Difference between revisions of "Part:BBa K2505030"
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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℃. | 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℃. | ||
==Result== | ==Result== | ||
| − | The result of C8 production using the TraI wild-type and the mutants is shown in Figure | + | [[File:http://2017.igem.org/File:TraImutationResults.jpg|thumb|left|600px| '''Figure 1:''' '''3OC8HSL production of TraI wild type and mutant'''<br style="clear: both" />Sender E. coli were grown at 37℃ in liquid LB medium with 1μM of SAM. ]]<br> |
| + | |||
| + | 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. | 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 | + | 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. | 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. | ||
| − | + | <br style="clear: both" /> | |
==Discussion== | ==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. | 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. | ||
Revision as of 19:25, 31 October 2017
Ptet-rbs-traI (K34G)-tt
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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℃.
Result
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.