Part:BBa_K950002

yeast anb1 promotor

There are many genes in yeast that are regulated by oxygen supply as a means to adapt to fluctuations in oxygen tension (Lowry and Zitomer, 1988). One of these genes is the ANB1 gene: under aerobic conditions the ANB1 gene is regulated by heme while it is expressed under anaerobic conditions (Lowry and Zitomer, 1984). Under aerobic conditions “the ROX1 gene product mediates the heme regulation of the ANB1 gene” (Lowry and Zitomer, 1988). Lowry and Zitomer (1988) found that this repression is regulated by the synthesis and elimination of ROX1 transcripts depending on the presence of oxygen whereby aerobic conditions induce the synthesis of heme which in turn stimulates the expression of ROX1. “ROX1 is a member of the HMG family of DNA-binding proteins” (Balasubramanian et al., 1993) and binds to a “12-bp consensus sequence repeated twice in two of the ROX1-responsive operators of ANB1” (Lowry et al., 1990). According to Lowry et al. (1990) the operator sequences (which are the targets for ROX1) are located in the upstream region of the ANB1 gene.

Short: This is the promoter of yeast ANB1 gene. Panb1 is a target of the ROX1 repressor, see Part BBa_K950001.

 

Increased promoter activity of Psuc2 and Panb1 in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).

 

Promoter activity of Psuc2 and Panb1 in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 124
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

HUST-China 2021 iGEM team

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.

Fig2 Colony PCR result of yeast.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.

Fig3. SDS-PAGE result of FMO after purification of yeast total protein extraction product through Nickel-affinity chromatography column.

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 53.96kDa but still within explainable and acceptable range of glycosylation modification. FMO could be confirmed as successfully expressed. The concentration of yeast total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its dark color and consistency among several times of elution, this band could be verified as our target FMO.