Difference between revisions of "Part:BBa K1696011"
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This part is based on the BBa_K1172303 in Part Registry constructed by 2013 Team Bielefelf-Germany which was also aimed at producing riboflavins.
 
This part is based on the BBa_K1172303 in Part Registry constructed by 2013 Team Bielefelf-Germany which was also aimed at producing riboflavins.
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<img src="https://static.igem.org/mediawiki/2015/2/29/Riboflavin_metabolism.png" width="300px"/>
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<br>Fig1.Pathway for engineered riboflavin and FMN production in ''E.coli''.
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As we learn from metabolic flux ( Fig.1), it reveals the relevant pathways of riboflavin production and engineering strategies for riboflavin production in ''E.coli''. In their previous research(Tao, et al) [1] ,they construct a high-yield'' E.coli'' strain with a yield of 229.1 mg/L.
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Based on their study, we constructed a flavin producing part (ribABDEC cluster) named EC10.(Figure 2).
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<img src="https://static.igem.org/mediawiki/parts/a/ac/屏幕快照_2015-09-17_下午2.47.14.png" width="300px"/>
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Fig2. The design of this part.
  
 
The riboflavin biosynthesis genes ribABDEC from E. coli are overexpressed under the control of the inducible trc promoter. Enzymes encoded by the genes shown are: ribA, GTP cyclohydrolase II; ribB, 3,4-dihydroxy-2-butanone 4-phosphate synthase; ribD, fused diaminohydroxyphosphoribosylaminopyrimidine deaminase/ 5-amino-6-(5-phosphoribosylamino)uracil reductase; ribE, 6,7-dimethyl-8-ribityllumazine synthase; ribC, riboflavin synthase gene.
 
The riboflavin biosynthesis genes ribABDEC from E. coli are overexpressed under the control of the inducible trc promoter. Enzymes encoded by the genes shown are: ribA, GTP cyclohydrolase II; ribB, 3,4-dihydroxy-2-butanone 4-phosphate synthase; ribD, fused diaminohydroxyphosphoribosylaminopyrimidine deaminase/ 5-amino-6-(5-phosphoribosylamino)uracil reductase; ribE, 6,7-dimethyl-8-ribityllumazine synthase; ribC, riboflavin synthase gene.
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The RBS here is a weak syn-RBS to make the metabolic flux accumulate in riboflavin instead of FMN.
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<img src="https://static.igem.org/mediawiki/parts/c/c2/Ribo-3.jpg" width="300px"/>
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</html>
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Fig3. The production of EC10 in tubes.
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The part we designed, compared with BBa_K117230, has been well optimized and the yield of that reached 17 mg/L( as shown in Fig.4). We can see from the results, the functionality of their parts has successfully improved.
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<img src="https://static.igem.org/mediawiki/2015/2/25/Ribo-4.png" width="300px"/>
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</html>
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Fig4. The yield of riboflavin in different strains :EC10, Rf02S (Δpgi+EC10), EC10*(BBa_K1696010)
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For riboflavin measurements, culture samples were diluted with 0.05 M NaOH to the linear range of the spectrophotometer and the A444 was immediately measured, according to Chen’s method[1].
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:41, 17 September 2015

Riboflavin producing parts

This part is based on the BBa_K1172303 in Part Registry constructed by 2013 Team Bielefelf-Germany which was also aimed at producing riboflavins.


Fig1.Pathway for engineered riboflavin and FMN production in E.coli.

As we learn from metabolic flux ( Fig.1), it reveals the relevant pathways of riboflavin production and engineering strategies for riboflavin production in E.coli. In their previous research(Tao, et al) [1] ,they construct a high-yield E.coli strain with a yield of 229.1 mg/L.

Based on their study, we constructed a flavin producing part (ribABDEC cluster) named EC10.(Figure 2).

Fig2. The design of this part.

The riboflavin biosynthesis genes ribABDEC from E. coli are overexpressed under the control of the inducible trc promoter. Enzymes encoded by the genes shown are: ribA, GTP cyclohydrolase II; ribB, 3,4-dihydroxy-2-butanone 4-phosphate synthase; ribD, fused diaminohydroxyphosphoribosylaminopyrimidine deaminase/ 5-amino-6-(5-phosphoribosylamino)uracil reductase; ribE, 6,7-dimethyl-8-ribityllumazine synthase; ribC, riboflavin synthase gene.

The RBS here is a weak syn-RBS to make the metabolic flux accumulate in riboflavin instead of FMN.

Fig3. The production of EC10 in tubes.

The part we designed, compared with BBa_K117230, has been well optimized and the yield of that reached 17 mg/L( as shown in Fig.4). We can see from the results, the functionality of their parts has successfully improved.

Fig4. The yield of riboflavin in different strains :EC10, Rf02S (Δpgi+EC10), EC10*(BBa_K1696010)

For riboflavin measurements, culture samples were diluted with 0.05 M NaOH to the linear range of the spectrophotometer and the A444 was immediately measured, according to Chen’s method[1].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3410
    Illegal BamHI site found at 1370
    Illegal XhoI site found at 64
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 262
    Illegal AgeI site found at 992
    Illegal AgeI site found at 1034
    Illegal AgeI site found at 2752
    Illegal AgeI site found at 3163
  • 1000
    COMPATIBLE WITH RFC[1000]