TraI Improvement Assay

Introduction

In previous study (TraI Assay), we found that the amount of C8 production heavily depend on culture temperature. But to construct co-culture system, current TraI’s C8 production in 37℃ is not enough to send AHL signal to mammalian cells. So, we mutate TraI gene and tried to improve the amount of C8 production in 37℃.
A report says LuxI’s C6 production got 72 folds compared to wildtype by mutating at 34th amino position and 63th amino position. We focused that LuxI gene and TraI gene have homology and mutated at 34th amino position and 63th amino position (1).
After experiment in various condition, we found that TraI gene mutated at 34th amino position shows 3 folds of RFU compared to wild type in LB medium with 1μM of SAM (S‐adenosylmethionine).
AHL is derived from SAM and TraI involved in a reaction of SAM and ACP (acyl carrier protein) to produce AHL (2).
At last we also found that C8 production is depend on strain.But experiment missed iGEM presentation

Summary of experiment

At first, we designed primer to introduce mutation at 34th position and 63th position and mutate TraI gene. Primer sequence is shown Figure. 1.

Then we added 1μM of SAM (structure is shown Figure.2) to sender E. coli’s culture because SAM is ingredients of AHL.

At last, we confirmed that TraI gene mutated at 34th amino position shows 3 folds of RFU compared to wild type in 37℃ and performed same experiment in 25℃.
The plasmids we used are shown in Fig.4~6. Same reporter E. coli as the TraI assay was used. We made E. coli with wild type TraI or mutated TraI at 34th amino position as Sender E. coli and confirmed the difference in C8 production. The sequence of TraI mutant and wild-type is shown in Fig. 3.

Results

C8 production of TraI wildtype and mutant is shown in Figure. 2.
RFU value of mutant is about 3 folds larger than wildtype.
Calculated from the graph obtained in the reagent assay,
3OC8HSL concentration of TraI Wild type culture was nM and TraI mutant culuture was nM.

But mutant advantage is disappeared by lowering culture temperature to 25℃.
The value of RFU exceeded the detection limit of the graph obtained in the reagent assay. Since the value of RFU peaks at more than 100 nM, both cultures are considered to synthesize more than 200nM of 3OC8HSL.

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.

Discussion

In previous study, it is found that LuxI gene mutation at 34th amino position most likely enhances the interactions between the enzyme and the acyl ACP substrate. Therefore we thought that this TraI gene mutation at 34th amino position also enhances the interactions between the enzyme and the acyl ACP substrate. But in 25℃ of culture, the effect of interaction improvement is disappeared because it is thought that thermal motion of protein become calm and the acyl ACP substrate stably bind the enzyme in case of TraI wildtype. Consequently, we improved TraI gene’s C8 production in 37℃ condition same as temperature of human body.
We also found that MG1655hapb strain produce more C8 than DH5αstrain.
It is thought that strain dependence of C8 production resulted from permeability of E. coli’s cell
membrane because MG1655hapB strain has higher permeability compared to its wildtype MG1655.
We expect further improvement of C8 production to send a signal to mammalian cells. I hope the day in which human can talk with microorganism as a same living thing.

Materials and Methods

Supernatant assay
1.Cultivate Sender E. coli in LB medium for about 15hours
2.Centrifuge the culture 16,000rpm and 5minutes
3.Follow Reagent assay process (1~4) and Prepare Reporter culture.
4.Mix 250μL of sender culture’s supernatant with Reporter culture in micro tube.
5.Incubate the micro tube for 5 hours with Small shaking incubator in 37℃.
6.Take 100μL of culture and Measure fluorescent (excitation wave length is 495nm, Measurement wavelength is 520nm gain is 45) and absorbance (Measurement wavelength is 600nm).

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