Difference between revisions of "Part:BBa K4815000"

PYPH1 targets at s. cerevisiae (saccharomyces cerevisiae), the most studied eukaryotic expression system in synthetic biology. ===Loci=== PYPH1 consists of 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 PYPHs. It is a structure that we learned and utilized from 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, PYPH1 can drive extremely high expression of downstream products as is predicted by our excellent AI model Pymaker. Furthermore, our experiments have abundantly validated the ability of PYPH1. Under same circumstance, PYPH1 can drive expression rate 5 times that of natural high promoters (pTEF, pGAP, pADH), which are now most widely used high expression constitutive promoters in entrepreneurship. Meanwhile, our experiments proof that this ability of driving extremely high expression is preserved among different downstream products, which make PYPH1 an excellent and practical substitute for natural promoters.

Besides, PYPH1 is anti-mutant as our excellent AI model predicts (as is represented by the red line labeled 'high' in the figure bellow). 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 extremely high, are highly anti-mutant. This character of anti-mutant makes PYPH1 a highly competitive merchant among promoter parts used in yeasts fermentation.

What’s more, PYPH1 shows a highly constant expression rate among yeast population, which is not the case considering the natural promoters. This character of PYPH1 is totally out of expectation but is of vital importance in yeasts fermentation on a large commercial scale.

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 PYPH1-7 from our dual-fluorescence reporter plasmids, using colony PCR with designed primers (primers sequences are shown in the figure below, and spacer H1-7 stands for PYPH1-7)