Difference between revisions of "Part:BBa K2084002"
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+ | <p>This part contains a pT7 and a 5'UTR toehold switch that blocks translation of a gene placed downstream unless it is bound and unfolded by a DNA/RNA trigger molecule. As the data below suggests, this toehold switch provides a powerful regulatory element in any circuit and expression of the reporter is dependent on the concentration of the toehold switch in a cell-free reaction. For this specific trigger, see <a href="http://www.cell.com/abstract/S0092-8674(14)01291-4" target="_blank">Pardee <i>et. al.</i> 2014</a>. The sequence of the activating oligo trigger is AAGACAATGGTAAGTAGTAATAGATA.</p> | ||
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<p>The switch is activated with a complementary RNA strand in the Collins paper. As shown in the figure below, it can also be activated with a complementary DNA strand, though protein expression is lower. Activation is measured by the expression of the reporter <i>lacZ</i> in absorbance at 570 nm.</p> | <p>The switch is activated with a complementary RNA strand in the Collins paper. As shown in the figure below, it can also be activated with a complementary DNA strand, though protein expression is lower. Activation is measured by the expression of the reporter <i>lacZ</i> in absorbance at 570 nm.</p> | ||
<img src="https://static.igem.org/mediawiki/2016/d/d2/T--Pittsburgh--Parts_PT7Toehold_DNAvRNA.jpg" style="display:block; margin:auto; width:50%"> | <img src="https://static.igem.org/mediawiki/2016/d/d2/T--Pittsburgh--Parts_PT7Toehold_DNAvRNA.jpg" style="display:block; margin:auto; width:50%"> | ||
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<img src="https://static.igem.org/mediawiki/2016/f/f7/T--Pittsburgh--Parts_PT7Toehold_DNAtitration.jpg" style="display:block; margin:auto; width:50%"> | <img src="https://static.igem.org/mediawiki/2016/f/f7/T--Pittsburgh--Parts_PT7Toehold_DNAtitration.jpg" style="display:block; margin:auto; width:50%"> | ||
− | <p>In PURExpress, an <i>in vitro</i> protein synthesis reaction, maximum expression of the LacZ reporter protein regulated by the switch is reached | + | <p>In PURExpress, an <i>in vitro</i> protein synthesis reaction, maximum expression of the LacZ reporter protein regulated by the switch is reached within 2 hours.</p> |
<img src="https://static.igem.org/mediawiki/2016/f/f1/T--Pittsburgh--Parts_PT7Toehold_Kinetics.jpg" style="display:block; margin:auto; width:60%"> | <img src="https://static.igem.org/mediawiki/2016/f/f1/T--Pittsburgh--Parts_PT7Toehold_Kinetics.jpg" style="display:block; margin:auto; width:60%"> | ||
Latest revision as of 03:27, 30 October 2016
pT7 Toehold
Regulatory ribosome binding site with T7 RNAP promoter.
This part contains a pT7 and a 5'UTR toehold switch that blocks translation of a gene placed downstream unless it is bound and unfolded by a DNA/RNA trigger molecule. As the data below suggests, this toehold switch provides a powerful regulatory element in any circuit and expression of the reporter is dependent on the concentration of the toehold switch in a cell-free reaction. For this specific trigger, see Pardee et. al. 2014. The sequence of the activating oligo trigger is AAGACAATGGTAAGTAGTAATAGATA.
The switch is activated with a complementary RNA strand in the Collins paper. As shown in the figure below, it can also be activated with a complementary DNA strand, though protein expression is lower. Activation is measured by the expression of the reporter lacZ in absorbance at 570 nm.
![](https://static.igem.org/mediawiki/2016/d/d2/T--Pittsburgh--Parts_PT7Toehold_DNAvRNA.jpg)
The activation of the switch also increases with increasing concentration of trigger as shown below. Maximum activation is reached at a concentration of about 100 nM of DNA oligonucleotide trigger.
![](https://static.igem.org/mediawiki/2016/f/f7/T--Pittsburgh--Parts_PT7Toehold_DNAtitration.jpg)
In PURExpress, an in vitro protein synthesis reaction, maximum expression of the LacZ reporter protein regulated by the switch is reached within 2 hours.
![](https://static.igem.org/mediawiki/2016/f/f1/T--Pittsburgh--Parts_PT7Toehold_Kinetics.jpg)
References
Pardee K, Green AA, Ferrante T, Cameron DE, DaleyKeyser A, Yin P, Collins JJ. Paper-based synthetic gene networks. Cell 159 (4): 940-954, 2014.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]