Part:BBa_K4121002
pPGK1
We paid attention to an existing basic part in the iGEM part library, the S. Cerevisiae promoter PGK1, and tried to improve it. pPGK1 is a promoter in Saccharomyces cerevisiae, and has relatively strong expression strength. As we know, an excessively long target sequence is not conducive to molecular experiments, sequencing and bioinformatics analysis, which will cause a lot of trouble to the experimenters. At present, the length of the PGK1 promoter in iGEM component library is 1497bp. We truncate 1100bp, 800bp, 600bp, 400bp or even 200bp in the existing sequence of PGK1 as the promoter sequence, and then use the red protein mCherry characterizes the startup strength of the PGK1 promoter of different lengths, trying to find the optimal PGK1 promoter with the same strength and shorter sequence compared the original promoter.
1 Find PGK1 promoter in iGEM component library
2 Truncate the existing sequence We truncate 1100bp, 800bp, 600bp, 400bp or even 200bp in the existing sequence of PGK1 as the promoter sequence, and then use the red protein mCherry characterizes the startup strength of the PGK1 promoter of different lengths, trying to find the optimal PGK1 promoter with the same strength and shorter sequence compared the original promoter.
3 Design primers According to the recognition site and cleavage site of BsaI/BsmbI restriction endonuclease, we designed the modified base at the end of primers. The length of primer fragments are generally 59bp. The annealing temperature of promoters is 55 ℃, and the annealing temperature of upstream and downstream primers is basically the same.
4 Amplification of promoter of different length by PCR The PGK1 promoter sequence was amplified from the Saccharomyces cerevisiae genome with the lengths of 200bp, 400bp, 600bp and 800bp.The specific system and procedure are as follows.
5 Purification of PCR products
6 Goldengate ligation We use Goldengate ligation to connect the promoter fragments with the TU11-ccdB-mcherry-T4 fragment. The specific system and procedure are as follows.
7 Saccharomyces cerevisiae transformation Transformation the products of Goldengate ligation to Saccharomyces cerevisiae.
8 Expression intensity of mCherry After Saccharomyces cerevisiae transformation, the single Saccharomyces cerevisiae colony was shaken in SD-trp liquid medium to OD600 about 1.0 and added to 96-well plate (200ul per well). The fluorescence intensity of mCherry was determined by enzyme labeling instrument.
9 Data analysis We used mCherry/OD600 (the ratio of mCherry to OD600) as the refere nce value for the expression intensity of promoter. We analyzed and compared the expression intensity of promoters of different lengths.
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]
| None |