Fatty acid consumption Module

In our project, in order to treat acne, we need to improve the fatty acid decomposition ability of our engineering bacteria. In order to test whether the construction of our system is effective, we must find a suitable measurement method to measure the fatty acid concentration in the engineering bacteria culture environment, and judge the fatty acid decomposition ability by measuring the decline of fatty acid in the culture environment.

Here, we used extraction spectrophotometry to detect the concentration of free fatty acids in the culture environment of engineering bacteria. The basic principle is that free fatty acids can combine with copper ions to form fatty acid copper salts, which are dissolved in chloroform. Copper dissolved in chloroform can catalyze the oxidation of diphenylcarbazide by hydrogen peroxide to diphenylcarbazone; The color products produced by copper and diphenylcarbazone can be extracted by chloroform, so as to increase the absorbance of the organic phase at 550 nm, and then can be measured by spectrophotometer or enzyme labeling instrument.

Reference tube Measuring tube Blank tube Standard tube
Distilled water (μL) 30
Sample (μL) 30
Chloroform (μL) 30
Standard (μL) 30
Reagent I (μL) 50 50 50 50
Reagent II (μL) 120 120 120 120
After full oscillation for 10min, centrifuge at 3000rpm for 10min
Upper solution (μL) 50 50 50 50
Reagent III (μL) 200 200 200 200
After fully oscillating for 2 min, stand for 15min, take 0.2 mL into 96 well plate, measure the absorbance value at 550 nm, and record it as reference tube, measuring tube, blank tube and standard tube respectively. (1-2 tubes for reference tube and blank tube)

(Reagent I:N-heptane: anhydrous methanol: chloroform = 24:1:25; Reagent II copper solution; Reagent III: Diphenylcarbohydrazide solution )

Fig. 1 Determination of fatty acid concentration

Fatty Acid Sensing Module

In our project, we decided to perform directed evolution on the fatty-acid sensitive promoter FadR, aiming to increase its sensitivity towards fatty-acids, and at the same time decrease its leaking activity.

Thus, to quantify the result of directed evolution, we decided to use this promoter to drive the expression of a GFP gene. In this way, by examining the fluorescence result in conditions of varying concentrations of fatty acids, we can quantitatively see how well the promoter has been evolved. To examine the fluorescence, we decided to use a microplate reader as a means of collecting data.

Microplate-based measurements detect light signals which are produced by, converted by or transmitted through a sample. The signal is measured by a detector, usually a photomultiplier tube (PMT). PMTs convert photons into electricity that is then quantified by the microplate reader. The output of this process is numbers by which a sample is quantified.

Depending on the nature of the optical signal changes during a reaction and consequently on the detection mode, samples may need to be excited by light at specific wavelengths. In order to allow excitation of the sample only by specific wavelengths, the light produced by the lamp is selected by a specific excitation filter or monochromator. To increase the sensitivity and specificity, filters or monochromators are equally employed on the emission/detection side. These are usually placed between the sample and the detector.

The procedure of our experiment is as follows:

1. Grow the bacteria that has GFP expressed under the control of the fatty-acid sensitive promoter in LB medium.

2. Add fatty-acid to the culture to induce fluorescence.

3. Add the culture into a microplate.

4. determine the fluorescent signal using the microplate reader.

The results are as follows:

We first used already existing fatty-acid sensitive promoter, and did some improvement on the RBS region. The plasmids we’ve designed are as what the following diagram shows.

Fig. 2 The Maps of pDSW208-99-1 and pDSW208-99-2
(a) pDSW208-99-1(pFadD_Lac+B0030+GFP) contains RBS without modification, and can test promoter function. (b) pDSW208-99-2(pFadD_Lac+B0035+GFP) contains RBS with modification, and can test promoter function.

After consulting a large amount of RBS data, we found that most RBS with higher binding efficiency had the repetitive sequence of "aggg", or "aaa" after the initiation codon. B0030 was the one with the highest expression efficiency among the four existing RBS, with the sequence of "aaa" but no sequence of "aggg". Therefore, we modified the sequence to change "aagagg" to "aggagg", so that it could simultaneously have the characteristics of two sequences with high binding efficiency. In later experiments, we wanted to compare the performance of the two sequences.

Fig. 3 The system to test the performance of the Fatty Acid Sensing Module

After successful transfection of the plasmid, we induced it with fatty acids under different concentrations and different duration. And by measuring fluorescence with a microplate reader, we measured the level of expression of eGFP of the organism, and thus obtained data about the efficiency of the promoter. The result are as follows:

pDSW208-99-2(pFadD_Lac+B0030+GFP) contains RBS without modification
pDSW208-99-2(pFadD_Lac+B0035+GFP) Contains RBS with modification
(OA concentration unit: mmol/L)
Fig. 4 Measurement results of eGFP expression

From the result, we can see that there are improvements on the sensitivity of our fatty acids-sensitive promoter. Take the fatty acid under 0.125× concentration for example, at the 6th hour, the fluorescent intensity of 99-1 has bypassed 11000, while the promoter of the 99-2 plasmid didn’t reach 10000 under all three concentrations of fatty acids. However, as we can see, there are still problems of high leaking and low levels of expression in short time period. But we still considered this result sufficient in proving the function of the fatty acids-sensitive promoter, and proved in part the effeteness and reasonableness of our design.

Inhibition Module

Since we had purified the precursor of interested proteins PctA in cytosol, which has a sec-dependent signal peptide in its N-terminal and a His-tag in its C-terminal, we wanted to verify our design further more. So we used microplate reader to test the inhibition ability against P. acnes. The principles of microplate reader have been described above.

We planned to measure the absorbance value at 600 nm of the solutions after culturing for a certain time and incubated in a certain condition to examine our purified protein’s inhibition ability. If it can inhibit P. acne, after incubating for a certain time, the OD600 of the processed group should be lower than the negative reference group’s. The following table is our protocol.

Reference tube 1 Reference tube 2 Reference tube 3 Test tube 1 Test tube 2 Test tube 3
H2O(µl) 200 80 100 100
Elution buffer(µl) 200
5mg/ml Kan(µl) 120
Sample(µl) 200 100 20
P. acne solution(ml) 1.8 1.8 1.8 1.8 1.8 1.8
Total(ml) 2.0 2.0 2.0 2.0 2.0 2.0
Cultured at 37˚c,160rpm,and take out 200µl into 96 well plate every 2h to measure absorbance value at 600 nm.

After the measurement, we gained the following data.

Fig. 5 Measurement results of inhibition ability

As the figure shows, the inhibition ability of precursor Propionicin T1 which has a sec-dependent leader peptide is insignificant. Our experiment needs improvement in many respects. For example, the volume of P. acnes we cultured is so small that the solution remained heterogeneous, which caused a lot of random errors. And we may treated the P. acnes in low concentration of our samples, which may be too diluted to work. This experiment demonstrated that precursor Propionicin T1 can’t inhibit P. acnes. We hope to purify high concentration of mature Propionicin T1 after optimizing the signal peptide, and test its function again.