Difference between revisions of "Part:BBa J63005"
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[2] Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters, 464(3), 123–128. https://doi.org/10.1016/S0014-5793(99)01698-1 | [2] Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters, 464(3), 123–128. https://doi.org/10.1016/S0014-5793(99)01698-1 | ||
| − | + | <br><br> | |
| − | < | + | <h2>Tianjin's 2021 characterization</h2> |
| − | < | + | <b><h3>Characterization of ADH1 Promoter’s GFP expression in SY14 yeast</h3></b> |
| − | <b>< | + | |
<p>Group: Tianjin 2021<br> | <p>Group: Tianjin 2021<br> | ||
| − | Author: Ruiqi Liu<br> | + | Author: Ruiqi Liu, Lingwei Ding<br> |
| − | Summary: we characterized ADH1 Promoter’s | + | Summary:we characterized ADH1 Promoter’s strength in 4742 yeast.</p> |
<i><h3>Background</h3></i> | <i><h3>Background</h3></i> | ||
| − | + | SY14 is a special kind of Artificially modified yeasts which contains only one chromosome fused by 16 chromosomes. SY14 yeast differs from normal yeast in many properties: growth rate, metabolic activity etc. We use GFP as reporter gene to test the ADH1 promoter’s strength in SY14. We constructed a GFP fragment that was controlled by the ADH1 promoter and bound it to the chromosome genome of SY14 yeast. We measured the GFP expression levels of SY14 yeast at 15h, 20h,25h and 30h by using microplate analyzer. Meanwhile, we used SY14 yeast (no GFP fragment) as control group. | |
<i><h3>Result</h3></i> | <i><h3>Result</h3></i> | ||
| − | <p>We | + | <p>We use GFP as reporter gene to test the intensity of ADH1 promoter, and draw a bar chart with fluorescence intensity /OD as the ordinate and time as the abscissa.The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. The strength of ADH1 promoter reaches its peak at 15h. </p> |
| − | [[File:T--Tianjin--Parts4.png| | + | [[File:T--Tianjin--Parts4.png|600px|thumb|left|Figure 1.Characterization of ADH1 promoter’s strength]] |
Latest revision as of 05:39, 21 October 2021
yeast ADH1 promoter
ADH1 promoter from S. cerevisiae
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 180
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 830
Wroclaw’s 2019 characterization
Comparison of the ADH1 and TEF promoters activity
Background
In order to analyze the activity of ADH1 and TEF promoters[1] and their usefulness to express sugar transporters from Yarrowia lipolytica, S. cerevisiae EBY.VW4000 (strain deleted for all hexose transporters, kindly provided by Prof. Eckhard Boles [2]) was used as a host organism. The genes encoding sugar transporters (YALI0C06424g, YALI0C08943g, YALI0F19184g) were cloned into two types of pRS426 plasmid: with S. cerevisiae ADH1 promoter or TEF promoter. The replicative plasmids were cloned into S. cerevisiae.
The transformants were cultivated in YNB medium with maltose as carbon source. After 48 h of culture, cells were centrifuged, washed twice with distilled water and its OD600 was standardized to 10. Several decimal dilutions were prepared: 100, 10-1, 10-2, 10-3, 10-4, 10-5. After the dilutions, cells were spoted in drop test on YNB agar plated with 2% glucose.
Results
The analysis of promoter strength and its usefulness for expression of hexose transporters from Y. lipolytica in S. cerevisiae EBY.VW4000 was analyzed. The 2µ plasmid (pRS426) was used with ADH1 or TEF promoter. After incubation of S. cerevisiae transformants for 48 h at 30°C growth of transformants expressing each of the three transporters under TEF promoter was observed. In turn, expression of only YALI0C06424g transporter was observed when ADH1 promoter was used. The results clearly indicated that AHD1 promoter is not strong enough and thus not appropriate for the analysis of heterologous hexose transporters in the S. cerevisiae EBY.VW4000.
Source
genomic DNA of ADH1 from S. cerevisiae
References
Wroclaw's 2019 references
[1] Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955–964. doi:10.1002/yea.1806
[2] Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters, 464(3), 123–128. https://doi.org/10.1016/S0014-5793(99)01698-1
Tianjin's 2021 characterization
Characterization of ADH1 Promoter’s GFP expression in SY14 yeast
Group: Tianjin 2021
Author: Ruiqi Liu, Lingwei Ding
Summary:we characterized ADH1 Promoter’s strength in 4742 yeast.
Background
SY14 is a special kind of Artificially modified yeasts which contains only one chromosome fused by 16 chromosomes. SY14 yeast differs from normal yeast in many properties: growth rate, metabolic activity etc. We use GFP as reporter gene to test the ADH1 promoter’s strength in SY14. We constructed a GFP fragment that was controlled by the ADH1 promoter and bound it to the chromosome genome of SY14 yeast. We measured the GFP expression levels of SY14 yeast at 15h, 20h,25h and 30h by using microplate analyzer. Meanwhile, we used SY14 yeast (no GFP fragment) as control group.
Result
We use GFP as reporter gene to test the intensity of ADH1 promoter, and draw a bar chart with fluorescence intensity /OD as the ordinate and time as the abscissa.The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. The strength of ADH1 promoter reaches its peak at 15h.
