Part:BBa_K553001

A. tumefaciens TraI - OC8 HLA synthase

The Agrobacterium tumefaciens TraI synthase generates the OC8 HLA.

The gene is located on the Ti plasmid together with TraR (BBa_K553000), the other regulatory component of A. Tumefaciens quorum sensing.

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Improvement and Characterization

Group: [http://2017.igem.org/Team:TokyoTech Tokyo Tech 2017]

Author: Takuma Yasue

Summary of Improvement and Characterization

We found that the wild type TraI(BBa_K553001) did not suit for our genetic circuits because enzyme activity was weak. Thus, we constructed the improved TraI(BBa_K2505033) appropriate to our final circuits.

Result

Figure 1: 3OC8HSL production of TraI wild type and mutant
Sender E. coli were grown at 37℃ in liquid LB medium with 1μM of SAM. E. coli introduced empty vector was used as Negative Control.

The result of C8 production using the wild type TraI and mutants is shown in Figure 6. The RFU value of the TraI (K34G)-expressing cells was approximately 3-fold higher than that of the TraI-expressing cells. Other mutant didn’t show improvement of 3OC8HSL production. When these RFU values were converted to 3OC8HSL concentrations using the calibration curve obtained in the reagent assay (see the TraI Assay), they were calculated as 28 nM and 42 nM, respectively.

For more information, visit our page[http://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement TraI_Improvement page].

Discussion

In the previous study, it was considered that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the acyl-ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect.
Taken together, we conclude that increasing the productivity of C8 at 37℃ was successful. Notably, generation and functional identification the mutant traI gene (TraI-K34G) meet the medal criteria of ”parts improvement”, because the wild-type traI parts was registered in iGEM parts collection earlier. However, further improvement of C8 production is necessary to transmit the signal from bacteria to mammalian cells. Such improvement is possible through tuning the experimental conditions further.