Composite

Part:BBa_K2593008

Designed by: Huimin Li   Group: iGEM18_SSTi-SZGD   (2018-09-21)
Revision as of 09:47, 17 October 2018 by YuhangYang (Talk | contribs)

P43-RBS-tuaD-gtaB-T1

This part is a functional composite part, It consists of a P43 promotor, an RBS, and an operon of tuaD-gtaB which encode UDP-glucose dehydrogenase and UDP-gulcose pyrophosphoryase, respectively, to participate in the biosynthesis of HA precursor UDP- GlcUA.
Promoter P43(BBa_K1628006):P43 is a constitutive promoter that common use in Bacillus subtilis168.
RBS(BBa_K2593005): A strong ribosome binding site commonly used in Bacillus species, it is part of the pP43NMK shuttle vector.
tuaD(BBa_K1469002): tuaD is one of native gene of B. subtilis, it encodes UDP-glucose 6-dehydrogenase has 461 amino acid (also know as: UDP-GlcDH in megaterium).
gtaB(BBa_K1469005): gtaB gene encodes UTP--glucose-1-phosphate uridylyltransferase in Bacillus megaterium. The enzyme catalyzes the conversion of glucose 1-phospahate to UDP-glucose 1-phosphate.
T1 terminator(BBa_B0010):it is the most used terminator in bacteria.

Usage

This is a improved part of operon tuaD-gtaB (tuaD also known as: UDP-GlcDH in B.megaterium). we firstly characterized the tuaD and gtaB from 2014 saarland iGEM team by co-overexpressing tua-D and gtaB genes to further increase the accumulation of HA.
In our project, expression of the operon tuaD-gtaB was regulated under the control of a constitutive promoter P43, this operon is used for further increasing the production of the HA in B.subtilis, tuaD and gtaB gene, products regulate the last two steps in the synthetic pathway of UDP-GlcUA(Fig1).Cloning of operon tuaD-gtaB into B.subtilis 168E were confirmed by colony PCR polymerization .(Fig2)


In our experiment, by conducting CTAB experiments that form turbidity from a reaction between HA and CTAB solution, the results showed a remarkable increase in HA production when co-overexpressed tuaD-gtaB together (488mg/L, a 38% increase) (Figure3),In addition, Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Figure4 ).


T 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

Peng Jin, Zhen Kang, Panhong Yuan. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168[J].Metabolic Engineering, 2016:22-25

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