Team:TokyoTech/Project/Basic Parts
<!DOCTYPE html>
Basic Parts
TokyoTech 2017 iGEM Team Basic Parts
| Name | Type | Description | Design | Length(bp) |
|---|---|---|---|---|
| BBa_K2505000 | Transrational unit | rbs-ahk4 | Kohei Umedera | 3197 |
| BBa_K2505002 | Coding | atIPT4 | Takuma Yasue | 960 |
| BBa_K2505003 | Transrational unit | IVS-IRES-log1 | Hazuki Hasegawa | 1571 |
| BBa_K2505006 | Regulatory | Pcps-rbs(native) | Takuma Yasue | 708 |
| BBa_K2505033 (Best Basic Part) |
Coding | traI (K34G) | Kazunori Motai | 639 |
| BBa_K2505034 | Coding | traI (Q63G) | Kazunori Motai | 639 |
| BBa_K2505035 | Coding | traI (K34G, Q63G) | Kazunori Motai | 639 |
traI (K34G)
Introduction
In the TraI Assay page, we describe that the productivity of C8 in E. coli depends on the culture temperatures. However, to complete our co-culture system, the current 3OC8HSL (hereafter C8) productivity at 37℃ was not enough to transmit the AHL signal to human cells, because human cells are usually grown at 37℃. Therefore, we tried to mutate the traI gene and increase the productivity of C8 at 37℃ (Read TraI Imptovement page).
Results
When amino acid sequences of TraI and LuxI were aligned using the clustal W program (1), the E34 and E63 residues of LuxI were found to correspond to K34 and Q63 residues of TraR. According to this information, oligonucleotide primers to create TraI-K34G, TraI-Q63G, and TraI-K34G,Q63G mutants were designed. The primer sequences are shown in Fig. 1. The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.
The sequences of traI mutants and wild-type are shown in Fig. 1. The Sender and the Reporter strains were prepared in the same way as described in the TraI Assay page.
The result of C8 production using the TraI wild-type and the mutants is shown in Fig. 2. "W.T." means native traI.
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 did not show improvement of C8 production (data was not shown).
When these RFU values were converted to C8 concentrations using the calibration curve obtained in the reagent assay (Read TraI Assay page), they were calculated as 28 nM and 42 nM, respectively.
