Part:BBa_K3730000
sPrcn+T7+RBS
sPrcn+T7+RBS is a cobalt-activated promotor which integrates T7 promotor. It is improved from part BBa_K540001 (Prcn) using one-step recombination. The expression level of the promotor is significantly higher than that of part BBa_K3730000 even cobalt molecules are given.
Characterization
Experiment regarding the response of sPrcn to different concentrations of cobalt and IPTG was carried out. Comparison of part kinetics between sPrcn and Prcn was also considered in the experiment.
This figure shows the expression of eGFP controlled by Prcn (the left panel) and sPrcn (the right panel) promotor upon the cobalt chloride of different concentrations and of different treating time (with sufficient oxygen). For sPrcn, The fluorescence/OD600 increased rapidly when we raised the IPTG concentration, implying that the expression of eGFP could be controlled by T7 promoter in sPrcn, and that about 1mM IPTG is enough to induce high expression.
At the meantime, the result shows that sPrcn could respond to Co2+ as well, even with a higher sensitivity and efficiency. Cells treated with 60μM Co2+ or higher concentration would manifest a downward fluorescence/OD600 trend, and that dosage for Prcn might be around 150μM. And the peak value of fluorescence/OD600 for sPrcn reached more than 400000, which was 15000 higher than the peak value for Prcn, which means that sPrcn provides the expression of downstream protein with a wider mediating amplitude. In contrast, high concentration of Co2+ did not necessarily translate into high fluorescent activity of the Prcn-gfp, and might exert a toxic effect on cells. In conclusion, sPrcn possessed higher sensitivity and efficiency than Prcn, perfectly in line with our expectations, overcoming the paradox of GFP expression caused by toxicity of metal ions.
We caveat that anaerobic environment is generally NOT allowed when applying this promotor. For example, culturing E.coli in 1.5mL-Eppendorf for several hours would lead to incorrect results due to lack of oxygen. Under such conditions, the expression of eGFP could still be regulated by IPTG, whereas it became insensitive to the Co2+ of different concentrations, suggesting that sPrcn cannot function as a cobalt-specific promoter under oxygen-deficient environments. However, fluorescence activity of sPrcn is still higher than that of Prcn in anaerobic environment. This indicates that T7 promotor significantly enhanced the efficiency of sPrcn.
Protocol
ZJU China 2021 uses the following protocol to construct this part:
1. Linearize pSB1c3 plasmid using PCR. (F: TGCCACCTGACGTCTAAGAA, R: ATTACCGCCTTTGAGTCAGC).
2. Amplifying RBS+EGFP sequence using PCR. (F: agaaagaggagaaatactagATGAGCAAGGGC, R: tctcctctttctgaagaagagttgt)
3. Construction of pSB1c3_GFP plasmid with sPrcn promotor using homologous recombination.
4. Transformation of improved plasmid into E. coli DH5α. Incubate bacterial culture in LB + Cm (Chloramphenicol) agar plate for 12h-18h.
5. Check for growth and select 10 single colonies from plate using pipette tip. Drop the tip into the liquid LB + Cm in tubes and swirl.
6. To characterize improved parts, transfer bacteria medium to new tubes into new tubes with LB + Cm medium. After incubation for 3–4h, Add IPTG and/or metal ions with a specified concentration based on experimental design.
7. After induction, incubation time varies from 3h, 6h, 9h, 12h, and 15h.
8. Measure the fluorescence (excitation wavelength: 485nm; detection wavelength: 528nm) and OD600.
Conclusion
1. sPrcn is constructed by introducing of T7 promotor and preserving the rcn operator.
2. The downstream expression controlled by sPrcn could be regulated by IPTG, and the concentration of 1.0-2.0mM is recommended.
3. The improved part (sPrcn) possessed higher sensitivity and efficiency than Prcn, enhancing the expression of GFP by around 100,000 Fluorescence/OD600. Less Co2+ could be applied to reach a high expression of GFP, thus causing less harm to cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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