Difference between revisions of "Part:BBa K4815009"

PYPL3 targets at s. cerevisiae (saccharomyces cerevisiae), the most studied eukaryotic expression system in synthetic biology.

Loci

PYPL3 consists two parts: the core promoter and the scaffold. The core promoter is an 80 bp sequence and is seated at approximately -170 to -90 upstream to the codon (which is the presumed transcription start site-TSS and is where most transcription factors binding sites lie). The scaffold is a preserved sequence in all PYPLs. It is a structure that we learned and utilized from a previous research that can link the core promoter with the codon and provide restriction sites of BamH I and Xho I which make it possible for the plasmids with the scaffold to be inserted by various core promoter sequences at ease. .

Usage and Biology

To begin with, PYPL3 can drive extremely low expression f downstream products as is predicted by our excellent AI model Pymaker. Furthermore, our experiments have abundantly validated the ability of PYPL3. Under same circumstance, PYPL3 can drive expression lower than that of natural promoters (pTEF, pGAP, pADH), which can be related with a series of dynamic events, such as the regulation of some metabolic pathway. Meanwhile, our experiments proof that this ability of driving low expression is preserved among different downstream products, which make PYPL3 an excellent and practical substitute for natural promoters. Besides, PYPL3 is anti-mutant as our excellent AI model predicts. Our AI model Pymaker outperforms competitors on all sample size datasets, and its ability to predict the specific expression rate of the core promoters is proved by experiments. On this basis, we are very confident that by introducing 1-3 random mutations per generation and imitating 100 generations, those promoters, whose expression rates predicted by Pymaker remain low, are highly anti-mutant. .

What’s more, PYPL3 shows high variance expression rate among yeast population, which is similar to the natural promoters instead of the PYPHs. The low and unstable expression character of PYPL3 is as expected.

Sequence and Features

Experiments

Extraction

We design to extract promoter sequences from synthesized whole dual-fluorescence reporter plasmids, and then insert them into the LTB-eGFP expression plasmids.We extract PYPL1-3 from our dual-fluorescence reporter plasmids, using colony PCR with designed primers (primers sequences are shown in the figure below, and spacer L1-3 stands for PYPL1-3)

Validation

We synthesized the dual-fluorescence reporter plasmids where PYPL3 is already placed in, comparing to the natural promoter GAP. We transform the plasmids into targeted yeasts and use fluorescence-activated cell sorting (FACS) strategy and record the fluorescence density through a flow cytometer. The figure above shows the result.

Proof In different scenario

PYPL3 has shown its ability in tackling real challenges. Currently, significant breakthroughs have been achieved in the application of brewing yeast in the field of biotechnology. One of them is the production of the heat-labile toxin B subunit (LTB) of Escherichia coli using brewing yeast, an important oral vaccine adjuvant widely used to prevent various diseases such as cholera, traveler’s diarrhea, and E. coli infection. We use the same plasmid framework of BBa_K4815011 and change the YeGFP with our part BBa_K4815021, aimed at using PYPL3 to drive the expression of fusion protein LTB-eGFP. We test the expression rate from three different dimensions: flow cytometry, protein density through western blot, transcription accumulation through quantitative RT PCR, and the results are as follows.

We then checked the quantitative gene expression levels using quantitative RT-PCR, and the results indicated that PYPL3 drive a much lower transcript accumulation than natural promoters. The result gives a strong validation that it is our generated promoters that play a fundamental role in driving a extremely low promoter sequences.

Also, the flow cytometry result suits the result above, expression rate of LTB-eGFP driven by PYPL3 remain the sharp waveform, which means the ability to drive constant expression even better than natural constitutive promoters of PYPL3 remains when the downstream codon changes.