Part:BBa_K1442006
Anti-theophylline Aptazyme
An aptazyme is an allosteric ribozyme acting as a regulatory riboswitch. It self-splices in response to theophylline, a drug used to treat asthma and other chronic obstructive pulmonary diseases acting as a bronchodilator. This differs from caffeine by one methyl group and a nitrogen atom creating the structure seen below. This is not endogenous to the body therefore we used this element as a "kill switch" for our system.
RNA-nased switchesregulating gene expression have become exceedingly useful in order to post-transcriptionally regulate genetic circuits and can process information and respond accordingly, for example to the concnetration of theophylline present. We use ribozymes in our construct for two purposes, firstly accosiated with an aptamer as the RNA kill switch and secondly preceeding and following the 5' and 3'UTRs respectively in order to provide a clean splicing with no scars following transcription in vitro. As programming of the circuits generated in synthetic biology become exponentially more complex these will be essential to regulate the replication and easily reprogram the circuit in vivo will be indispensable hence we thought this was one of the features most useful to characterize for RNA experimentation.
Group I aptazymes as genetic regulatory switches The aptazyme we used was from the Group I intron-splicing type. 5' and 3' splice sites are indicated with arrows and indicated where the ribozyme self-splices following binding of a theophylline molecule.
One RNA molecule binds to theophylline with a dissociation constant of 0.1microM and a significant conformational change occurs on binding. The addition of the aptamer to the ribozyme significantly increases the activation of the ribozyme when theophylline is added, creating a ribozyme activated 1600 times in saturating theophylline concentrations. [
THompson et al. 2002] When included in a system they can be used as knockouts for certain mRNAs or to make more economical gene therapies.Usage
This aptazyme was included as an RNA kill switch We put this structure centrally in our replicon in order to cause the maxmimum disruption when activated with theophylline.
A diagram of the sequence generated by Geneious can be seen below:
Mathematical Model
This part was determined to work via these models:
Where R= concentration of positive strand RNA
t=transfection time
and X=concentration of theophylline
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]
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