Part:BBa_K2505030
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 our wiki page ([http://2017.igem.org/Team:TokyoTech/Experiment/TraI_ 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
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. It was caluculated from calibration curve that the wild type TraI produced 28nM of C8 and TraI (K34G) produced 42nM of C8.
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 showned 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 we could successfuly increase the productivity of C8. Notably, generation and functional identification the mutant TraI, 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.
Reference
(1). Pavan Kumar Reddy Kambam, Daniel J. Sayut, Yan Niu, Dawn T. Eriksen, Lianhong Sun (2008) Directed evolution of LuxI for enhanced OHHL production. Biotechnology and Bioengineering Volume 101, Issue 2 1 October 2008 Pages 263-272
(2). MATTHEW R. PARSEK, DALE L. VAL, BRIAN L. HANZELKA, JOHN E. CRONAN, E. P. GREENBERG (1999) Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4360-4365, April 1999 Biochemistry
| None |