Generator

Part:BBa_K3264025

Designed by: Boxiang Wang   Group: iGEM19_GreatBay_SZ   (2019-10-21)


tnaA

tnaA



BJU-China 2021 iGEM TEAM

TnaA is endogenous tryptophanase from E. coli that can convert tryptophan to indole. MaFMO is monooxygenase from Methylophaga aminisulfidivorans which could effectively synthesize indigo by oxidation of indole.

Description

In our project, TnaA-MaFMO is a combination used in 6BrIG production strategy in E. coli BL21(DE3). Since E. coli inherently carries TnaA, to prevent the influence of endogenous expression, we knock out TnaA in the genome, and then compensate TanA on the pET28b plasmid together with MaFMO. The expression of TnaA-MaFMO was controlled by T7-LacO inducible promoter, and IPTG acts as inducer.

T--BJU China--Fig.1 TanA-MaFMO1.png

Figure 1. Gene circuit of TnaA-MaFMO

Experiments and Results

We obtained TnaA gene and MaFMO gene by PCR and then assembled them to pET28b backbone by Gibson. Colony PCR and gene sequencing were use to verify the sequences. The following was the agarose gel electrophoresis of the results (Figure 2).

T--BJU China--Fig.2 TanA-MaFMO2.png

Figure 2. Agarose gel results of TnaA-MaFMO

Next, we want to test the expression of these enzymes with IPTG induction. For better expression of enzymes, we optimize the induction condition of gene expression: three different temperatures (18℃, 30℃, 37℃) and inducer concentrations (0mM, 0.1mM, 1mM) were set for test. We successfully detected TnaA-MaFMO enzyme after IPTG induction using SDS-PAGE (Figure 3). In the gel picture, we can clearly see TanA-MaFMO bands (about 52.8 KDa）. And we can find that TanA-MaFMO expresses significantly better at 30℃ and 37℃ than 18℃. When the inducer concentration is lower (0.1mM), the protein bands are more obvious, and even when there is no inducer, the gene also have a basic expression, that is also called leakage. The leakage may caused by high gene expression using high copy plasmid pET28b.

T--BJU China--Fig.3 TanA-MaFMO3.png

Figure 3. SDS-PAGE results of TnaA-MaFMO

In summary, we successfully constructed a plasmid including two enzymes TnaA and MaFMO, and detected their expression through the induction of IPTG.


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 1262
  • 1000
    COMPATIBLE WITH RFC[1000]


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