Difference between revisions of "Part:BBa K2084002"

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.

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.

In PURExpress, an in vitro protein synthesis reaction, maximum expression of the LacZ reporter protein regulated by the switch is reached within 2 hours.

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

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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+<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>
 <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%">
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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%">
-<p>In PURExpress, an <i>in vitro</i> protein synthesis reaction, maximum expression of the LacZ reporter protein regulated by the switch is reached by 2 hours.</p>
+<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%">