TraI Improve Assay

Introduction

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℃.

Summary of experiment

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 (2). 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.

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 Figure 1. The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method described elsewhere, and successful introduction of the mutations were confirmed with Sanger sequencing.

Figure. 1 Sequences of the primers. Note that each primer set is divergent for inverse-PCR

The sequences of traI mutants and wild-type are shown in Figure 2. The plasmids we used are shown in Figures 3 and 4. The “Sender” and “Reporter” strains were prepared in the same was as described in the previous wiki page (TraI Assay).

Fig. 2 Sequence of traI wild type gene and mutant

Figure. 3 Structure of the plasmids used for creating the “Reporter” strain.

Figure. 4 Structure of the plasmids used for creating the “Sender” and “Mutated Sender” strains. One of these plasmids was used for preparing each transfromant

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 (3), we expected that the addition of SAM may increase the productivity.

Figure. 5 Chemical structure of SAM (S‐adenosylmethionine)

The result of C8 production using the TraI wild-type and the mutants is shown in Figure 6.
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.
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.

Figure. 6 Improvement of C8 production by the K34G mutant (37℃ culture)

Also, the other modification was made concerning the host strain of the Sender. The preceding iGEM study has shown that the amount of AHL produced by the luxI gene highly depends on the host strain; depending on the used strains as the Sender, there was approximatly100-fold difference in cell number for obtaining the same activation level of the lux promoter (4). Therefore, we here used the other strain, MG1655hapB, as the Sender.
Note that, in the experiments of the previous wiki page (TraI Assay), only the DH5α strain was used as a host. The MG1655hapB strain is a mutant of MG1655 (the representative wild-type K12 strain) and has higher membrane permeability for hydrophobic compounds compared to the parent.
As a result (Figure 8), we found that amount of C8 produced is dependent on strains. The RFU value in the MG1655hapB strain was about 3-fold higher than that in the DH5α strain. E. coli introduced empty vector was used as Negative Control
. When these RFU values were converted to C8 concentrations using the calibration curve obtained in the reagent assay (see the TraI Assay), they were calculated as 28 and 37 nM, respectively.

Strain dependence of AHL production
We found that Amount of C8 production is depend on E. coli’s strain. RFU is 2 folds larger than DH5α.
Calculated from the graph obtained in the reagent assay, 3OC8HSL concentration of DH5α culture was nM and MG1655hapB culture was nM.

Figure. 8 Strain dependence of C8 production

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.

Appendix: Material and Method

Reference

(1). http://www.genome.jp/tools-bin/clustalw
(2). 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
(3). 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
(4). https://2007.igem.org/wiki/index.php/Chiba/Quorum_Sensing