoxin-MSMEG_5998 (Synthetic MSMEG_5998) at each time spot.


Discussion

Figure 4 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.

According to the data shown above, the growth curve of E.coli BL21 with Synthetic MSMEG_5998 reached the ceiling when the O.D. value was approximately at 2 while the amount of Synthetic MSMEG_5998 were still increasing.

Though the amount of Synthetic MSMEG_5998 increased consistently with time, we could not jump to conclusions that it was proper to incubate E.coli as long as possible. Another consideration was the time it would take. Just as our expected, it growed fast at the first 2.5 hours. That’s why we also chose 2.5hr after induced by IPTG when we extracted Synthetic MSMEG_5998 from total cell lysate in other experiments.

Figure 5 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.

Based on previous experience, if the E.coli was incubated over 4 hours, the protein that it expressed may be degraded or mis-folded, leading to malfunction. As a result, it was also an important issue for this modeling. However, because of the lack of F420, we did not have the chance to check the enzyme activity of each time spot. It was still unknown whether the titer of the Synthetic MSMEG_5998 would change or not and awaited further research.

Figure 6 : 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.