Overview

After successful production of indigo, tyrian purple, and tyrian red in our E. coli. We needed a way to quantitively determine the titer of our two-celled system. Furthermore, we needed to determine the enzymatic rates of Fre-SttH and TnaA-Fmo to perfect our model (visit the modeling page for more). Thus, we performed HPLC and created standard calibration curves for indigo and two types of tyrian purple (6,6’-di-bromo-indigo and 6, 6’-di-chloro-indigo) to numerically assess the production of different compounds in our dye production.

Introduction

Qualitative data has always been something we needed but had trouble acquiring. It is extremely important — for comparing our different strains of TnaA-Fmo and comparing our titres to the titres of other researchers. Here, we utilize several different techniques to complete our measurements and acquire data on the different steps of our dye production process.

HPLC analysis of Fre-SttH

Fre-SttH is an enzyme which converts Tryptophan (Trp) into 6-Cl-Trp or 6-Br-Trp with the addition of Cl and Br salts (NaCl and NaBr in our case) respectively (visit our results page for more info). To find out its rate of conversion of Trp to 6-X-Trp, we cultured our ptac-Fre-SttH and took samples every 6 hours for 24 hours. We then performed HPLC analysis of our samples. We determined the peaks for trp and 6-X-trp using trp and 6-X-trp standards.

Figure 1. HPLC results for Fre-SttH samples. A) HPLC results for Trp, 6-Cl-Trp standards, and ptac-Fre-SttH with NaCl at 0h, 6h, 12h, 18h, and 24h from top to bottom respectively B) HPLC results for Trp, 6-Br-Trp standards, and ptac-Fre-SttH with NaBr at 0h, 6h, 12h, 18h, and 24h from top to bottom respectively.

We then turned our HPLC results into a concentration-time graph.

Figure 2. A.Concentration of Trp and 6-Cl-Trp in ptac-Fre-SttH with NaCl B.Concentration of Trp and 6-Br-Trp in ptac-Fre-SttH with NaBr

It was surprising to find that the enzymatic rates of Fre-SttH differed when different halogen salts were added (we speculate that this is due to the different atomic sizes of Cl and Br), and this data was used in our model.

Determining a solvent for indigoid dyes

To quantify our production of dye, we constructed calibration curves for our three dyes (indigo, 6, 6’-di-chloro-indigo, and 6, 6’-di-bromo-indigo). As indigoid dyes are not water-soluble, we need to determine a suitable organic solvent. We first attempted dimethylformamide (DMF) as our solvent, and successfully constructed an indigo calibration curve using concentration of 0.4, 0.2, 0.1, 0.05, and 0.01mM.

However, 6, 6’-di-chloro-indigo and 6, 6’-di-bromo-indigo had lower solubility than indigo in DMF. In 0.4mM solutions, 6, 6’-di-chloro-indigo and 6, 6’-di-bromo-indigo would precipitate out. Furthermore, DMF will react with the plastic on our 96 well plate, rendering readings inaccurate.

Therefore, we switched the solvent from DMF to dimethyl sulfoxide (DMSO), and lowered the concentration for the calibration curve to 0, 0.0025, 0.005, 0.01, 0.0125, 0.015, 0.02, and 0.0225mM.

Standard calibration curves for indigo, tyrian purple, and tyrian red

We then determined the optimal wavelength to construct our calibration curve by performing an observance scan on all three DMSO-dye solutions. The optimal wavelengths are 620, 604, and 610nm for the indigo, 6, 6’-di-chloro-indigo, and 6, 6’-di-bromo-indigo solutions respectively.

Figure3. The absorption spectra of indigo,6, 6’-di-chloro-indigo and 6, 6’-di-bromo-indigo in DMSO solution

Afterwards, the calibration curve was constructed with concentrations of 0, 0.005, 0.01, 0.0125, 0.015, 0.02, and 0.0225 mM.

Figure4. The Standard substance of 6, 6’-di-bromo-indigo , 6, 6’-di-chloro-indigo and indigo after DMSO dilution.L1.The Standard substance of 6, 6’-di-bromo-indigo after DMSO dilution.L2.The Standard substance of 6, 6’-di-chloro-indigo after DMSO dilution.L3.The Standard substance of indigo after DMSO dilution.

Figure 5. Calibration curves for DMSO-dye solutions A. Calibration curve for DMSO-indigo solution.

Figure 5. Calibration curves for DMSO-dye solutions B. Calibration curve for DMSO-6, 6’-di-chloro-indigo solution.

Figure 5. Calibration curves for DMSO-dye solutions C. Calibration curve for DMSO-6, 6’-di-bromo-indigo solution.

Sample analysis

Samples were diluted 10.5 times and scanned according to the optimal wavelength. Then, the calibration curve was used to determine the concentration of the DMSO-dye solutions and concentrations of the original cell culture. Example results is portrayed below.

Figure 6. Comparison of production titers of TnaA-RL-FMO, TnaA-FL-FMO, and TnaA-RBS-FMO with 1mM Trp, 6-Cl-Trp, or 6-Br-Trp added. Error bars denote two standard deviations from the mean.

Significance of our measurement

Our calibration curves for indigo, 6, 6'-di-chloro-indigo and 6, 6’-di-bromo-indigo can be directly used by other iGEM teams dealing with the same pigments. In order to use our calibration curves, other teams only need to set a new point at 0mM concentration and use the slope of our calibration curve. This saves cost for other iGEM teams as 6, 6'-di-chloro-indigo and 6, 6’-di-bromo-indigo standards are expensive (1100usd/g).

Furthermore, we developed a systemic method of measuring natural pigment production. Similar methods can be used by other iGEM teams in the future which deal with quantification of any natural pigment production.

Protocol

Protocol for measuring dye production titre

Materials:

1 machine capable of measuring OD (preferably from 350 to 800nm)

2 96-well plate (more for more samples)

3 Scale (preferably accurate to 4 decimal places)

4 Ultrasound cell breaker

5 Centrifuge for 1.5mL centrifuge tubes

6 20-200ul Pipette

7 100-1000ul Pipette

Indigo, 6, 6’-di-chloro-indigo, and 6, 6’di-bromo-indigo standard powder

Various 50mL centrifuge tubes

Various 2mL centrifuge tubes

Various 200ul PCR tubes

DMSO

Ice and water

Bacterial culture samples

Constructing standard calibration curves for indigo, tyrian purple, and tyrian red

1. Weigh around 0.001Xg of indigo

2. Dilute with DMSO to a final concentration of 0.225mM dye in a 50mL centrifuge tube

3. Using the 0.225mM indigo solution, make solutions of 0.02, 0.015, 0.0125, 0.01, 0.005, 0.0025, 0mM dye

4. Pipette 200ul of the 0.225mM solution into a well on the 96-well plate

5. Scan the well with the 0.225mM solution for absorbance intensity between 350nm and 800nm to determine the optimal wavelength for constructing the calibration curve (lowest reading should mean highest absorbance)

6. For each 0.225, 0.02, 0.015, 0.0125, 0.01, 0.005, 0.0025, 0mM dye solution, pipette 200ul into a well on a 96-well plate

7. Using the previously determined optimal wavelength, measure the OD value of each well at that wavelength

8. Construct the calibration curve with the two axes being concentration and absorbance (which axis is which is not important)

9. Repeat for 6, 6’-di-chloro-indigo and 6, 6’-di-bromo-indigo

Preparing & measuring bacterial samples

1. For each bacterial culture sample, pipette 2mL into 2mL centrifuge tubes

2. Centrifuge at 12000rpm for 15 minutes

3. Pour off supernatant, leaving the pellet inside the tube

4. Resuspend sample using 1mL water

5. Transfer 10ul of suspended liquid into a 200ul PCR tube

6. Centrifuge at 4000rpm for 10 minutes

7. Resuspend pellet using 200ul DMSO

8. Centrifuge at 4000rpm for 10 minutes (the pellet should not have dyes, resuspend and centrifuge again if there are dyes; ultrasound can be used to lyse the cells if there still are dyes after multiple resuspensions and centrifuged)

9. Pipette 200ul into a 96-well plate and measure the OD value at the optimal wavelength for the dye in question

Team:LINKS China/Measurement