Regulatory

Part:BBa_K4238000

Designed by: Asako Kitai   Group: iGEM22_UTokyo   (2022-10-01)


ADH1 promoter short


Description

Constitutive promoter for S.cerevisiae. Improved part based on part BBa_K2365039
We attempted to create a shorter, and therefore easier to use for genetic engineering, and more powerful basic part of a promoter to make more options for easy-to-handle constitutive promoters. ADH1 is alcohol dehydrogenase I (ADH1), and its promoter of yeast, especially the medium-length one, is often used as a constitutive expression promoter for yeasts. The part called BBa_K2365039
is a medium-length ADH1 promoter whose length is 618 bp. We developed a promoter whose length is 409 bp by deleting the upstream 209 bp of this part.
The upstream sequence we deleted contained an activation sequence called UASrpg (upstream activation sequence of ribosome protein genes), so we could expect an increase in the expression level.[1]

Usage

This ADH1p short can be used as a constitutive promoter with a higher expression level than BBa_K2365039.

Biology

ADH1p short is a constitutive promoter derived from Saccharomyces cerevisiae, and you can get it from the genome of Saccharomyces cerevisiae as a part of ADH1p.

Improvement of existing BBa_K2365039 by iGEM17_NAU-CHINA (2017)

Many variations of the basic components of ADH1p already exist in iGEM's Parts Registry, but most of them are long ones, such as a full-length ADH1p or ADH1p with a regulation sequence. Among them, the BBa_K2365039 is a medium-length ADH1p that is relatively short and can be used as a constitutive expression promoter, but the expression level of the downstream gene is not so high. Our goal was to shorten the length and improve the expression of this part, so we deleted the upstream 209 bp sequence containing an activation sequence called UASrpg (upstream activation sequence of ribosome protein genes), making it shorter and easier to use for genetic engineering.

Characterization

We added a pair of forward and reverse primers which extract the full-length ADH1p (ADH1p long) from the yeast genome, to templates of two yeast colonies, both of which were transformed with plasmids containing ADH1p (Here we call them 1 and 2), and conducted PCR, and got products.
We conducted electrophoresis using the PCR product, and the bands of the length of the desired sequence were observed. (Figure 1)

Figure 1. The results of the electrophoresis. The desired bands are 668bp for ADH1p long, 467bp for ADH1p short.



This band was cut out and the DNA was extracted from the gel. The results of the spectrophotometer of the solution after gel extraction are as follows.

Table 1. The results of the spectrophotometer.


Based on this result, we decided to use ADH1p long 1 and ADH1p short 2. 3ul of either ADH1p long 1 or ADH1p short 2 was transformed into E. coli together with 6ul of mCherry + back, which is shown in the figure 2 below. Then the plasmids were extracted.

Figure 2. The sequence obtained by removing the "ADH1 p" part from this plasmid is mCherry + back.


This was transformed into a pre-cultured yeast BY4741 strain. Three colonies of each yeast transformed with ADH1p long and ADH1p short were taken and Zymolyase treatment was performed. It was then subjected to PCR, and electrophoresis was conducted using the product. (Figure 3)

Figure 3. The result of the electrophoresis. The expected band is 1762 bp for ADH1p_long and 1632 bp for ADH1p short.


The bands were consistent with expectations and the transformation appears to have been successful. Both of the yeast transformed with ADH1p short and ADH1p long was inoculated. Thereafter, fluorescence observation of these transformed yeasts was performed using a microscope. The observation conditions are as follows.

phase(Transmitted light): 50ms
TxRED(Light through a filter to observe mCherry): 500ms
Z-stuck(Width of the shooting break in the Z-axis direction): 1µm

Results

The results are shown in figure 4 and 5 below.

Figure 4. Distribution of fluorescence. 257 cells of ADH1p long and 418 cells of ADH1p short were observed.
Figure 5. Average of fluorescence. Error bars indicate standard errors. * denotes a significant difference at $p<0.05$, ** denotes a significant difference at $p<0.01$, and n.s. denotes no significant difference. The statistical test was conducted using the Wilcoxon rank sum test.



In order to compare the expression level of each promoter, the median and average were calculated as representative values for relative fluorescence intensity. The results are shown in the table 2 below.

Table 2. The median and average of relative fluorescence intensity.


From these figures and table, it can be concluded that the expression of the fluorescent protein increased by using ADH1p short.

Reference

[1] Arja E.I. Vainio. (1994). Effect of upstream sequences of the ADH1 promoter on the expression of Hormoconis resinae glucoamylase P by Saccharomyces cerevisiae FEMS Microbiology Letters, 121(2), 229-235

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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