The Optimal IPTG Concentration and Temperature for the Expression of Protein in E.coli BL21 (DE3) with the PLIac01 Hybrid Promoter (BBa_R0011) and pSB1C3 and the Relation between the Strength of BBa_R0011 and IPTG Concentration at the Transcription Level

Overview

In this year, we constructed an EGFP expression vector (with pSB1C3 as the plasmid skeleton) in which the expression of EGFP was controlled by the PLIac01 Hybrid Promoter (BBa_R0011) and transformed it into E.coli BL21 (DE3) which is commonly used to express protein. Then, by measuring the EGFP fluorescence, we investigate the optimal IPTG concentration and temperature for the expression of protein in E.coli BL21 (DE3) using the BBa_R0011 in pSB1C3. In the measurement, the E.coli BL21 (DE3) which contained plasmid only consisting of EGFP and pSB1C3 was used as the blank. Meanwhile, we carried out qRT-PCR to directly investigate the relation between the original strength of BBa_R0011 and IPTG concentration at the transcription level without interfering by context.

Results

Fluorescence Characterization Shows that the Optimal Protein Expression Condition: 0.1mM IPTG & 25℃

First of all, we found that when the IPTG concentration was 0 mM (no inducer was added), the expression of EGFP was visible to the naked eye (Fig. 1), indicating that when BBa_R0011 was used to express protein in E.coli BL21 (DE3) with pSB1C3 as the plasmid skeleton, it is “leaky”. We measured the EGFP green fluorescence of E.coli BL21 (DE3) when the concentration of IPTG was 0 mM, 0.05 mM, 0.1 mM and 0.5 mM at 18℃, 25℃ and 37℃, respectively. The results showed that the optimal condition for using BBa_R0011 to express protein in the given background was: 0.1 mM IPTG & 25℃ culture temperature. For IPTG concentration, no matter which culture temperature, the green fluorescence intensity increased with the increase of IPTG concentration and reached the maximum value when the IPTG concentration was 0.1mM, and then it decreased with the further increase of IPTG concentration. We speculated that this is because the IPTG greater than 0.1mM inhibited the growth of E.coli BL21 (DE3), therefore, the expression of EGFP decreased. For the culture temperature, except for the condition where IPTG concentration is 0 mM, the green fluorescence intensity at 25℃ is always higher than that at other two culture temperatures, which may be because the 25℃ is conducive to the growth of E.coli BL21 (DE3) compared with 18℃, and is conducive to the expression of foreign genes compared with 37℃.

Figure 1. The leakage of BBa_R0011 in E.coli BL21(DE3) (with pSB1C3 as plasmid skeleton). The Blank is the E.coli BL21 (DE3) which containes plasmid consisting of EGFP and pSB1C3 skeleton under the same culture conditions. The black arrow indicates the visible leakage expression of EGFP at each culture temperature without IPTG.

Figure 2. The different EGFP expression of E.coli BL21 (DE3) under the conditions of IPTG of 0 mM, 0.05 mM, 0.1 mM and 0.5 mM at 18℃, 25℃ and 37℃, respectively. The horizontal axis shows the different concentrations of IPTG, the vertical axis shows the green fluorescence per OD600 (excitation wavelength: 485 nm; detection wavelength: 528 nm), and segments and data points of different colors show the different culture temperature. The EGFP fluorescence per OD600 standardized with EGFP fluorescence per OD600 value of blank of each test group. Error bar indicates the SEM of three replicates.

The Relation between the Strength of BBa_R0011 and IPTG Concentration at the Transcription Level

From the results of fluorescence characterization, 25℃ seems to be a more favorable temperature for the opening of BBa_R0011 in p SB1C3 in E.coli BL21 (DE3), so we chose this temperature as the culture temperature for qRT-PCR. We selected two frequently used housekeeping genes RecA and 16S rRNA as reference genes to compare the relative expression level of EGFP controlled by BBa_0011 under different IPTG concentrations of 0 mM, 0.02 mM, 0.08 Mm, 0.3 mM and 0.5 mM at 25℃. At IPTG concentration between 0 and 0.08 mM, the strength of BBa_0011 increases significantly with the increase of IPTG concentration. The relative normalized expression of EGFP at the IPTG concentration of 0.08 mM is 1.32 times that at the IPTG concentration of 0 mM, which is the highest value in 5 test groups. After the IPTG concentration exceeds 0.08 mM, the strength of BBa_0011 gradually decreases with the increase of IPTG concentration.

Figure 3. The relative normalized expression level of EGFP of E.coli BL21 (DE3) under the conditions of IPTG concentration of 0 mM, 0.02mM, 0.08 mM, 0.3mM and 0.5 mM at 25℃.The horizontal axis shows the different concentrations of IPTG, the vertical axis shows the relative normalized expression level of EGFP of each test group.

Protocol

Fluorescence Characterization

1) Construct the expression vector of EGFP initiated by BBa_0011 and the vector containing only EGFP, both with pSB1C3 as the plasmid skeleton.

2) Transform these two kinds of plasmids into E. coli BL21(DE3) to obtain “blank” and “BBa_R0011”.

3) Pick a single colony from “blank” and “BBa_R0011”, respectively, and inoculate in 5mL liquid LB medium + 5uL Chloramphenicol (25mg/mL in EtOH) and grow the cells overnight at 37 °C and 220 rpm.

4) Inoculate 50uL of each overnight culture into 4.95 mL LB with Chloramphenicol to make 12 parallel groups of “blank” and “BBa_R0011”, respectively, and grow the cells at 37 °C and 220 rpm until OD600 reaches 0.6.

5) Add IPTG to final concentrations of 0(parallel 1-3), 0.05(parallel 4-6), 0.1(parallel 7-9), 0.5mM(parallel 10-12) and grow the cells at temperatures of 18℃(parallel 1,4,7,10), 25℃(parallel 2,5,8,11) and 37℃(parallel 3,6,9,12), 220 rpm for 6 hours. The IPTG concentration and culture temperature of “blank” and “BBa_R0011” with the same number are the same.

6) Read and record the fluorescence intensity (excitation wavelength: 485 nm; detection wavelength: 528 nm) and OD600.

7) Repeat each group for 3 times.

qRT-PCR

1) Pick a single colony from “BBa_R0011” and inoculate in 5mL liquid LB medium + 5uL Chloramphenicol (25mg/mL in EtOH) and grow the cells overnight at 37 °C and 220 rpm.

2) Inoculate 50uL of the overnight culture into 4.95 mL LB with Chloramphenicol to make 7 parallel groups and grow the cells at 37 °C and 220 rpm until OD600 reaches 0.6.

3) Add IPTG to final concentrations of 0(parallel 1), 0.02(parallel 2), 0.05(parallel 3), 0.08(parallel 4), 0.1(parallel 5), 0.2(parallel 6), 0.5mM(parallel 7) and grow the cells at temperature of 25℃ and 220 rpm for 6 hours.

4) Extracted RNA, make reverse transcription and carry out qRT-PCR with recA and 16S rRNA as reference genes.