Part:BBa_K2505030

Ptet-rbs-traI (K34G)-tt

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]

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

In the previous wiki page (TraI Assay), we describe that the productivity of 3OC8HSL in E. coli heavily depends on the culture temperatures. However, to complete our co-culture system, the current C8 productivity at 37℃ was not enough to transmit the AHL signal to mammalian cells; note that mammalian cells are usually grown at 37℃. Therefore, we tried to mutate the traI gene and increase the productivity of 3OC8HSL at 37℃.
TraI has not been characterized so extensively, and thus, it is unclear what kind of mutations is appropriate for the above purpose. A preceding study describes that, in the case of LuxI, the amino acid substitution at the 34th and 63rd positions (both are substitutions from glutamate to glycine; E34G and E63G) increase the productivity of C6 (1). Since TraI has homology to LuxI over the entire amino acid sequences, we speculate that the same amino acid substitutions in TraI can increase the productivity of 3OC8HSL. Also, we here describe the modification of the culture conditions and the host strain choice for increased 3OC8HSL production. In the experiments shown in this page, one additional modification was made in experimental conditions; 1 μM of SAM (S-adenosylmethionine; structure is shown in Figure 5) was added to the culture of the Sender. Since 3OC8HSL is synthesized from SAM and ACP (acyl carrier protein) through the action of TraI in bacterial cells (2), we expected that the addition of SAM may increase the productivity.

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.