Difference between revisions of "Part:BBa K3019014"
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| + | <b>Description</b><br><br> | ||
| + | One part of our project was connected to the improvement of a previously described part BBa_K2711000 added to the registry by UiOslo_Norway 2018 team. BBa_K2711000 is a bacterial gene from <i>Arthrobacter luteus</i> coding for glucan hydrolase. As our goal was to induce yeast self-lysis, we needed to improve the functioning of BBa_K2711000 when expressed in yeast. As the first step, we codon-optimized it for use in <i>Saccharomyces cerevisiae</i>. BBa_K2711000 protein is part of a commercial enzyme mixture Zymolyase commonly used for yeast cell wall dissolution. When expressed in bacteria, BBa_K2711000 is secreted from the cells due to the presence of Tat-type secretion signal. This type of secretion signal, however, is inactive in yeast, so presumably, when expressed in yeast, BBa_K2711000 will accumulate in the cytoplasm, where it cannot affect the cell wall. To prevent it from accumulating in the cell and to increase its concentration close to the cell wall we have added yeast secretion signals from α-factor and Ost1 to the N-terminus of BBa_K2711000.<br><br> | ||
| + | We tested the activity of different BBa_K2711000(BBa) constructs using time-lapse microscopy to follow the effect of BBa expression induced at the start of the experiment. In the microscopy experiments it can be seen that while expression of BBa_K2711000 does have an effect on cell viability, the effect is more pronounced if BBa is fused with eukaryotic secretion signals. From the movies, induced lysis of some cells can be seen in case of Ost1-BBa expression, confirming that BBa_K2711000 when fused with Ost1 secretion signal, is able to promote yeast self-lysis. Additionally, 13% of cells expressing Ost1-BBa were dead after 20 hours of induction, compared to 7% of cells expressing unmodified BBa_K2711000 (Fig. 6).<br> | ||
| + | Therefore, our team has contributed to the improvement and development of this biobrick for use in yeasts.<br><br> | ||
| + | Video 1 (https://2019.igem.org/wiki/images/b/b1/T--Tartu_TUIT--Ost1-BBa.mp4) Time-lapse microscopy. On the right side, there are induced Ost1-BBa_K2711000 expressing cells. The left side depicts uninduced Ost1-BBa_K2711000 expressing cells. Yellow arrows indicate cell lysis. | ||
Latest revision as of 01:31, 22 October 2019
Pre-Ost1-pro-α-factor signal BBa_K2711000
BBa_K2711000 yeast codon optimised
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 395
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 510
Illegal BamHI site found at 730 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 399
Illegal NgoMIV site found at 761 - 1000COMPATIBLE WITH RFC[1000]
Description
One part of our project was connected to the improvement of a previously described part BBa_K2711000 added to the registry by UiOslo_Norway 2018 team. BBa_K2711000 is a bacterial gene from Arthrobacter luteus coding for glucan hydrolase. As our goal was to induce yeast self-lysis, we needed to improve the functioning of BBa_K2711000 when expressed in yeast. As the first step, we codon-optimized it for use in Saccharomyces cerevisiae. BBa_K2711000 protein is part of a commercial enzyme mixture Zymolyase commonly used for yeast cell wall dissolution. When expressed in bacteria, BBa_K2711000 is secreted from the cells due to the presence of Tat-type secretion signal. This type of secretion signal, however, is inactive in yeast, so presumably, when expressed in yeast, BBa_K2711000 will accumulate in the cytoplasm, where it cannot affect the cell wall. To prevent it from accumulating in the cell and to increase its concentration close to the cell wall we have added yeast secretion signals from α-factor and Ost1 to the N-terminus of BBa_K2711000.
We tested the activity of different BBa_K2711000(BBa) constructs using time-lapse microscopy to follow the effect of BBa expression induced at the start of the experiment. In the microscopy experiments it can be seen that while expression of BBa_K2711000 does have an effect on cell viability, the effect is more pronounced if BBa is fused with eukaryotic secretion signals. From the movies, induced lysis of some cells can be seen in case of Ost1-BBa expression, confirming that BBa_K2711000 when fused with Ost1 secretion signal, is able to promote yeast self-lysis. Additionally, 13% of cells expressing Ost1-BBa were dead after 20 hours of induction, compared to 7% of cells expressing unmodified BBa_K2711000 (Fig. 6).
Therefore, our team has contributed to the improvement and development of this biobrick for use in yeasts.
Video 1 (https://2019.igem.org/wiki/images/b/b1/T--Tartu_TUIT--Ost1-BBa.mp4) Time-lapse microscopy. On the right side, there are induced Ost1-BBa_K2711000 expressing cells. The left side depicts uninduced Ost1-BBa_K2711000 expressing cells. Yellow arrows indicate cell lysis.