Designed by: Jiacheng Shi   Group: iGEM21_HUST-China   (2021-10-01)


PYNR071C is the promoter of aldose 1-epimerase superfamily protein YNR071C

A promoter originates from the yeast, which could be induced by xylose to activate genes related to material transportation. When the carbon source is mainly xylose, cell prefer to absorb extracellular nutrient. Xylose is the second most abundant saccharide in cellulose-like bio-staple, such as straw, right after glucose. Engineered yeast which is able to ferment xylose effectively, could be used to increase the availability of material and decrease the cost of ethanol extraction from cellulose fuel. As a kind of safe carbon source of relatively low cost, xylose could have been used in food industries, while there is few study about its promoter at present. Sequence and Features

Assembly Compatibility:
  • 10
  • 12
  • 21
  • 23
  • 25
  • 1000

Molecular cloning

AOX1 promoter is the strongest eukaryotic promoter currently known in yeast expression system. So we choose AOX1 as the primary promoter when we synthesized all these plasmid for the sake of more convenient expression. But noticing that methanol is hazardous, flammable, combustible and therefore, inappropriate to have direct contact with the hair, we need to substrate AOX1 for constitutive promoter Panb1 and xylose induced promoter Pynr071C to realize the projected regulation function as designed. Double-enzyme cleavage and rejointing is used to achieve this. We amplify the target gene located in the primarily synthesized plasmid without AOX1 promoter, and digest the acquired fragments and two kinds of plasmid, containing promoter only, with EcoR I and BamH I, then transform them into E.coli after linking the product together. Through this, we successfully substrate the primary AOX1 for Panb1 and Pynr071C.

Fig1. Plasmid construction and colony PCR results of reconstructed plasmid with Panb1 and Pynr071C promoter.

All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.
To verify whether our Pynr071c promoter could initiate the expression of downstream genes, we linked GFP it, then transfect it into yeast and test the exist of fluorescence.

We successfully construct the plasmid with the GFP and sequencing of which is correct. E.coli is used for amplification as well. Extract plasmid from E.coli, digest with Bgl II to get linear plasmid and concentrate for electroporation of yeast.
We induce the plasmid with confirmation of colony PCR to express. Adding xylose everyday for xylose induced promoter Pynr071C to induce synthesis of GFP. Using fluorescence microscopy to detect the exist of green fluorescence, which indicates whether our promoters function regularly.

Fluorescence characterization

Colonies after 24 hours of culture, of which we picked one and verified the expression of GFP
Observe the cells induced for 48 hours under a confocal microscope. From left to right: the field of view under the microscope (blank) is displayed; the field of view is marked after the light absorption value is detected; the field of view after the light absorption value is marked under dark background.