Difference between revisions of "Part:BBa K2505030"

Revision as of 19:50, 31 October 2017

Ptet-rbs-traI (K34G)-tt

Sequence and Features

This part constitutively produces C8. We introduced point mutation to traI gene, the productivity of C8 was improved by x fold. We introduced this part to E. coli then E. coli could produced enough C8 to induce transcription of human cells.

Characterization and improvement

Quorum sensing is the cell-to-cell communication system used by a variety of bacteria. Signal molecules used in quorum sensing are chemically diverse, and the acyl-homoserine lactone (AHL)-type molecules are the most studied and employed ones in synthetic biology. luxI (Vibrio fischeri) and traI (Agrobacterium fumigatus) encode the AHL synthases for 3OC6HSL and 3OC8AHL, respectively. Chemical structures of these molecules are shown in Fig. 1.

The luxR gene of V. fischeri encodes intracellular receptor for 3OC6HSL.The complex of LuxR and 3OC6HSL binds to the responsive promoter, Plux, and activates transcription of downstream genes. Note that the luxI gene is one of such downstream genes. A similar mechanism is present for 3OC8HSL that is produced in A. fumigatus, and in this case, the receptor is encoded by the traR gene. Therefore, for both cases, the positive feedback loop of transcription is formed, and when the concentration of AHLs exceeds a threshold level, specific transcription is induced rapidly. As a consequence, bacterial cells can sense their population density and carry out cell-density specific behaviors such as luminescence emission and pathogenicity exerting.
In a previous study, AHL-inducible eukaryotic gene expression system was developed based on TraR (1). In this system, expression from the eukaryotic promoter (CMV minimal promoter) is induced only in the presence of 3OC8HSL. Therefore, we chose 3OC8HSL as a signal molecule and tried to make E. coli cells produce 3OC8HSL.

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.

The result of C8 production using the TraI wild-type and the mutants is shown in Figure 1. The RFU value of the TraI-K34G-expressing cells was about 3-fold higher than that of the TraI-expressing cells. E. coli introduced empty vector was used as Negative Control. 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.

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. It was also showed that the MG1655hapB strain produced more C8 than the DH5α strain. We speculate that the difference in permeability of hydrophobic compounds through the cell membrane is the main reason for this result. 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.

Material and Method

Supernatant assay The procedure is the same as that described in the previous wiki page (TraI assay) except that (i) cells were cultured only at 37, (ii) 1 mM of SAM was added to the culture medium at the step1, and (iii) two strains (DH5α, MG1655hapB) was used.