Background

The precise and reliable expression of objective genes is a core step in Synthetic Biology. Different combinations of promoters and ribosome binding sites can influence the efficiency of gene expression and even cell growth. Meanwhile, repeatable and comparable measurement is needed in any testing to verify the consistency of results. However, due to different units or different ways of manipulation, it is hard to compare the data between labs.

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Aim

This year’s interlab aims to test some RBS devices that are intended to make gene expression more precise and reliable. Moreover, they establish a GFP measurement protocol to ensure teams to use this same protocol to produce common, comparable units for measuring GFP with different plate readers.

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Method

OD₆₀₀ reference

1. Add 100μl of LUDOX into wells A1, B1, C1, D1.
2. Add 100μl of H₂ O 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:

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Result

OD₆₀₀ reference

Fluorescein fluorescence standard curve

Fig 1: fluorescein fluorescence standard curve

Fig 1 shows the measured fluorescence over the concentration of fluorescence.
The slope is around 150 at low concentration and the function is gradually saturated as concentration increases.

Cell measurement

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Discussion

We can see from the data that, among the 6 combinations of these 3 promoters (J23101, J23106 and J23117) and 2 RBS (B0034 and BCD2), device 2 and 4 produced much fluorescein in cell culture and device 1 had the strongest ability to translate GFP in a single cell which was companied with low growth. Based on the statistics of Fluorescein/OD₆₀₀, whether with RBS B0034 or BCD2, the rank of promoter efficiency from strong to weak is J23101, J23106 and J23117. When the promoters are identical, the strength of BCD2 is weaker than that of B0034. It was quite typical that, although the culture of cells with device 1 has relatively low GFP amount, the Fluorescein/OD₆₀₀ value was quite high and even the highest (except 0h) among samples. However, when merely considering the total amount of GFP, obviously device 1 is not a good choice to accumulate GFP. That may because the overexpression of GFP will consume too many resources, leading to lack of material for cell growth.