Part:BBa_K5261021
CTR3 promoter strength characterisation pathway
mCherry is a red fluorescent dye currently widely used in biotechnology as a tracer and is commonly used to characterise promoter strength. Here we linked the copper ion repressive promoter CTR3 and terminator CYC1 to mCherry and characterised the strength of CTR3 at different copper ion concentrations by fluorescence intensity.
Design
Construction of the pCTR3-mCherry-tCYC1 expression plasmid
We constructed the mCherry gene expression cassette driven by the pCTR3 promoter on a free plasmid. We amplified the sequence of the endogenous copper-repressible promoter pCTR3 from the genome of Saccharomyces cerevisiae CEN.PK2-1C using PCR. Next, we amplified the mCherry gene sequence through PCR. At the same time, we amplified the plasmid vector fragment from the p416-pTEF1-Cas9-tCYC1-G418 plasmid, which carried the KanMX marker (conferring resistance to the antibiotic G418 in yeast). We verified the correctness of the three gene fragments using agarose gel electrophoresis.
The pCTR3 gene sequence, mCherry gene sequence, and p416 Vector were assembled by Gibson Assembly to construct the recombinant plasmid p416-pCTR3-mCherry-tCYC1-G418 according to the following diagram.
Figure 1. Plasmid map of p416-pCTR3-mCherry-tCYC1-G418
Characterization of the copper ion repression concentration of the PCTR3 promoter
To characterize the copper ion repression concentration of the pCTR3 promoter, we batch-cultivated Saccharomyces cerevisiae BY4741 strains transformed with the p416-pCTR3-mCherry-tCYC1-G418 plasmid in 50 mL YPD-G418 shake flasks, with an initial inoculation of OD600 = 0.1. At 0 hours, we added CuCl₂ to final concentrations of 0, 0.2, 0.5, 1, 2, 10, 20, 30, 40, 50, and 60 μM, and used Saccharomyces cerevisiae BY4741 strains as the wild-type control group.
Fluorescence measurements were performed approximately every 12 hours. We took a portion of the culture, washed it twice with ddH₂O, and then diluted it to OD600 = 1 with PBS. The fluorescence of mCherry was measured using a microplate reader with an excitation wavelength of 585 nm and an emission wavelength of 620 nm. We assessed the repression of the copper ion-repressible promoter pCTR3 based on the fluorescence intensity of each group. Measurements were conducted continuously for 72 hours.
Figure 2. Copper ion inhibition concentration characterization of the pCTR3 promoter
Result
Construction of the pCTR3-mCherry-tCYC1 expression plasmid
The electrophoresis results showed bands corresponding to the p416 Vector (5000+ bp), pCTR3 (700+ bp), and mCherry (700+ bp), which matched the theoretical lengths of 5733 bp, 777 bp, and 751 bp, respectively, indicating that we had successfully obtained the three fragments through PCR.
Figure 3. PCR results of p416 Vector, pCTR3, and mCherry gene fragments
The electrophoresis results showed that the bands of the four recombinant plasmids were approximately 1.5 kb, which was slightly different from the theoretical band length of 1801 bp (from promoter to terminator) for the designed plasmid. We thought this was likely due to unsuitable electrophoresis conditions, and further sequencing was required for verification.
Figure 4. Electrophoresis results of colony PCR
The sequencing results of plasmids 1, 3, and 4 were correct, but plasmid 2 was wrong, and discarded.
Figure 5. DNA manipulating methods from the literatures
Characterization of the copper ion repression concentration of the PCTR3 promoter
After adding copper ions at 0 hours, the fluorescence intensity of each group rapidly decreased to varying extents. Significant fluorescence repression was observed at a Cu(Ⅱ) concentration of 1 μM. After 72 hours of batch cultivation, groups with Cu(Ⅱ) concentrations greater than 30 μM showed more than 10-fold repression compared to the group without adding Cu(Ⅱ). These observations indicated that the pCTR3 promoter experienced effective repression. However, we also observed that there was still some level of leakage expression of the pCTR3 promoter even when Cu(Ⅱ) was added, which aligned with our previous speculation. In our design, we used the constitutive weak promoter pCYC1 to express the antitoxin gene RelB to counteract the leakage expression of RelE. Therefore, we could conclude that the design of our biosafety module was fundamentally reasonable.
Figure 6. Characterization of the copper ion repression concentration of the PCTR3 promoter. A. mCherry fluorescence values detected over 0-72 hours. B. mCherry fluorescence intensity for each group at 70 hours.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 100
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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