Background

Precise and reliable expression of the genes of interest is a core step in synthetic biology. Different combinations of promoters and ribosome binding sites (RBS) can influence the efficiency of gene expression and even cell growth. Meanwhile, repeatable and comparable measurement is very important in testing the consistency of experimental results. However, due to use of different equipment or different ways of manipulation due to idiosyncrasy, it is hard to compare the data between labs.

Aim

This year’s interlab study aims to test some RBS devices that are intended to make gene expression more precise and reliable. Moreover, the iGEM committe established a Green Fluorescence Protein (GFP) measurement protocol to ensure all iGEM teams to use this same protocol to produce common, comparable units for measuring the fluorescence signals from GFP with different types of plate readers.

Method

The OD₆₀₀ reference

1. Add 100μl of LUDOX into wells A1, B1, C1, D1.
2. Add 100μl of H₂O into wells A2, B2, C2 D2.
3. Measure absorbance 600 nm of all samples in a microplate reader and record the data.
4. Add 1ml of LUDOX into cuvette for the measurement of OD₆₀₀ in spectrophotometer.

Fluorescein fluorescence standard curve

1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
2. Prepare 2x fluorescein stock solution (100μM) by re-suspending fluorescein in 1ml pf 1x PBS.
3. Dilute the 2x fluorescein stock solution with 1x PBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50μM.
4. Add 200μl of 1x fluorescein stock solution into well A1, B1, C1 D1 of 96-well plate and 100μl of PBS to A2, B2, C2, D2…A12, B12, C12, D12.
5. Transfer 100μl of solution from A1 into A2 and mix by pipetting up and down three times, and then continue the same procedure and transfer from A2 to A3, from A3 to A4……Finally, transfer 100μl from A11 into liquid waste. Repeat dilution series for rows B, C, D.
6. Measure fluorescence of all samples in a microplate reader and record the data.

Cell measurement

1. Day 1: Transform Escherichia coli DH5α with these following plasmids:
   • Positive control
   • Negative control
   • Test Device 1: J23101+I13504
   • Test Device 2: J23106+I13504
   • Test Device 3: J23117+I13504
   • Test Device 4: J23101.BCD2.E0040.B0015
   • Test Device 5: J23106.BCD2.E0040.B0015
   • Test Device 6: J23117.BCD2.E0040.B0015
2. Day 2: Pick 2 colonies from each of plate and inoculate it on 5ml LB with 25μg/ml chloramphenicol for about 17 hours (3:00pm to 10am, next day), at 37℃ and 220rpm.
3. Day 3: Cell growth, sampling, and assay
   1. Take 200μl of each overnight culture mixed with 800μl water to make a 5-fold dilution.
   2. Measure OD₆₀₀ of the overnight cultures and record the data.
   3. Dilute the cultures to a target OD₆₀₀ of 0.02 in 12ml LB medium + Chloramphenicol in 50ml falcon tube.
   4. Incubate the cultures at 37°C and 220rpm.
   5. Take 500μl samples of the cultures at 0, 2, 4, and 6 hours of incubation.
   6. Place samples on ice.
   7. Take 100μl of each sample into 96-well plate to get values of fluorescence and OD₆₀₀. For each sample, there were 4 replicates.

We laid out sample as shown in following picture:

Result

The OD₆₀₀ reference

The average Abs₆₀₀ of LUDOX and H₂O were calculated and showed in the table. The corrected Abs₆₀₀ is obtained by subtracting the Abs₆₀₀ of H₂O from the Abs₆₀₀ of LUDOX. The reference OD₆₀₀ was measured from 1ml LUDOX using a reference spectrophotometer at 600nm. The value of OD₆₀₀/Abs₆₀₀ is 4.25.

Fluorescein fluorescence standard curve

Fig 1.    The fluorescence standard curve of fluorescein
The measured fluorescence was plotted against the concentration of fluorescein.

The fluorescence signal increased 150 A.U. with the concentration of fluorescein increased 1μM at low concentration, from 0 to 20μM, and the slope of the curve was gradually decreasing as the concentration increased.

Cell measurement

Fig 2.    OD₆₀₀ against the incubation time
This figure showed the OD₆₀₀ of 16 samples with different devices during the incubation.

The growth rate of DH5α transformed with device 1 was significantly less than that of DH5α transformed with other devices, while this might have been an effect of the gene expression of device 1, but this can also happen due to human error.

Fig 3.     Fluorescence signals from different devices over time
In this figure, we give a curve of fluorescence signal of each sample at specific points (0, 2, 4 and 6 hours).

The culture of DH5α transformed with device 2 produced the highest fluorescent signal and followed by device 4 less than the positive control.

Fig 4.     GFP productivity of different devices

The blue, orange, grey and yellow bars correspondingly represented the ratio of GFP concerntration to OD₆₀₀ value at 0, 2, 4, 6 hours. The concertation of GFP was converted from fluorescence signal (in Figure 3), with calibration curve in Figure 2.

Discussion

We observed that, among the 6 combinations of these 3 promoters (J23101, J23106 and J23117) and 2 RBS (B0034 and BCD2) in various devices, devices 2 and 4 produced high fluorescence signals in the bacterial cell population and device 1 had the strongest ability to produce GFP per cell which was companied with low growth. Based on the plot of [GFP]/OD₆₀₀, either with RBSs B0034 or BCD2, the rank of the promoter efficiency (from strong to weak) is J23101, J23106 and J23117. When the promoters are the same, the strength of BCD2 is weaker than that of B0034. It was quite typical that, although the population of bacterial cells with device 1 had relatively low GFP level, the [GFP]/OD₆₀₀ of device 1 was the highest (except 0h) among all the samples. However, when merely considering the total quantity of GFP in bacterial population, obviously device 1 is not a good choice to produce GFP. That may be due to the overexpression of GFP in comsuming too many resources, leading to the lack of materials for cell growth.